Patent Application: US-200913514987-A

Abstract:
the invention relates to fusion proteins comprising an amino acid sequence of a fragment h corresponding to a fragment of a calcium binding protein excreted - secreted by adult worms of fasciola hepatica , followed by an amino acid sequence corresponding to a unrelated protein or fragment of protein , pharmaceutical compositions , vaccines and adjuvants containing the immunogen , to a process for their preparation , another process for the production of antibodies and their use . the present invention relates to the preparation of immunogens by the addition of a peptide sequence . thus the present invention is useful for producing an immune response , with increases in specific antibody titers in serum against proteins or other antigens and can be applied in particular for the production of specific polyclonal antibodies , immunotherapy and immunoprophylaxis . the addition of the polypeptide to a target antigen , either through the production of recombinant proteins containing the polypeptide or by addition or fusion of this polypeptide with the target antigen , induces a significant increase in the immunogenicity of these molecules , amplifying the immune response elicited by injection of this molecule in a subject susceptible to produce antibodies .

Description:
the statements described to validate the application are based on the use of vector pqe ( qiagen ), commonly used for research purposes . the various constructs were subcloned into the expression vector in escherichia coli pqe ( qiagen ), which results in the production of recombinant proteins expressed in the following n - terminal sequence of histidines , this production performed in e . coli m15 ( prep4 ) ( qiagen ), allowing its isolation by affinity chromatography with a column of ninta agarose ( qiagen ), based on protocols provided by the manufacturer ( castro , 2001 , silva and al ., 2004 ). all products were made using this system , and we performed the isolation of the recombinant antigen under denaturing conditions to allow more effective isolation . for the production and isolation of the protein of interest it is possible to use any expression system and isolation of recombinant protein , provided that it does not compromise the exposure of the fragment h on to the immune system . the system used to obtain the constructions should be seen as the vehicle used in research to obtain high quantities of protein needed for experimental demonstrations . the same methodology can be used in other systems of protein production , in particular fungi or eukaryotic systems . the results refer to the examples using the fragment h from fh8 ( seq id no 1 and 2 ). one of the principles of the invention is characterized by the addition of the fragments h , corresponding to the sequence of the n - terminal fragment of fh8 , specifically the addition of the polypeptide seq id no 2 , by processes of molecular biology , before the polypeptide sequence intended to use as immunogen . this constructions can be accomplished by inclusion of this sequence by molecular biology techniques , including using appropriate restriction enzymes to add these fragment in appropriate restriction sites , methodology used in the described demonstrations , or by other processes such as adding dna fragments with the sequence of interest ( linkers ) to a pcr product , or other approaches . the reduced amplitude of the fragment h allows the use of a variety of strategies for the merger with the polypeptide of interest . another possibility for the construction is the preparation of a fused protein using the polypeptide corresponding to the sequence of the polypeptide fh8 to be manufactured . the process of inserting the following fh8 can be accomplished using the techniques of molecular biology , including the use of restriction enzymes , a methodology used in the process of demonstration . the invention has been applied to various fragments and proteins with different immunogenic characteristics , including proteins or fragments described as non or poorly immunogenic as cwg , cd4 , or fragments that , due to their characteristics , including its molecular weight would be less immunogenic , such as il5 , pfsp and ent , proteins described as being very immunogenic as pal , and proteins and proteic fragments moderately immunogenic , such as cp12 and lec , as well as other targets with unknown characteristics such as bg and toxo . we also applied throughout the experiments several different protocols varying in protein concentration of administrations given to mice and the time periods between them . these various protocols were used to demonstrate the versatility of the invention . we also evaluated the possibility of administering the antigen under denaturing conditions , as in the case of the lec or in non - denaturing , as the case of the remaining fragments and proteins . for all the demonstrations performed we used mice as experimental models and performed the administration of antigens via intra peritoneal . the production of antibodies to targets cwp , il5 , ent , toxo , bg and cp12 was also evaluated in rabbits using subcutaneous administration with similar results ( not shown ). the antigens were produced and isolated under denaturing conditions using the same conditions with ninta agarose resin ( qiagen ). the antigens were prepared for immunization after dialysis against pbs and sterilization by filtration through a 0 . 22μ filter . to demonstrate the specific effect exerted by the fragment at the level of immunogenicity we used two targets , namely : cwg : cwp ( cyst wall protein ) protein of giardia lamblia cysts . on completion of the work we used a part of the sequence of cwp2 of 427 bp , the original sequence has 1089 bp and the region to be amplified is located 527 bp to 931 bp ( genbank access no . xm — 001710190 ). the fragment was amplified by pcr and subcloned into the vector pqe ( cwp ) were also prepared constructs containing the fragment h followed by the cwp ( hcwp ) and containing the sequence of fh8 fused with the sequence of the cwp ( fh8cwp ). inocula were administered with the same amount of protein ( 50 μg ) at intervals described in table 2 . the results showed the production of significant levels of anti - cwp only in group hcwp visible after the 5 ip whose presence was maintained throughout the remainder of the experiment , including 83 days after the last administration of protein . the blottings made using the antigen fh8cwg confirm the results of elisas and demonstrate the specificity of antibodies produced . the immunofluorescence assay with the parasite showed that the antibodies produced recognize the native protein that exists in the wall of this structure . cp12 : the cp12 is a surface protein of cryptosporidium parvum . the fragment cp12 ( genbank no . xm — 625821 ) used in this work has 213 bp and corresponds to the nucleotide sequence of the protein cp12 without its transmembrane domain . the fragment was amplified by pcr and subcloned into the vector pqe ( cp12 ,) we also prepared constructs containing the fragment h followed by cp12 ( hcp12 ). the antigens were produced and isolated under denaturing conditions using the same conditions with ninta agarose resin ( qiagen ). inocula were administered with the same amount of protein ( 20 μg ) with the periodicity showed in table 2 . the results showed a significant increase in production of antibodies between the cp12 and hcp12 groups , both in intensity and speed , these levels remained significantly increased throughout the experiment . the blottings made with fh8cp12 confirm the results of elisas and demonstrate the specificity of antibodies produced . the immunofluorescence assay with the parasite showed that the antibodies produced recognize the native protein exists in the wall of this structure . for the remaining targets have prepared groups immunized with the construction containing the h tag to demonstrate the presence of an immune response . for the examples described below , fragments were amplified by pcr with the exception of fragment lec that was provided by other institutions , and subcloned in the pqe vector containing the fragment h followed by the target fragment . the conditions for obtaining such fragments are described in table 1 . bg : we cloned the complete sequence of the gene of β - giardina of giardia lamblia which has a size of 850 bp , with a deletion ( 691 bp to 787 bp ) ( genbank access no . x85985 ), encoding a protein of 33 kda . we administered inoculations with the same amount of protein ( 20 μg ) with the periodicity shown in table 2 . the results showed a significant increase after the 3rd ip leveling after the 4th ip . the blottings made with protein bg confirm the specificity of antibodies produced . the maintenance of antibody titers was detected even 47 days after the last inoculation made . immunofluorescence tests with the parasite showed that the antibodies produced recognize the native protein existing in the wall of this structure . ent : the amplicon at work has a size of 163 bp encoding a protein with 5 kda and is the region between 291 bp - 453 bp of a gene with a size of 456 bp ( no access genbank xm 13 645825 ) from entamoeba histolytica cyst wall specific glicoprotein jacob . inocula were administered with the same amount of protein ( 50 μg ) with the periodicity shown in table 2 . the results showed a significant increase after the 4th ip . the blottings made with protein fh8ent confirm the specificity of the antibodies produced . we were able to confirm the maintenance of antibody titers even 90 days after the last inoculation made . the immunofluorescence assay with the parasite showed that the antibodies produced recognize the native protein exists in the wall of this structure . pfsp : in this study we used a small part of the sequence ( 165 bp ) of falcipaina - 1 , which is inserted into the sequence of 3d7 plasmodium falciparum trophozoite cysteine proteinase precursor ( 1423 bp - 1587 bp ) ( no . access genbank xm 13 001348691 . 