Patent Publication Number: US-2004053221-A1

Title: Methods for analyzing antibody response in an individual to major surface glycoprotein (Msg), immunological assays, and recombinant MsgA, MsgB and MsgC fragments

Description:
RELATED APPLICATION  
     [0001] This application claims priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 60/389,632 filed Jun. 18, 2002. 
    
    
     GOVERNMENT INTERESTS  
     [0002] This invention was made, at least in part, with funds from the Federal Government, awarded through grant numbers: National Institute of Health AACTG.27.Pc01, National Institute of Health Contract AI-753A, Veterans Administration Research Grant, and National Institute of Health Research Grant R01 HL-64570, SP-A. 
    
    
     
       FIELD OF THE INVENTION  
       [0003] The present invention is directed toward methods of analyzing antibody response to major surface glycoprotein (Msg) in an individual. The present invention is also directed to immunological assays for analyzing antibody response to major surface glycoprotein (Msg) in an individual. The present invention is further directed to recombinant MsgA, MsgB and MsgC fragments.  
       BACKGROUND OF THE INVENTION  
       [0004] Pneumocystis jiroveci  ( P. jiroveci ), a fungal opportunistic pathogen of humans previously known as  Pneumocystis carinii  fsp. hominis, is the causative agent of Pneumocystis pneumonia (PcP), a leading cause of serious illness in immunocompromised patients. The use of potent anti-human immunodeficiency virus (HIV) drugs has dramatically reduced the frequency of opportunistic infections, including  P. jiroveci , in HIV +  patients. Furthermore, the use of highly active anti-retroviral therapy (HAART) is associated with reconstitution of the immune system. Although the effects of HAART are generally measured by the increase in T cell numbers, data on reconstitution of organism-specific immune responses have been limited.  
       [0005] There is a high prevalence of anti- P. jiroveci  antibodies in normal healthy adults, with greater than 70% of sera positive for reactivity to  P. jiroveci  antigens. This level of reactivity is consistent with widespread exposure to  P. jiroveci  organisms, which happens during childhood. Serum antibodies reactive with  P. jiroveci  antigens are commonly found in children of less than 4 years of age. It is known that many proteins of  P. jiroveci  can be recognized by serum antibodies, but little information exists on the responses to specific  P. jiroveci  antigens, how those responses change over time and whether these responses are protective. This is, in part, due to the fact that the antigen preparations used in previous studies were crude homogenates from human or rodent lungs, and did not allow the identification of specific antigenic proteins. Furthermore, sera isolated in different parts of the world exhibited different patterns of reactivity to  P. jiroveci  antigens in Western blot analysis, suggesting that there may be variability between different  P. jiroveci  preparations, or that there are geographic differences in reactivity to  P. jiroveci  antigens.  
       [0006] One of the  P. jiroveci  proteins that is consistently recognized by serum antibodies and helper T cells is a 95 kD protein called the major surface glycoprotein (Msg) or glycoprotein A (gpA). This antigenic protein shares epitopes with the 120 kD antigen of rodent  Pneumocystis carinii  (P.c.), elicits protective B and T cell responses, and plays an important role in the interaction of Pneumocystis organisms with the host lung. Analysis of antibody reactivity to Msg has been hampered by the crude antigen preparations described above, and the inherently diverse nature of Msg molecules. For example, native Msg is recognized in only 30-40% of serum specimens from healthy individuals.  
       [0007] Accordingly, there is a need to simplify antigen preparations to analyze the immune response to a single Msg fragment. The identification of antigenic epitopes on a single isoform of Msg would allow tracking of the response to that specific epitope during a Pneumocystis infection, in convalescence following an episode of PcP, and in evaluating the antigen specific immune reconstitution of HIV+patients following HAART.  
       SUMMARY OF THE INVENTION  
       [0008] Accordingly, it is an object to provide methods to simplify antigen preparations to analyze the immune response to a single Msg fragment. In accordance with one aspect of the invention, there are provided methods of analyzing antibody response in an individual to major surface glycoprotein (Msg). The methods comprise analyzing a pattern of reactivity of a sample with a recombinant Msg fragment.  
       [0009] In accordance with another aspect of the invention, there are provided immunological assays for analyzing antibody response to major surface glycoprotein (Msg) in an individual. The immunological assays comprise recombinant Msg fragments.  
       [0010] In accordance with yet another aspect of the invention a recombinant MsgA fragment is provided. In accordance with a further aspect of the invention a recombinant MsgB fragment is provided. In accordance with yet a further aspect of the invention a recombinant MsgC fragment is provided. 
     
    
    