1 ). inocula were administered with 50 μg with the periodicity shown in table 2 . the results showed a significant increase after the 4th ip reaching the maximal title after the 7 th ip . we can confirm the presence of specific antibodies against the fragment pfsp 82 days after the last inoculation . the blottings made with protein fh8pfsp confirm the specificity of antibodies produced . il5 : the human interleukin 5 is a hematopoietic growth factor , having a nucleotide sequence of 816 bp coding for 134 amino acids ( genbank no . bc069137 . 1 ). the fragment used for evaluation il5 is a very small part of il5 , consisting of 144 bp corresponding to an exon of the 5 ‘ end of il5 coding for 48 amino acids . inocula were administered with the same amount of protein ( 20 μg ) with the periodicity shown in table 2 . the results showed a significant increase after the 4th ip . the blottings made with protein fh8il5 confirm the specificity of antibodies produced . toxo — toxo protein is a protein of the oocyst wall of toxoplasma gondii with 1846 bp coding for 499 amino acids ( genbank no . eu851867 . 1 ). the toxo fragment corresponds to exon 2 , between the 2875 and 3238 bp . inocula were administered with the same amount of protein ( 20 μg ) with the periodicity shown in table 2 . the results showed a significant increase after the 4th ip . the blottings made with protein toxo confirm the specificity of antibodies produced . cd4 : the cd4 protein is a recipient of the wall of lymphocytes of dicentrarchus labrax ( genbank no . amb849812 . 1 ). the cd4 fragment corresponds to two domains of this receptor between 193 and 714 bp . inocula were administered containing 30 μg with the periodicity shown in table 2 . the results showed a significant increase after the 4th ip . the blottings made with the cd4 protein confirm the specificity of antibodies produced . pal : pal protein ( peptidoglycan - associated lipoprotein precursor ) of legionella pneumophila is a protein from the wall of this bacterium ( genbank no . yp001250824 ). pal corresponds to the complete protein . we administered an inoculum with 30 μg with the periodicity shown in table 2 . the results showed a significant increase after the 2nd ip . the blottings made with protein pal confirm the specificity of antibodies produced . lec : the dna fragment of the lectin from artocarpus incisa with 846 bp was containing the local for the enzymes sac i and kpni . since this antigen is potential hemagglutinating activity when in a non - denaturing form , we proceeded with the preparation of protein inocula under denaturing conditions . to remove the maximum amount of urea ( final concentration less than 10 mm ) we proceeded with the dialysis against pbs buffer with 50 mm urea and at the time of preparation of sample we diluted the antigen in pbs and filtered through 0 , 22μ . we administered with an inoculum of 12 . 5 μg with the periodicity shown in table 2 . the results showed a significant increase after the 4th ip . the blottings made with protein hlec confirm the specificity of antibodies produced . in the examples described above evaluation of the response was performed by elisa using the corresponding antigen . in blotting procedures we evaluate the response to the antigen whose production was more efficient . for blotting performed with recombinant proteins containing the tag fh8 , due to the possibility of forming polymers , we proceeded to the location of the recombinant protein with polyclonal antibody specific for fh8 . in most cases we included in the blot a nitrocellulose strip containing an antigen with the fragment h , usually the recombinant fh8 , for evaluation of the response against this fragment and , apart from the response obtained in the group inoculated with fh8cwp , we didn &# 39 ; t detected the presence of significant levels of antibodies anti - h . in the examples above the inoculations were always made only by the antigen diluted in pbs , with the exception of hlec whose inocula consistained 10 mm urea in pbs . the antigen fh8 was previously isolated and characterized by elements in the list of inventors ( castro , 2001 , silva et al ., 2004 , eguino et al ., 1999 ) ( fig1 ). the isolation of fh8 was carried out from the screenin of a f . hepatica cdna bank ( fig1 ). the clones coded for a polypeptide of 69 amino acids with a calculated molecular mass of 8 kda , which was designated by fh8 or fasciolina ( genbank number af213970 ). the recombinant protein fh8 is produced at high levels of protein in e . coli expression systems with vector pqe (& gt ; 5 mg / liter of culture ). studies with fh8 mutants led to hypothesize that the n - terminal sequence of this antigen have an important role in protein stability . demonstration of this hypothesis originated the invention described in patent no . 20091000005031 . another characteristic was its high immunogenicity , this feature extends to another family of calcium binding proteins , present in the extract excreted secreted by adult worms of fasciola hepatica , the family of fh22 ( embl number aj003821 , embl number aj003822 ). both antigens proved to be capable of inducing an immune response with high specific antibody titers ( castro , 2001 , silva et al ., 2004 ). these results also suggested its use as a tag for recombinant protein production with the aim of producing antibodies . the demonstration that the fragment h was essential to the stability of the antigen and that the addition of this fragment to other unrelated proteins or fragments allowed an increase in protein production , presumably due to increased stability of the fused protein , suggested the hypothesis that the addition of fragment h , for the same reasons , would increase the immunogenicity of that antigen . this inference appears from the fact that the stability of a protein is oftenly related to its immunogenicity . this hypothesis was confirmed by the demonstrations described above in this study , we used strains escherichia coli xl1 blue ( stratagene ) and escherichia coli m15 [ prep4 ] ( qiagen ) for the cloning of the plasmids pgem - t easy ( promega ) and plasmid pqe30 ( qiagen ), respectively . for protein expression we used the escherichia coli strain m15 [ prep4 ]. the plasmid dna was isolated and purified by kit wizard ® plus sv minipreps dna purification system from promega , from bacterial cultures grown at 37 ° c . overnight , following the instructions provided by the manufacturer . the layout of the buildings used to evaluate the structural element of 11 amino acids ( fragment h ) as a factor in the induction of immunogenicity of recombinant proteins is shown in fig2 . the constructs shown in fig2 were obtained by polymerase chain reaction ( pcr ) and cloned into pgem and then into pqe are shown in table 1 , as indicated below . the constructs referred to other antigens used to evaluate the immune response were obtained by polymerase chain reaction ( pcr ) and cloned into pgem and then into pqe are shown in table 1 . to obtain the fragments h and fh8rsac , containing the restriction sites bamhi and saci , we used as template for the pcr reaction , the pqe30 vector containing the gene coding for the polypeptide fh8 ( castro , 2001 , silva et al ., 2004 ). the pcr reaction began with a denaturation step of 1 minute at 95 ° c ., followed by 30 amplification cycles , with 45 seconds denaturation at 94 ° c ., 30 seconds of annealing at 50 ° c . and 45 seconds of polymerization at 72 ° c . we made a step further polymerization for 11 minutes at 72 ° c . the fragments chosen ( cwg , cp12 , bg , ent , pfsp , il5 , toxo , cd4 , pal and lec ) to assess the ability of recombinant proteins prepared by the merge of unrelated polypeptides with the h fragment , to produce an immune response , as measured the appearance of specific antibodies against the protein or fragment in question , were amplified by pcr . this pcr reaction also added to the restriction enzymes saci and kpni to their fragments . pcr reactions , as well as dna templates used for the preparation of the various fragments were performed under the conditions described in table 1 . for the preparation of genomic dna was used the kit to extract dna qiaamp dna mini kit from qiagen following the manufacturer &# 39 ; s protocol . the thermal cycler used for all pcr reactions was the my cycler ™ thermal cycler ( biorad ). the mixture of pcr reactions carried out consisted of 1 μl of sample ( template dna ), 2 μl of magnesium chloride , 1 μl dntps ( roche ), 1 μl of forward primer and 1 μl of reverse primer , 5 μl of buffer taq polymerase ( thermo scientific ), 1 unit / reaction of taq polymerase ( thermo scientific ) and distilled water to complete a final volume of 50 μl . the pcr products were