     DETAILED DESCRIPTION OF THE FIGURES  
     [0011]FIG. 1 illustrates Msg recombinants as compared with the full length Msg gene.  
     [0012]FIG. 2 shows a comparison of the deduced amino acid sequences of MsgA and the corresponding sequences of the known Msg gene products, wherein “*” indicates identity and “.” indicates space is introduced for optimal alignment.  
     [0013]FIG. 3 shows a purification profile of MsgB. Inclusion bodies containing MsgB were dissolved in urea (crude) and purified on HIS tag affinity columns, wherein PT is pour though, B is binding buffer, W is wash solution with the numerical values representing the molar strength of urea in the solution, E is protein eluted from the column and S is the protein stripped from the column at the end of the purification process.  
     [0014]FIG. 4 shows specificity of the Msg constructs as shown by antibody reactivity with MsgA, MsgB and MsgC but not with the control (pET). A: Reactivity of a polyclonal rabbit antiserum, raised against a cell wall preparation of  Pneumocystis jiroveci , for the Msg constructs. Clear reactivity to MsgC was seen after increase development time (MsgC + ) B: Rabbit antibodies eluted from MsgB (aMsgB) but not antibodies eluted from an irrelevant protein (neg.) react with a 95 kD protein (*) in a crude homogenate of infected human lung. C: the reactivity of human serum for each of the Msg recombinants.  
     [0015]FIG. 5A shows the frequency of reactivity of blood donor sera and sera from human immunodeficiency virus (HIV) +  patients to the Msg fragments. P=0.003* and 0.0086** when HIV −  and HIV + sera are compared.  
     [0016]FIG. 5B shows the frequency of reactivity of HIV+donors with prior PcP and those without exposure to PcP to the Msg fragments. P=0.0046* when PcP −  and PcP +  sera are compared. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0017] Pneumocystis jiroveci  ( P. jiroveci ), a fungal opportunistic pathogen of humans previously known as  Pneumocystis carinii  fsp. hominis, is the causative agent of Pneumocystis pneumonia (PcP), a leading cause of serious illness in immunocompromised patients. As noted above, one of the  P. jiroveci  proteins that is consistently recognized by serum antibodies and helper T cells is a 95 kD protein called the major surface glycoprotein (Msg) or glycoprotein A (gpA). This antigenic protein shares epitopes with the 120 kD antigen of rodent  Pneumocystis carinii  (P.c.), elicits protective B and T cell responses, and plays an important role in the interaction of Pneumocystis organisms with the host lung.  
     [0018] Msg is a family of related proteins that is encoded by multiple genes in the Pneumocystis genome. While not wishing to be bound by theory, it appears that only one Msg is expressed at a given time, suggesting that Pneumocystis species may evade the host immune system by switching the isoform of the protein expressed. Such antigenic variation is commonly used by microorganisms such as trypanosomes and the spirochete Borrelia. Analysis of antibody reactivity to Msg has been hampered by the crude antigen preparations described above and the inherently diverse nature of Msg molecules. For example, native Msg is recognized in only 3040% of serum specimens from healthy individuals.  
     [0019] The present inventors have found that identification of antigenic epitopes on a single isoform of Msg allow tracking of the response to that specific epitope during a Pneumocystis infection, in convalescence following an episode of PcP, and in evaluating the antigen specific immune reconstitution of HIV +  individuals following HAART. Accordingly, the inventors have generated a panel of recombinant Msg fragments that correspond to a single Msg molecule for use as a standardized set of reagents in immunological assays. The inventors have characterized the specificity of the fragments, and have further analyzed the patterns of reactivity of healthy and HIV +  serum samples to the recombinants.  
     [0020] In accordance with these findings, the present invention is directed to methods for analyzing antibody response in an individual to major surface glycoprotein (Msg). The methods comprise analyzing a pattern of reactivity of a sample with a recombinant Msg fragment. Additionally, the present invention is also directed to immunological assays for analyzing antibody response to major surface glycoprotein (Msg) in an individual. The immunological assays comprise recombinant Msg fragments. Furthermore, the present invention is directed to recombinant MsgA, MsgB and MsgC fragments.  
     [0021] In this work, the inventors have generated three overlapping recombinant fragments of human  P. jiroveci -derived Msg for use as standardized reagents in immunological assays. Each recombinant represents a single Msg molecule which can be analyzed independently. As used herein, “fragment” is intended to refer to a sequence of nucleic acids and/or amino acids encoding DNA, RNA, protein or combinations thereof. One skilled in the art will appreciate the various techniques for generating a recombinant Msg fragment, any of which may be employed herein. In one embodiment, recombinant Msg fragments are generated by cloning More specifically, oligonucleotides are designed based on the known sequence of  P. jiroveci  Msg, and are used in PCR to generate three overlapping fragments of a Msg gene. The sequences of the oligonucleotides used are as follows: 5′ AACTACCTTTAAAACTTTCAA 3′ (forward), 5′ TTAGGATCTGGATTCTGA 3′ (reverse) to generate msg 15-1119 , 5′ CGAAGCTTATGACTGCGCGAG 3′ (forward), 5′ TCCTCGGAGCTCTACTTTGAG 3′ (reverse) to generate msg 729-2282 , 5′ TTCCAAAACGCTACGTGTAA 3′ (forward), 5′ TCAATTGATGCTGAAGAGATG 3′ (reverse) to generate msg 2015-3332 . The sequence of the PCR products is confirmed, and they are cloned into the pET30 expression vector (Novagen, Madison, Wis.) in the correct reading frame and expressed in  E. coli . The 3 recombinant fragments are called MsgA, MsgB and MsgC which are encoded by the gene fragments msg 15-1119 , msg 729-2282  and msg 2015-3332 , respectively.  
     [0022] Given the difficulty in obtaining sufficient quantities of antigens from  P. jiroveci , and the inherent variability in Msg isotypes, these generated Msg fragments represent a unique opportunity to analyze immune responses to a single Msg molecule. The usefulness of these fragments for serological studies is underscored by the fact that each fragment can be recognized in a specific manner by both human and rabbit serum antibodies.  
     [0023] In order to analyze antibody response of an individual with major surface glycoprotein (Msg), a sample is obtained from an individual. As used herein, “individual” is intended to refer to an animal, including but not limited to humans, mammals, and rodents. Samples include, but are not limited to blood samples, tissue samples, serum samples, or combinations thereof. The obtained sample of the individual is contacted with a recombinant Msg fragment and a pattern of reactivity is analyzed. As used herein, “pattern of reactivity” is meant to refer to the representation of molecules, including but not limited to genes, proteins or combinations&#39; thereof, in an antibody response. One skilled in the art will appreciate the various techniques for analyzing a pattern of reactivity, any of which may be employed herein.  
     [0024] In a further embodiment, the step of analyzing the pattern of reactivity comprises the steps of determining the pattern of reactivity exhibited by the sample with the recombinant Msg fragment and comparing the pattern of reactivity to a predetermined pattern of reactivity. One skilled in the art will appreciate the various techniques for determining the pattern of reactivity, any of which may be employed herein. In one embodiment, the pattern of reactivity is determined by Western Blot analysis. As shown in the Example, the inventors have analyzed the patterns of healthy and HIV +  serum samples to the recombinant Msg fragments.  
     [0025] As further illustrated in the Example, western blot analysis of 95 randomly obtained serum samples from anonymous healthy donors demonstrates that each of the constructs can be recognized by human serum. Eighty of the 95 samples (84%) reacted with at least one of the recombinant Msg fragments. MsgB is recognized by 65% of the sera tested, whereas MsgA and MsgC are recognized by 40% and 41% of the sera respectively. Seventy-six percent (29/38) of the sera that recognizes MsgA and 69% (27/39) of the sera that recognizes MsgC, also recognizes MsgB. While not wishing to be bound by theory, this data suggests that MsgB may contain more commonly recognized epitopes.  
     [0026] This high level of reactivity in a healthy population of adults is consistent with prior exposure to Msg as an antigen, probably as a consequence of early childhood exposure to  P. jiroveci . Further, while the inventors do not wish to be bound by theory, it is interesting that such a large percentage of healthy samples reacted positively with the recombinant fragments (84%), because it suggests that either the variability in Msg epitopes recognized by B cells is restricted and biased towards cross-reactive epitopes, or that the responders in this study were exposed to the Msg that is represented in the recombinant fragments or were exposed to a dominant, localized population of  P. jiroveci  expressing a single Msg. However, the fact that serum samples collected from different global locations, and sera collected locally 10 years apart, exhibit essentially the same patterns of reactivity to the Msg recombinants, supports the former model theory. It has been suggested that Pneumocystis has the potential to evade the immune system by switching the isoform of Msg that is expressed, but in the absence of a continuous culture system for the organism, this theory is difficult to test.  
     [0027] While not wishing to be bound by theory, the inventors&#39; data, as set forth in the Example, suggests that there is a limited number of Msg epitopes that are recognized by serum antibodies, and that exposure to Msg results in cross-reactive antibodies being produced. This is shown by isolating a panel of recombinants that represent different Msgs, and analyzing the patterns of reactivity to those new fragments. Since reactivity to different Msgs can simply mean exposure to those Msgs, and not the presence of cross-reactive antibodies, antibody elution experiments elucidate the extent of cross-reactivity between different isoforms of Msg.  
     [0028] While not wishing to be bound by theory, the inventors believe that the titer of anti-Msg antibodies is important as a measurable parameter of infection. Further, the use of the Msg recombinants would be enhanced by the development of an ELISA system to measure quantitative differences in antibody titer among groups of subjects. Additionally, serum specimens may be obtained over time from patients who do and do not develop episodes of PcP to determine whether epitope recognition and the magnitude of the antibody response can be correlated with susceptibility to and recovery from PcP.  
     [0029] Analysis of the serum samples from four different countries shows that there is no significant difference in recognition patterns of the Msg fragments based on the geographical origin of the sera. These sera had previously shown differential reactivity to various high molecular weight  P. jiroveci  antigens, including Msg. However, while not wishing to be bound by theory, the inventors believe that the lack of variability in the response to the recombinant Msg fragments suggests that there is a limited number of Msg epitopes that can be seen by serum antibodies.  
     [0030] The ability of the inventors to correlate the reactivity of a given serum sample to a recombinant Msg fragment with its reactivity to the crude preparation of antigen previously analyzed suggests inherent differences in the preparations of antigen. While not wishing to be bound by theory, the inventors believe that the difference in results may reside in the fact that in the current study, the inventors directly analyze the response to recombinant fragments of Msg, whereas previous studies have analyzed the response to a complex mixture of  P. jiroveci  antigens derived from infected human lungs. Definitive analysis of a singe protein is very difficult when complex mixtures of proteins such as a lung extract are used.  
     [0031] Alternatively, while not wishing to be bound by theory, the inventors believe that the native Msg expressed in  P. jiroveci  is glycosylated, and the variability in reactivity to the native Msg is due to regional variations in glycosylation of the proteins. The present recombinants are expressed in  E. coli  and are not post-translationally modified. Therefore, the responses the inventors measure are directed at epitopes in the peptide backbone of Msg and are independent of glycosylation patters. Msg fragments may be engineered for expression in  Pichia pastoris , a yeast system that allows glycosylation of expressed proteins. While the glycosylation pattern of  P. pastoris  may not be the same as that of  P. jiroveci , expression of the Msg recombinants in this system enables the analysis of the effect of glycosylation on recognition by serum antibodies. Further, the patterns of serum reactivity to the Msg fragments expressed in two different systems can be compared.  
     [0032] The present data shows that healthy adults have a significantly higher prevalence of serum antibodies to recombinant Msg than HIV +  patients, but this frequency of recognition mainly resides in the MsgB fragment. HIV +  patients who recovered from PcP recognize MsgC significantly more frequently than HIV +  patients who never experienced PcP. Nine HIV +  patients who had a serum specimen drawn before they developed PcP did not recognize MsgC. These results are important because they demonstrate how the use of different fragments of a single recombinant antigen can reveal previously unrecognized differences among population groups.  