cloned into pgem vector and after digestion with restriction enzymes saci and kpni were subcloned into the vector pqe30 , pqe30 containing the fragment h ( pqeh ) or pqe30 containing the fragment fh8 ( pqef ), digested with saci and kpni . to isolate pcr products and dna bands resulting from digestion with restriction enzymes from gel electrophoresis , we used the illustra ™ gfx pcr dna & amp ; gel band purification kit ( ge healthcare ), following the procedure described by the manufacturer . the binding reaction to the vector pgem - t easy consisted in the mixing of 3 μl of dna sample ( pcr product or digestion with restriction enzymes ), with 1 μl of the vector pgem - t easy ( promega ), 5 μl of enzyme buffer 2 × dna ligase ( promega ) and 1 μl of enzyme t4 dna ligase ( promega ) to a final volume of 10 μl . this reaction occurred at room temperature overnight or for 1 hour and 30 minutes at 37 ° c . after the ligase reaction to the vector pgem , we transformed e . coli xl1 blue with the product . the cells were then spread on plates of lb / ampicillin / x - gal / iptg and incubated overnight at 37 ° c . the transformed clones that were used to prepare liquid cultures in lb / ampicillin and subsequently to perform the extraction of plasmid dna from e . coli . the presence of targeted dna fragments was performed by digestion with restriction enzyme ecori ( promega ), for each reaction we used 7 μl of the plasmid dna , 2 μl of h 10 × buffer and 1 μl of ecori , giving a final volume of 10 μl . the reaction occurred for 2 to 3 hours at 37 ° c ., and the result of digestion was displayed on agarose gel with appropriate percentage ( w / v ). the inserts resulting from digestion with restriction enzymes were inserted into the vector pqe , pqeh or pqefh8 by mixing 6 μl of insert with 2 μl of vector pqe , 1 μl of 10 × ligase buffer ( promega ) and 1 μl enzyme t4 dna ligase ( promega ). this reaction occurred at room temperature overnight or for 1 hour and 30 minutes at 37 ° c . after connecting the insert to the vector , e . coli m15 [ prep4 ] were transformed with thereacyion product . the cells were then spread on plates of lb / ampicillin / kanamycin and incubated overnight at 37 ° c . the transformants were transferred to liquid cultures of lb / ampicillin / kanamycin and subsequently used to extract plasmid dna from e . coli . confirmation of transformants was performed by digestion with restriction enzymes kpni and bamhi ( promega ). first , digestion with kpni was performed , mixing 26 μl of plasmid dna , 3 μl of buffer j 10 × ( promega ) and 1 μl of kpni ( promega ) for a final volume of 30 μl . 10 μl of digestion was analyzed on agarose gel and afterwards we proceeded to the second digestion with bamhi , for that purpose we added to the remaining first reaction , 2 μl of 10 × buffer k ( promega ) and 1 μl of bamhi ( promega ). the result of digestion was visualized on agarose gel with appropriate percentage ( w / v ). all the constructions made with the inserts in pgem and pqe vectors were confirmed by sequencing at eurofins mwg operon ( germany ). a pre - culture of 200 ml , were put to grow overnight at 37 ° c . with stirring , and used to prepare 2 liters of induced culture by placing 100 ml of saturated culture and 900 ml of lb medium containing 100 g / ml ampicillin , 50 g / ml kanamycin and 1 mm iptg . after 5 hours incubation we proceeded to collect the cells by centrifuging 20 minutes at 4000 rpm at 4 ° c . the cell lysis was performed by incubation of cells with 40 ml of 8 m urea , ph 8 . 0 , leaving under stirring overnight . the extract was centrifuged at 13 , 000 rpm for 15 minutes at room temperature and the supernatant collected . after recovery , the supernatant was filtered by a column of glass wool and applied to the column of ni - nta ( amersham biosciences ), pre - equilibrated with 8m urea , ph 8 . 0 . the supernatant was passed by the column by gravity , the column was washed with 5 cv ( column volumes ) of buffer 8 m urea , ph 8 . 0 , followed by 5 cv of buffer 8 m urea with 10 % glycerol , ph 6 . 5 . elution was done with buffer 8 m urea , ph 4 . 5 , and 4 ml fractions were collected . the protein content of the eluted fractions was quantified by the bradford method and fractions were analyzed in sds - page tris - tricine , as is described below . the protein quantification was performed by bradford method , with the protein assay reagent ( biorad ) diluted 1 : 5 , and to read the optical density at a wavelength of 595 nm . the calibration curve was obtained by reading the optical density at 595 nm of solutions of known concentration of bovine serum albumin ( bsa ) with this reagent . the recombinant proteins used for the demonstrations described , except hlec were isolated under denaturing conditions in 8 m urea . after protein quantification and analysis of the fractions , we proceeded to extensive dialysis against pbs buffer prepared with nonpyrogenic water . after dialysis we performed the filtration of protein ( under non - denaturing ) using a 0 . 2 u filter to sterilize . the volume of inoculum was hit with a 500 ul sterile nonpyrogenic pbs . the amount of protein administered varied between different samples , between 10 and 50 μg , as described above for each case . the recombinant protein hlec was prepared in 8m urea and was dialysed against pbs buffer containing 50 mm urea , prepared with apyrogenic water , afterwards the antigens was concentrated using centricon ( amicon ) membrane with cut off of 3 kda . inocula were prepared extemporaneously by diluting the concentrated protein in the appropriate volume of sterile pbs , non - pyrogenic , to ensure that the concentration of urea is less than 10 mm , and held the filtration of the inoculum through a filter pyrogenic 0 , 2μ . experiments carried out in this work were performed in models of cd1 mice obtained from charles river sa barcelona . the animals were housed and maintained with food and drink ad libitum . the maintenance and care of animals were made in accordance with existing directives . each group consists of 3 mice and inoculation was administered intraperitoneally periodically , according to the protocols described in table 2 , blood sampling have been conducted periodically at the tail vein , according to the protocols described in table 2 . after collecting the blood , serum was obtained by centrifugation at 2500 rpm for 10 min and kept in the at − 20 ° c . the tris - tricine gels used to analyze the collected fractions were based on tris - tricine system of schägger , h . and jagow , g . ( 1987 ) and sds - page of laemmli ( 1970 ). thus , the system adopted consisted of two gels : one resolvent gel of 15 % and a packaging gel of 4 %. the resolvent gel contained 3 . 3 ml 30 % acrylamide , 2 . 205 ml of gel , 705 ml of glycerol , 367 . 5 ml of water , 150 μl of psa 10 % and 9 μl of temed . the gel packing contained 700 μl of 30 % acrylamide , 1 . 25 ml of gel , 3 ml of water , 200 μl of 10 % psa and 5 μl of temed . the electrophoresis system used was composed of two reservoirs , higher ( from the gels ) and bottom , in which were placed the cathode buffer and anode buffer , respectively . we applied a potential difference ( ddp ) of 100 v to the gel packaging and a ddp of 150 volts for the resolvent gel . samples ( in native or denaturing conditions ) before being applied to the gel , were treated with sample buffer tris - tricine 1 ×. samples in native conditions were also placed in the bath at 100 ° c . for 2 minutes , after getting to 4 ° c . until loaded on the gel . we dipped into transfer buffer ( 25 mm tris , 0 . 2 m glycine , 100 ml methanol ), 2 filter papers , the nitrocellulose membrane , the sds - page in which proteins ran , and sponges needed for assembly of the sandwich . after soaked in buffer we proceeded to mount the sandwich , and transfer was performed using system te 80 ( hoefer ). the transfer took place in transfer buffer , for 1 h at a constant potential difference of 80v . after the transfer , 0 . 45 μm , μ - nitrocellulose membrane ( schleicher & amp ; schell ) was saturated with pbs - milk 5 %, for 1 h at room temperature . he washed the membrane with 2 × pbs - 0 . 3 % tween ( pbs - t ). incubated the membrane with sera diluted in pbs - milk with the appropriate concentration , overnight at 4 ° c . we washed the membrane 3 × with pbs - t . the conjugate protein g - peroxidase ( bio rad ) was added to diluted to 1 / 1000 in pbs - milk , and incubated at room temperature for 2 h . we washed the membrane 3 × with pbs - t and revealed with 15 mg 4 - chloro - 1 - naphthol dissolved in 5 ml cold methanol , 20 ml pbs and 25 μl 30 % h2o2 . the “ coating of microplates ( nunc ) polystyrene was performed with 100 μl / per well l of antigen at 10 μg / ml antigen in carbonate / bicarbonate buffer 0 . 1 m ph 9 . 5 on at 4 ° c . the wells were washed with pbs - t 0 . 3 %, then saturated with 200 μl pbs - 0 . 1 % gelatin per well at 37 ° c . for 30 min in moist chamber and washed again with pbs - t . we added to each well 100 μl of diluted sera at 1 / 400 in pbs - t and put to incubate in moist chamber overnight at 4 ° c . the plates were washed 3 × with pbs - t . we added to each well 100 μl of protein g - coupled peroxidase ( biorad ) diluted at 1 / 2000 in pbs - t , and put to incubate for 1 h at 37 ° c . in moist chamber . the wells were washed 3 × with pbs - t . the reaction of substrate contained 1 mg of opd per ml 0 . 