     [0033] While not wishing to be bound by theory, the inventors believe that it is possible that independent recognition of Msg fragments is of value in analyzing the diversity of epitopes during various stages of HIV infection or following HAART and further, that antibodies play a role in host defenses against PcP in HIV +  patients. Although there have been studies in humans, the most direct evidence for the importance of antibodies in the resolution of Pneumocystis infection has come from animal models of PcP. While not wishing to be bound by theory, the inventors believe that their results suggest that the B cell epitopes that elicit these antibody responses may reside in MsgC. Mapping of the epitopes recognized by such antibodies will be facilitated by localization within a single Msg fragment.  
     EXAMPLE  
     [0034] Materials and Methods  
     [0035] Cloning, Expression and Purification of Msg Fragments  
     [0036] Oligonucleotides are designed based on the known sequence of  P. jiroveci  Msg, and are used in PCR to generate three overlapping fragments of a Msg gene. The sequences of the oligonucleotides used are as follows: 5′ AACTACCTTTAAAACTTTCAA 3′ (forward), 5′ TTAGGATCTGGATTCTGA 3′ (reverse) to generate msg 15-1119 , 5′ CGAAGCTTATGACTGCGCGAG 3′ (forward), 5′ TCCTCGGAGCTCTACTTTGAG 3′ (reverse) to generate msg 729-2282 , 5′ TTCCAAAACGCTACGTGTAA 3′ (forward), 5′ TCAATTGATGCTGAAGAGATG 3′ (reverse) to generate msg 2015-3332 . The template used to generate msg 729-2282  and msg 2015-3332  is a λgt11 clone of human-derived Msg, whereas the template used to make msg 15-1119  is genomic DNA from  P. jiroveci -infected human lung.  
     [0037] The sequence of the PCR products is confirmed, and they are cloned into the pET30 expression vector (Novagen, Madison, Wis.) in the correct reading frame and expressed in  E. coli . The 3 recombinant fragments are called MsgA, MsgB and MsgC which are encoded by the gene fragments msg 15-1119 , msg 729-2282  and msg 2015-3332 , respectively. Recombinant protein expressed from the pET30 vector without insert is used as a control antigen. The recombinant proteins are expressed in inclusion bodies within  E. coli , and are purified using standard methods.  
     [0038] Briefly, bacterial cultures expressing recombinant Msg fragments are harvested by centrifugation, the cell pellet are sonicated and washed three times in binding buffer without urea (5 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9), and the final pellet is dissolved in binding buffer with 6M urea. The recombinant preparations are purified by affinity chromatography using HIS-binding resin (Novagen, Madison, Wis.) with the urea being removed in the wash stages. Eluted proteins are dialyzed overnight against PBS pH 7.4, filter sterilized and frozen at 70° C. Protein concentration is determined by A 280  using a standard curve generated with bovine serum albumin.  
     [0039] Western Blot Analysis  
     [0040] Recombinant Msg fragments are run on SDS-PAGE gels (Invitrogen, Carlsbad, Calif.), transferred to nitrocellulose and blocked in 1% non-fat milk in TTBS (20 mM THAM, 0.5M NaCl, 0.05% Tween-20) for 1 hour at room temperature. Blots are cut into strips and individual strips are incubated either overnight at 4° C., or for 2 hours at room temperature, with sera from healthy donors or HIV +  patients ({fraction (1/50)} dilution in TTBS), or with a rabbit polyclonal antiserum ({fraction (1/5000)} dilution in TTBS) generated against a cell wall prep of  P. jiroveci . The strips are washed three times with TTBS, and incubated either with horseradish peroxidase-labeled goat anti-human IgG or horseradish peroxidase-labeled goat anti-rabbit IgG (Kirkegaard &amp; Perry Laboratories, Gaithersburg, Md.) ({fraction (1/5000)} dilution in TTBS) for 1 hour at room temperature. The strips are washed three times in TTBS, color is developed with TMB membrane substrate (BioFX, Owings Mills, Md.), and the reactions are stopped with H 2 O.  
     [0041] To identify the appropriate bands for analysis and to serve as a positive control in our assay, one strip from each gel is blocked in milk as above, probed with horseradish peroxidase-labeled S protein (Novagen, Madison, Wis.) ({fraction (1/5000)} dilution in TTBS) for 1 hour at room temperature, washed three times and color developed as above. S protein reacts with the S•tag which is encoded in the pET30 vector and expressed as a fusion protein in our constructs, and therefore serves as a positive control. It does not react with  E. coli  proteins. As a negative control, one strip from each gel is blocked in milk as above, incubated with horseradish peroxidase-labeled goat anti-human IgG, washed and developed as above. The strips probed with human serum are compared to the positive and negative control strips. Each assay is read by two independent readers. Results are usually unequivocal, but in cases of ambiguous results, the assay is repeated at least once, and the predominant result from all reads of those samples is reported.  
     [0042] Samples from Volunteers  
     [0043] Samples are obtained from several different sources. Sera from 95 healthy adult blood donors are obtained from the Hoxworth Blood Bank in Cincinnati, Ohio. These samples are stored at −20° C. Four cohorts of serum samples, which had been obtained in 1990 from persons residing in different geographic regions, are also tested. These included sera from HIV seronegative Haitian women involved in a study of transmission of HIV infection; serum samples from South Africa obtained from healthy HIV seronegative men from a rural area; sera from South Korea obtained from HIV seronegative patients attending an outpatient clinic at the University Hospital, College of Medicine, Seoul National University; and serum samples from healthy blood donors from Hoxworth Blood Bank, Cincinnati, Ohio. These geographical samples have previously been tested for reactivity to a crude preparation of  P. jiroveci  isolated from human lung. Sera from 94 HIV +  patients are obtained from banked sera from the University of Cincinnati Infectious Diseases Center. The medical records of these subjects are abstracted to obtain the CD4 +  lymphocyte counts, HIV RNA levels closest to the time the samples (+/−3 months) and clinical history.  
     [0044] Antibody Elution Assay  
     [0045] Recombinant MsgB is run on SDS-PAGE gels, transferred to nitrocellulose and blocked in 1% non-fat milk in TTBS for 1 hour at room temperature. The location of the recombinant protein is identified by reactivity with S protein, and strips corresponding to the molecular mass of the recombinant are cut out and incubated either overnight at 4° C., or for 2 hours at room temperature, with a rabbit polyclonal antiserum (diluted {fraction (1/50)} with TTBS) generated against a cell wall preparation of human  P. jiroveci . The antiserum is removed and stored, and the strips are washed three times with TTBS. After the final wash, the strips are incubated with 0.1M glycine pH 2.5 for 5 minutes to release the bound antibodies. The released antibodies are removed, and the pH brought to neutrality with Tris-Cl pH 8.5. The blots are rinsed once with TTBS, the antiserum is reapplied and the bound antibodies are eluted as above a total of 3 times. The eluted antibodies are tested for reactivity to a crude preparation of  P. jiroveci  isolated from infected human lung using Western blot analysis.  
     [0046] Statistics  
     [0047] Statistical analysis is performed using Graphpad Instat (San Diego, Calif.). Sera from healthy blood donors are compared to those with HIV infection to determine the rates of reactivity. Sera from HIV seronegative subjects from Haiti, South Africa, Korea and Cincinnati are compared to determine the rates of reactivity. The inventors calculate that a sample size of 22 subjects per group is expected to have an 80% power to detect a 50% difference in the rate of reactivity (P&lt;0.05). The samples are correlated using a Kappa test. Statistical significance for categorical variables is determined by using a chi-square or Fisher&#39;s exact test where appropriate (P&lt;0.05).  
     [0048] Results  
     [0049] Specificity of the Msg Constructs  
     [0050] Using oligonucleotides specific for the sequence of a cloned msg gene, the inventors use PCR to generate 3 overlapping fragments spanning the entire length of the msg gene, illustrated in FIG. 1. Whereas msg 729-2282  and msg 2015-3332  are generated using the cloned msg gene as template, msg 15-1119  is generated using  P. jiroveci  DNA isolated from infected human lung as template. The nucleotide sequence of msg 15-1119  (Genbank AY072779) is 86% identical to the corresponding sequence of the cloned msg gene used as a template for the remaining fragments, and the deduced amino acid sequence is 74% identical to that of the cloned gene. The sequence of msg 15-1119  also exhibits 67-70% identity at the nucleotide level and 57-60% identity at the deduced amino acid level with the corresponding portion of other cloned msg genes, as illustrated in FIG. 2.  
     [0051] The 3 recombinant fragments of Msg (MsgA, MsgB and MsgC) are expressed in  E. coli , and are purified away from contaminating bacterial proteins using affinity chromatography. A typical purification profile for MsgB is shown in FIG. 3, with similar profiles being obtained for the other fragments and for the control protein (pET expression vector without insert. Whereas the purification of MsgA and MsgB consistently gives a single band on SDS-PAGE analysis, the profiles for purification of MsgC always has multiple bands in addition to the full length fragment. Presumably these are breakdown products that are co-purified along with the full length fragment because they express the S•tag used for purification. These extra bands are not taken into consideration when scoring reactivity to the MsgC fragment; only reactivity to the full length band is recorded.  
     [0052] To investigate the utility of the recombinant fragments in analysis of human serum antibody reactivity, the inventors demonstrate the specificity of the fragments and that human serum can recognize all 3 fragments. The specificity of the Msg fragments is illustrated in three ways in FIG. 4. First, a polyclonal rabbit antiserum that is raised against a cell wall preparation of human  P. jiroveci  that includes native Msg, is tested for reactivity to the recombinant fragments in Western blot. All three fragments are recognized by the rabbit antiserum, even though reactivity with MsgC is weak. Second, antibodies from the rabbit antiserum that recognized MsgB are eluted from the Msg fragment and are tested for reactivity to the cell wall preparation used to generate the antiserum. The eluted antibodies show specific reactivity with a protein band whose molecular mass corresponded with that of native Msg and not with any other bands. Finally, both rabbit and human sera (more than 300 human sera tested) show no reactivity to the control protein encoded by the expression vector without insert, showing that the reactivity seen in the inventive assays is directed at the Msg fragments and not at the portion of the recombinants encoded by the expression vector. The reactivity profile of a sample of human serum that recognizes all 3 recombinant Msg fragments but not the control protein, is shown in FIG. 4C.  
     [0053] Recognition of Msg Fragments by Human Serum  
     [0054] The inventors test 95 sera from healthy volunteers for the ability to recognize the Msg constructs by Western blot analysis. Eighty of the 95 samples (84%) recognize at least one of the constructs, whereas 15 (16%) of the sera do not recognize any of the fragments, as depicted in Table 1. Thirty-eight of the serum samples (40%) recognize MsgA whereas 61 sera (64%) and 39 sera (41%) recognize MsgB and MsgC, respectively. Each of the fragments is recognized independently of the others, yielding 8 patterns of recognition. None of the sera tested reacts with the control protein expressed by the empty vector.  
     [0055] Comparison of Sera from Different Geographic Locations  
     [0056] To determine if recognition of the Msg fragments is a function of the geographical source of the serum samples, the inventors test four panels of sera that originated in different geographic locations for the ability to recognize the recombinant fragments. Twenty-two sera each from the US, Haiti, South Africa and South Korea, respectively, are tested by Western blot analysis for the ability to recognize the Msg fragments as depicted in Table 1.  
               TABLE 1                          Recognition of Msg fragments by human immunodeficiency virus       (HIV) negative sera from 4 distinct geographic regions                                                     South   South           USA a     USA b     Haiti b     Africa b     Korea b         Fragment recognized:   (n = 95)   (n = 22)   (n = 22)   (n = 22)   (n = 22)               MsgA alone    7%   4.5%     9%     0%     0%       MsgB alone   21%   4.5%     9%     14%     9%       MsgC alone   10%     9%     9%     18%    4.5%       MsgA + MsgB   15%    14%   4.5%     9%     0%       MsgA + MsgC    2%     0%     9%     9%     9%       MsgB + MsgC   13%    27%    14%     14%     23%       MsgA + MsgB +   16%    18%   4.5%    4.5%     9%       MsgC       None   16%    23%    41%   31.5%   45.5%       Total MsgA   40%    36%    27%     23%     18%       Total MsgB   64%    64%    32%     41%     41%       Total MsgC   41%    55%    36%   45.5%   45.5%                                          
 