2 m phosphate ph 5 . 6 . for each ml of this solution we added 1 μl of h2o2 30 %. 100 μl of substrate was added per well and reaction was stopped with 100 μl of 3m hcl per well . the optical density was read at 490 nm in a elisa plate model 680 ( biorad ). the biological material for tests , immunofluorescence was obtained from water samples and faeces , in the case of cryptosporidium parvum ( cp12 ) and giardia lamblia ( cwg ), for entamoeba histolytica ( ent ) slides were obtained from the supplier biomérieux diagnosis , in the case of β - giardina ( bg ) we used axenic cultures of trophozoites . to prepare slides with parasitary material , sample of parasites were added to each well of the slide for immunofluorescence and left to dry in the oven until the sediment remain fixed ; were added two drops of acetone to dry completely . left to dry at room temperature plus five more minutes in the oven 37 ° c . for the immunofluorescence we added 10 μl of serum ( primary antibody ) in the corresponding dilution , and left in a moist chamber at 37 ° c . about 1 hour and washed 3 × with pbs . the conjugate anti - mouse igg labeled with fitc ( sigma ) diluted in pbst was added to the slide and incubated in a moist chamber for 1 h at 37 ° c . the slides were washed . we added the contrasting ( evans blue ) solution and then 10 μl of mounting medium . the slides were observed under a microscope nikon optiphot immunofluorescence . for an easier understanding of the invention are described below preferential examples of application of the invention , which , however , are not intended to limit the scope of this invention . the recombinant antigen under study in this work is characterized by inducing an immune response that can be assessed by the production of specific polyclonal antibodies . studies have previously indicated that the fragment h played a key role in stability and immunological characteristics of antigen fh8 : antigens derived from fh8 whose sequence h had been deleted showed a drastic reduction in their stability and immunogenicity . the strategies presented had , as starting point these assumptions , fragments were chosen in order to vary widely in heir origin , nature and immunological characteristics , as well as different application protocols intended to evaluate the use of this application in the induction of specific immune responses without the use of another constituent ( adjuvant ) than the antigen itself . to this end we proceeded to the selection of fragments whose immunological characteristics in the presence of adjuvants , had previously been evaluated , such as cd4 and fragments cwg that had proven to be poorly immunogenic , the fragments cp12 and lec , which were shown to have intermediate immunogenic characteristics or the pal that is a very immunogenic antigen , or fragments , as fragments ent , il5 and pfsp whose biochemical characteristics , including family protein , molecular weight and amino acid sequence , determined that they would be poorly or non - immunogenic . finally we also used fragments , such as toxo and bg which immunological characteristics were completely unknown . to assess the actual impact of fragment h on the immunogenicity of the antigen to which is added we conducted demonstrations assessing the ability to induce an immune response by fragments cwg and cp12 in the absence and presence of fragment h . evaluation of the immune response in proteins resulting of the constructs with the fragment cwg after obtaining the b constructs pqecwg , pqehcwg and pqefcwg we proceeded to the production and analysis of the respective recombinant antigens under denaturing conditions ( fig3 a ). in the analysis of sds - page tris - tricine gels ( fig3 a ) we can see that the protein cwg has a molecular weight of 16 kda , as expected , while the fusion protein hcwg has a weight of about 17 kda and the recombinant antigen fcwg has a weight of about 24 kda . demonstration of the effect of the presence of the fragment h on the induction of a specific immune response against the cwg has been performed by inoculation with 3 groups of cd1 mice with 50 μg of antigen cwg , hcwg and fcwg , regularly ( table 2 ) by ip administration . he used also a group of 3 cd1 with the same characteristics that received no inoculation . there has been regular blood collection ( table 2 ) for further evaluation of the presence of ig anti - cwp . the evaluation of the presence of specific antibody response was performed by elisa with plates containing antigens cwg ( fig3 b . a ) hcwg ( fig3 b . b ) and fcwg . there is the appearance of significant immunoglobulin g ( igg ) anti - cwg from the 4th inoculation onwards but