     [0057] Each of the three Msg fragments can be recognized by sera from each country, and the frequency of recognition did not vary significantly between the groups of sera. Seventeen of 22 sera from the US (77%) react with one or more of the fragments, whereas 59% of Haitian sera, 68% of South African sera and 55% of South Korean sera reacts with one or more of the fragments. Furthermore, the recognition of any one of the eight possible patterns is not statistically different between the panels of serum samples when compared using a Kappa test; for example 36% of US sera (n=8/22), 27% of Haitian sera (n=6/22), 23% of South African sera (n=5/22) and 18% of South Korean sera (n=4/22) recognized MsgA. To determine if the apparent variation in reactivity to the Msg fragments is due to the relatively small number of samples tested, the inventors analyze additional 45 sera from Korea for reactivity to MsgA. When all 67 Korean sera are analyzed together, 39% (26/67) are positive for reactivity to MsgA, compared with 36% and 38% of US sera (1990 and 2000 respectively).  
     [0058] Analysis of HIV +  Sera  
     [0059] To determine if the pattern of recognition of the Msg recombinants seen in healthy volunteers (Table 1) is similar in HIV +  patients, the inventors assay serum from 94 HIV +  donors, illustrated in FIG. 5A The median CD4 +  lymphocyte count within 3 months of the collection of sera is 94 cells/mm 3  (n=86). The median HIV-1 RNA level is 87,000 copies/ml (n=67). Few subjects, 4.4% (n=3/67), with HIV infection have an HIV-1 RNA level &lt;400 copies/ml. Thirty-five subjects have a prior episode of histologically proven PcP.  
     [0060] The overall reactivity of the HIV +  sera is lower than the healthy sera, in that 32/94 of the HIV +  sera (34%) do not react with any of the fragments, whereas only 15/95 (16%) of the healthy sera showed lack of reactivity (P=0.003). The reactivity to individual fragments of Msg varies depending on the source of the serum, and the identity of the fragment being tested. Both MsgA and MsgB are recognized more frequently by healthy sera than by HIV +  sera. MsgA is recognized by 38 out of 95 healthy sera (40%) but by only 25/94 HIV +  sera (P=0.064). However, the difference in recognition of MsgB is significant in that 61/95 of healthy sera (64%) and 42/94 of HIV +  sera (45%) recognize the fragment (P=0.0086). There is no difference in frequency of recognition of MsgC as 39/95 healthy and 37/94 HIV+sera (41% and 39% respectively) recognize the fragment.  
     [0061] The reactivity of HIV +  sera to individual fragments of Msg varies depending on whether the patient had a previous bout of PcP (FIG. 5B. Twenty of 34 PcP+patients (59%) recognize MsgC compared with only 28% of 60 PcP −  patients (P=0.0046). Of particular interest, none of the 0.9 HIV +  patients who went on to develop PcP after their serum specimen is obtained had antibodies to MsgC. There is no significant difference in the rates of reactivity to the other Msg fragments between the groups of sera (FIG. 5B. There is also no significant difference in frequency of reactivity to any of the fragments when the HIV +  sera are separated based on CD4 +  T cell count (comparing those with &lt;100 CD4 +  T cells/mm 3  to those with &gt;100 CD4 +  T cells/mm 3 ) or when separated based on viral titer (comparing those with &lt;100000 viral copies/ml to those with &gt;100000 viral copies/ml). The data provided in the Example illustrates that these novel recombinant Msg fragments are useful for analyzing antibody response in an individual.  
    
     
       
         1 
         
           
             13  
           
           
             1  
             336  
             PRT  
             Human  
           
            1 

Leu Ala Arg Ala Val Ala Arg Ala Val Lys Arg Arg Gly Ala Ala Ala 
1               5                   10                  15 

Gln Gly Thr Ser Val Tyr Asn Asp Glu Glu Ile Leu Leu Ala Leu Ile 
            20                  25                  30 

Leu Lys Glu Asp Gly Leu Glu Glu Lys Lys Cys Lys Glu Lys Leu Lys 
        35                  40                  45 

Arg Tyr Cys Glu Ala Leu Lys Asn Val Thr Leu Thr Ala Asp Lys Ile 
    50                  55                  60 

His Glu Lys Leu Lys Asp Phe Cys Asn Asn Gly Asn Glu Glu Lys Lys 
65                  70                  75                  80 

Cys Lys Gly Leu Lys Asp Lys Ile Glu Lys Lys Cys Asn Asp Phe Lys 
                85                  90                  95 

Lys Asp Lys Leu Glu Lys Lys Leu Thr Asn Pro Ser Asp Asn Asp Cys 
            100                 105                 110 

Lys Glu His Glu Arg Gln Cys Leu Phe Leu Glu Gly Ala Cys Pro Ser 
        115                 120                 125 

Asp Leu Ile Glu Asn Cys Asn Lys Leu Arg Asn Lys Cys Tyr Gln Lys 
    130                 135                 140 

Lys Arg Asp Arg Val Ala Glu Glu Val Leu Leu Arg Ala Leu Arg Gly 
145                 150                 155                 160 

Asp Leu Glu Lys Glu Thr Glu Cys Glu Lys Lys Ile Lys Asp Val Cys 
                165                 170                 175 

Pro Lys Ile Gly Gln Glu Ser Asp Glu Leu Thr Leu Leu Cys Leu Asp 
            180                 185                 190 

Gln Lys Lys Thr Cys Met Asn Leu Ile Thr Ala Arg Glu Lys Lys Cys 
        195                 200                 205 

Asn Lys Leu Glu Glu Asp Val Lys Lys Ala Leu Glu Asn Lys Asn Asn 
    210                 215                 220 

Leu Leu Gly Lys Cys Leu Pro Leu Leu Glu Gln Cys Tyr Phe His Arg 
225                 230                 235                 240 

Gly Asp Cys Lys Lys Lys Ala Ser Gln Cys Lys Pro Pro Asn Lys Asp 
                245                 250                 255 

Cys Glu Asp Tyr Leu Pro Lys Cys Asp Glu Leu Ala Glu Glu Cys Gly 
            260                 265                 270 

Lys Lys Gly Ile Ile Tyr Ile His Pro Gly Pro Asp Phe Asp Pro Thr 
        275                 280                 285 

Lys Pro Glu Pro Thr Val Ala Glu Asp Ile Gly Leu Glu Glu Leu Tyr 
    290                 295                 300 

Lys Lys Ala Ala Glu Asp Gly Val His Ile Gly Lys Pro Pro Val Arg 
305                 310                 315                 320 

Asp Ala Thr Ala Leu Leu Ala Leu Leu Ile Gln Asn Pro Asp Pro Lys 
                325                 330                 335 

 
           
             2  
             329  
             PRT  
             Human  
           
            2 

Val Ala Arg Ala Val Lys Arg Gln Val Ala Gly Val Lys Asn Asn Glu 
1               5                   10                  15 

Ala Glu Glu Arg Leu Phe Ala Leu Ile Met Arg Ala Asp Tyr Lys Asp 
            20                  25                  30 

Glu Ser Lys Cys Lys Asn Lys Ile Lys Glu Tyr Cys Asp Gly Leu Lys 
        35                  40                  45 

Asn Ala Ser Leu Thr Ser Glu Glu Val His Lys Glu Leu Lys Asp Phe 
    50                  55                  60 

Cys Lys Asp Gly Ser Gln Gly Lys Lys Cys Glu Glu Leu Lys Lys Asn 
65                  70                  75                  80 

Val Glu Ala Lys Cys Asn Asn Phe Lys Thr Lys Leu Glu Gly Leu Val 
                85                  90                  95 

Lys Lys Asp Ala Ser Gly Leu Thr Asn Asp Asp Cys Lys Glu Asn Glu 
            100                 105                 110 

Arg Gln Cys Leu Phe Leu Glu Gly Ala Cys Pro Asp Leu Val Glu Asp 
        115                 120                 125 

Cys Ser Lys Leu Arg Asn Leu Cys Tyr Gln Lys Lys Arg Glu Gly Val 
    130                 135                 140 

Ala Glu Glu Val Leu Leu Arg Ala Leu Arg Gly Asp Leu Gly Asn Lys 
145                 150                 155                 160 

Thr Glu Cys Glu Lys Lys Ile Lys Asp Val Cys Pro Lys Ile Gly Gln 
                165                 170                 175 

Glu Ser Asp Glu Leu Thr Leu Leu Cys Leu Asp Gln Lys Lys Thr Cys 
            180                 185                 190 

Thr Asn Leu Met Thr Ala Arg Asp Lys Lys Cys Asn Thr Leu Glu Glu 
        195                 200                 205 

Asp Val Lys Lys Ala Leu Glu Asn Lys Asn Asn Leu Leu Gly Lys Cys 
    210                 215                 220 

Leu Pro Leu Leu Glu His Ala Thr Phe Thr Glu Gly Thr Ala Lys Lys 
225                 230                 235                 240 

Ala Ser Gln Cys Thr Pro Asn Lys Asp Cys Glu Asp Tyr Leu Pro Lys 
                245                 250                 255 

Cys Asp Glu Leu Ala Glu Glu Cys Gly Lys Lys Gly Ile Ile Tyr Ile 
            260                 265                 270 

His Pro Gly Pro Asp Phe Asp Pro Thr Lys Pro Glu Pro Thr Val Ala 
        275                 280                 285 

Glu Asp Ile Gly Leu Glu Glu Leu Tyr Lys Lys Ala Ala Glu Asp Gly 
    290                 295                 300 

Val His Ile Gly Lys Pro Pro Val Arg Asp Ala Thr Ala Leu Leu Ala 
305                 310                 315                 320 

Leu Leu Ile Gln Asn Pro Asp Pro Lys 
                325 

 
           
             3  
             331  
             PRT  
             Human  
           
            3 

Ala Arg Ala Val Lys Arg Arg Ala Lys Gly Ala Gln Asn Ser Ile Asp 
1               5                   10                  15 

Glu Glu His Val Leu Ala Leu Ile Leu Lys Lys Asn Gly Leu Glu Asp 
            20                  25                  30 

Thr Lys Cys Lys Thr Lys Leu Glu Glu Tyr Cys Lys Thr Leu Thr Asn 
        35                  40                  45 

Ala Gly Leu Asn Pro Glu Lys Val His Glu Lys Leu Lys Asp Phe Cys 
    50                  55                  60 

Asp Asn Gly Lys Arg Asn Glu Lys Cys Gln Asp Leu Lys Asn Lys Val 
65                  70                  75                  80 

Asn Gln Lys Cys Ile Lys Phe Gln Gly Lys Leu Gln Thr Ala Ala Gly 
                85                  90                  95 

Lys Lys Ile Ser Glu Leu Thr Asp Glu Asp Cys Lys Lys Asn Glu Gln 
            100                 105                 110 

Gln Cys Leu Phe Leu Glu Gly Ala Cys Pro Thr Glu Leu Lys Asp Asp 
        115                 120                 125 

Cys Asn Lys Leu Arg Asn Asn Cys Tyr Gln Lys Glu Arg Asn Asn Val 
    130                 135                 140 

Ala Glu Glu Val Leu Leu Arg Ala Leu Arg Gly Asp Leu Asn Glu Thr 
145                 150                 155                 160 

Lys Thr Cys Glu Lys Lys Leu Lys Glu Val Cys Pro Lys Leu Glu Arg 
                165                 170                 175 

Glu Ser Asp Glu Leu Thr Glu Leu Cys Leu Tyr Gln Lys Thr Thr Cys 
            180                 185                 190 

Val Ser Leu Val Thr Lys Gly Lys Ser Lys Cys Asp Thr Leu Glu Lys 
        195                 200                 205 

Glu Val Glu Glu Ala Leu Lys Lys Asn Glu Leu Arg Glu Lys Cys Leu 
    210                 215                 220 

Leu Leu Leu Glu Gln Cys Tyr Phe His Arg Gly Asn Cys Glu Gly Asp 
225                 230                 235                 240 

Lys Ser Lys Cys Asn Lys Pro Asn Asn Lys Asp Cys Lys Glu Tyr Val 
                245                 250                 255 

Pro Glu Cys Asp Glu Leu Ala Glu Lys Cys Gly Lys Glu Asn Ile Val 
            260                 265                 270 

Tyr Met His Pro Gly Ser Asp Phe Asp Pro Thr Lys Pro Glu Pro Thr 
        275                 280                 285 

Leu Ala Glu Asp Ile Gly Leu Glu Glu Leu Tyr Lys Arg Ala Glu Glu 
    290                 295                 300 

Asp Gly Ile Phe Val Gly Arg Gln His Val Arg Asp Ala Thr Ala Leu 
305                 310                 315                 320 

Leu Ala Leu Leu Leu Lys Lys Thr Leu Lys Lys 
                325                 330 

 
           
             4  
             331  
             PRT  
             Human  
           
            4 

Ala Arg Ala Val Lys Arg Gln Ala Lys Gly Ala Gln Asn Ser Ile Asp 
1               5                   10                  15 

Glu Glu His Val Leu Ala Leu Ile Leu Lys Lys Asn Gly Leu Glu Asp 
            20                  25                  30 

Thr Lys Cys Lys Thr Lys Leu Glu Glu Tyr Cys Lys Thr Leu Thr Asn 
        35                  40                  45 

Ala Gly Leu Asn Pro Glu Lys Val His Glu Lys Leu Lys Asp Phe Cys 
    50                  55                  60 

Asp Asn Gly Lys Arg Asn Glu Lys Cys Gln Asp Leu Lys Asn Lys Val 
65                  70                  75                  80 

Asn Gln Lys Cys Ile Lys Phe Gln Gly Lys Leu Gln Thr Ala Ala Arg 
                85                  90                  95 

Lys Lys Ile Ser Glu Leu Thr Asp Glu Asp Cys Lys Lys Asn Glu Gln 
            100                 105                 110 

Gln Cys Leu Phe Leu Glu Gly Ala Cys Pro Thr Glu Leu Lys Asp Asp 
        115                 120                 125 

Cys Asn Lys Leu Arg Asn Asn Cys Tyr Gln Lys Glu Arg Asn Asn Val 
    130                 135                 140 

Ala Glu Glu Val Leu Leu Arg Ala Leu Arg Gly Asp Leu Asn Glu Thr 
145                 150                 155                 160 

Lys Thr Cys Glu Lys Lys Leu Lys Glu Val Cys Pro Lys Leu Glu Arg 
                165                 170                 175 

Glu Ser Asp Glu Leu Thr Glu Leu Cys Leu Tyr Gln Lys Thr Thr Cys 
            180                 185                 190 

Val Ser Leu Val Thr Lys Gly Lys Ser Lys Cys Asp Thr Leu Glu Lys 
        195                 200                 205 

Glu Val Glu Glu Ala Leu Lys Lys Asn Glu Leu Arg Glu Lys Cys Leu 
    210                 215                 220 

Leu Leu Leu Glu Gln Cys Tyr Phe His Arg Gly Asn Cys Glu Gly Asp 
225                 230                 235                 240 

Lys Ser Lys Cys Asn Lys Pro Asn Asn Lys Asp Cys Lys Glu Tyr Val 
                245                 250                 255 

Pro Glu Cys Asp Glu Leu Ala Glu Lys Cys Gly Lys Glu Asn Ile Val 
            260                 265                 270 

Tyr Met His Pro Gly Ser Asp Phe Asp Pro Thr Lys Pro Glu Pro Thr 
        275                 280                 285 

Leu Ala Glu Asp Ile Gly Leu Glu Glu Leu Tyr Lys Arg Ala Glu Glu 
    290                 295                 300 

Asp Gly Ile Phe Val Gly Arg Gln His Val Arg Asp Ala Thr Ala Leu 
305                 310                 315                 320 

Leu Ala Leu Leu Leu Lys Lys Thr Leu Lys Lys 
                325                 330 

 
           
             5  
             279  
             PRT  
             Human  
           
            5 

Lys Pro Glu Lys Val His Lys Lys Leu Lys Glu Phe Cys Glu Asn Lys 
1               5                   10                  15 

Lys Ala Asp Ser Lys Cys Lys Glu Leu Lys Glu Lys Leu Thr Gln Lys 
            20                  25                  30 

Cys Thr Ala Ile Lys Gly Lys Leu Thr Glu Ala Ile Lys Lys Lys Asn 
        35                  40                  45 

Ser Asp Leu Thr Asp Glu Asp Cys Lys Glu Asn Glu Gln Gln Cys Leu 
    50                  55                  60 

Phe Leu Glu Gly Ala Cys Pro Ala Glu Leu Lys Asp Asp Cys Asn Thr 
65                  70                  75                  80 

Leu Arg Asn Lys Cys Tyr Gln Lys Lys Arg Asp Lys Val Ala Glu Glu 
                85                  90                  95 

Ala Leu Leu Arg Ala Val Arg Gly Gly Leu Ile Asn Glu Thr Thr Cys 
            100                 105                 110 

Glu Gly Lys Leu Lys Glu Val Cys Ile Glu Leu Ser Gln Glu Ser Asp 
        115                 120                 125 

Glu Leu Thr Lys Leu Cys Leu Tyr Gln Lys Met Thr Cys Lys Thr Phe 
    130                 135                 140 

Val Leu Glu Lys Gln Lys Lys Cys Asn Ala Leu Lys Gln Asp Val Asn 
145                 150                 155                 160 

Ala Ala Leu Glu Lys Lys Asp Glu Leu Arg Gly Lys Cys Leu Pro Leu 
                165                 170                 175 

Leu Glu Arg Cys Tyr Phe Tyr Arg Gly Asn Cys Glu Asp Ile Ser Lys 
            180                 185                 190 

Cys Asn Lys Ser Ser Glu Asp Cys Tyr Glu Tyr Leu Pro Val Cys Asp 
        195                 200                 205 

Thr Leu Ala Val Lys Cys Glu Glu Asn Lys Ile Ile Tyr Thr His Pro 
    210                 215                 220 

Gly Ser Asp Phe Asn Pro Thr Lys Ser Lys Pro Thr Val Ala Glu Asp 
225                 230                 235                 240 

Ile Gly Leu Glu Glu Leu Tyr Lys Lys Ala Ala Glu Glu Gly Val His 
                245                 250                 255 

Ile Gly Lys Pro Pro Val Arg Asp Ala Thr Ala Leu Leu Ala Leu Leu 
            260                 265                 270 

Ile Gln Asn Leu Asp Pro Lys 
        275 

 
           
             6  
             316  
             PRT  
             Human  
           
            6 

Ala Arg Ala Val Lys Arg Gln Val Thr Gly Ala Ser Gly Gln Tyr Asp 
1               5                   10                  15 

Asp Glu Val Asn Ile Leu Ala Leu Ile Leu Gln Glu Asp Ala Met Glu 
            20                  25                  30 

Asp Thr Lys Cys Lys Lys Ser Leu Glu Lys Tyr Cys Glu Glu Leu Lys 
        35                  40                  45 

Lys Ala Ser Leu Asp Met Glu Lys Val His Lys Met Leu Lys Asp Phe 
    50                  55                  60 

Cys Gly Asn Gly Lys Ala Ser Lys Ala Asn Thr Lys Cys Gln Gly Leu 
65                  70                  75                  80 

Gln Ala Lys Val Thr Gly Lys Cys Thr Asn Phe Lys Thr Gln Lys Leu 
                85                  90                  95 

Gly Pro Ala Leu Thr Asn Pro Ser Asp Asp Asn Cys Lys Glu Ser Glu 
            100                 105                 110 

Arg Gln Cys Leu Phe Leu Glu Gly Ala Cys His Asn Leu Val Glu Asp 
        115                 120                 125 

Cys Asn Lys Leu Arg Asn Leu Cys Tyr Gln Lys Lys Arg Asp Gly Val 
    130                 135                 140 

Ala Glu Glu Val Leu Leu Arg Ala Leu Arg Ser Asp Leu Asn Lys Thr 
145                 150                 155                 160 

Glu Thr His Glu Lys Lys Leu Lys Glu Ile Cys Pro Val Leu Gln Arg 
                165                 170                 175 

Glu Ser Asn Glu Leu Thr Asp Leu Cys Leu Asn Gln Lys Lys Thr Cys 
            180                 185                 190 

Glu Asn Ile Ile Lys Glu Lys Asp Lys Lys Cys Thr Thr Leu Lys Ala 
        195                 200                 205 

Asn Val Ala Thr Ala Leu Gly Ser Phe Lys Lys Glu Ile Cys Leu Glu 
    210                 215                 220 

Leu Leu Glu Gln Cys Tyr Phe Tyr Ile Gly Asn Cys Gly Asp Asp Asp 
225                 230                 235                 240 

Ile Ile Lys Cys Ile Glu Leu Gly Gly Lys Cys Gln Glu Gln Asn Ile 
                245                 250                 255 

Val Tyr Ile Pro Pro Gly Pro Asp Phe Asp Pro Thr Arg Pro Glu Ala 
            260                 265                 270 

Thr Leu Ala Glu Asp Ile Asp Leu Asp Glu Leu Tyr Lys Lys Ala Glu 
        275                 280                 285 

Glu Asp Gly Val Phe Ile Gly Lys His His Leu Arg Asp Ala Thr Ala 
    290                 295                 300 

Leu Leu Thr Leu Leu Val Lys Lys Asp Asp Thr Gly 
305                 310                 315 

 
           
             7  
             316  
             PRT  
             Human  
           
            7 

Ala Arg Ala Val Lys Arg Gln Ala Ala Gly Thr Gln Asn Ser Ile Asp 
1               5                   10                  15 

Glu Glu His Val Leu Ala Leu Ile Leu Lys Glu Asp Gly Leu Ser Glu 
            20                  25                  30 

Gln Glu Cys Lys Lys Lys Leu Lys Lys Tyr Cys Gln Glu Leu Thr Glu 
        35                  40                  45 

Ala Lys Leu Asn Ile Glu Gln Val His Arg Lys Leu Lys Gly Phe Cys 
    50                  55                  60 

Glu Asp Gly Lys Ala Asp Thr Lys Cys Lys Glu Leu Lys Ala Asn Ile 
65                  70                  75                  80 

Glu Lys Lys Cys Thr Thr Ile Lys Gly Lys Leu Lys Glu Ala Ile Lys 
                85                  90                  95 

Lys Lys Ile Gln Ile Ile Thr Asp Lys Asp Cys Lys Glu Asn Glu Gln 
            100                 105                 110 

Gln Cys Leu Phe Leu Glu Gly Val Cys Ser Lys Glu Leu Lys Asp Asp 
        115                 120                 125 

Cys Asn Thr Leu Arg Asn Lys Cys Tyr Gln Lys Lys Arg Asp Lys Val 
    130                 135                 140 

Ala Glu Glu Val Leu Leu Arg Ala Leu Arg Ser Asp Leu Asn Gly Ser 
145                 150                 155                 160 

Val Ile Cys Glu Lys Lys Leu Lys Glu Ile Cys Pro Val Met Gly Arg 
                165                 170                 175 

Glu Ser Asp Glu Leu Thr Asn Leu Cys Leu Asn Gln Lys Glu Thr Cys 
            180                 185                 190 

Lys Asn Ile Leu Ile Glu Lys Asp Lys Lys Cys Gly Thr Leu Lys Thr 
        195                 200                 205 

Asp Val Ser Ala Ala Leu Gly Ser Phe Lys Lys Glu Thr Cys Leu Glu 
    210                 215                 220 

Leu Leu Glu Gln Cys Tyr Phe Tyr Ile Gly Asn Cys Gly Asp Asp Asp 
225                 230                 235                 240 

Ile Ile Lys Cys Ile Glu Leu Gly Gly Lys Cys Gln Glu Gln Asn Ile 
                245                 250                 255 

Ala Tyr Met Pro Pro Gly Pro Asp Phe Asp Pro Thr Arg Pro Glu Ala 
            260                 265                 270 

Thr Ile Ala Glu Asp Ile Gly Leu Glu Glu Phe Tyr Lys Lys Val Glu 
        275                 280                 285 

Glu Asp Gly Val Phe Ile Gly Lys Asn His Leu Arg Asp Ala Thr Ala 
    290                 295                 300 

Leu Leu Ala Leu Leu Ile Gln Asp Ser Ser Leu Lys 
305                 310                 315 

 
           
             8  
             21  
             DNA  
             Human  
           
            8 

aactaccttt aaaactttca a                                               21 

 
           
             9  
             18  
             DNA  
             Human  
           
            9 

ttaggatctg gattctga                                                   18 

 
           
             10  
             21  
             DNA  
             Human  
           
            10 

cgaagcttat gactgcgcga g                                               21 

 
           
             11  
             21  
             DNA  
             Human  
           
            11 

tcctcggagc tctactttga g                                               21 

 
           
             12  
             20  
             DNA  
             Human  
           
            12 

ttccaaaacg ctacgtgtaa                                                 20 

 
           
             13  
             21  
             DNA  
             Human  
           
            13 

tcaattgatg ctgaagagat g                                               21