only in the group hcwg . in group fcwg we verified the presence of ig anti - fh8 but could not confirm the presence of specific igg against fragment cwg ( data not shown ). the presence of ig g anti - cwg , in hcwg group , was detected even 83 days after the last administration indicating the existence of a specific memory for this antigen . to assess the specificity of polyclonal antibodies we performed blots using as antigen the fcwg . the location of the recombinant protein fcwg as well as possible polymers was carried out with the antisera produced against fh8 ( fig3 d . i ), diluted 1 / 100 , that allows the viewing of polymers fcwg . using pools of serum from negative group , cwg group and hcwg group obtained 9 days after the 5th ip and post 6th ip , diluted 1 / 200 , the appearance of precipitates corresponding to fcwg . the realization of immunoblottings with the same dilutions using antigen fh8 ( to evaluate the production of igg anti - fragment h ) didn &# 39 ; t show no appearance of any precipitate . all these results shows that the antibodies developed by the group hcwg are specific of fragment cwg . the presence of significant cross - reactions with antigens of e . coli or the presence of ig anti - fragment h is not observed . to assess whether the ig produced were capable of recognizing the native protein existing in the wall of giardia cysts we proceeded to the realization of immunofluorescence with sera from group hcwg which revealed the presence of fluorescent wall structures ( fig3 e ). evaluation of the immune response for proteins of the constructs with the cp12 fragment after obtaining the constructs pqecp12 and pqehcp12 we proceeded to the production and analysis of the respective recombinant antigens under denaturing conditions ( fig4 a ). in the analysis of sds - page tris - tricine gels ( fig3 a ) we can see that the cp12 protein has a molecular weight of 9 kda , as expected , while the fusion protein hcp12 has a weight of approximately 10 kda and the recombinant antigen fcp12 presents an antigen with weight of about 29 kda . having regard to the pm calculated for the recombinant antigen , the band of 29 kda may be a polymer fcp12 . demonstration of the effect of the presence of the fragment h on the induction of a specific immune response against the cp12 has been performed by inoculation with 2 groups of cd1 mice with 20 μg of antigen cp12 and hcp12 periodically ( table 2 ) by ip administration . we also used a group of 3 cd1 with the same characteristics that received no inoculation . there has been regular blood collection ( table 2 ) for further evaluation of the presence of ig anti - cp12 . the evaluation of the presence of specific antibody response was performed by elisa with plates containing the cp12 antigen ( fig4 b . a ) hcp12 ( fig4 b . b ). there is the appearance of anti - cp12 from the 4th inoculation onwards in group hcp12 , being also visible the appearance in cp12 group of ig anti - cp12 from the 6th ip onwards . in both groups the titles of ig anti - cp12 evolve throughout the experiment . in this example the increase of immunogenicity can be observed by the earlier immune response and the higher amount of igg anti - cp12 present in group hcp12 . to assess the specificity of polyclonal antibodies produced we performed blots using as antigen the fcp12 . the location of the recombinant protein fcp12 was carried out with the fh8 specific antisera ( fig4 c ), diluted 1 / 100 , that allows the viewing of fcp12 polymers , whose locations are indicated by arrows . there is , using sera from groups : negative , cp12 and hcp12 , harvested post 8 a ip , and diluted 1 / 1000 , the appearance of precipitates corresponding to proteins identified by serum anti - fh8 . the highest intensity present in hcp12 group , when compared with the cp12 group , confirms the increase of immune response that occurs in group hcp12 . immunoblottings performed with the same sera using antigen fh8 didn &# 39 ; t show that appearance of any precipitate . all these results shows that the antibodies developed by the group hcp12 are specific to the cp12 fragment since the presence of significant cross - reactions with antigens of e . coli or the presence of anti - ig fragment h is observed . to assess whether the ig produced were capable of recognizing the native protein existing in the wall of cryprosporidium oocysts we proceed to the realization of immunofluorescence with sera from group hcp12 which revealed the presence of fluorescent in wall structures ( fig4 d ). evaluation of immune response proteins and fragments resulting from the construction with the fragment h for each of the fragments described we proceeded to demonstration on the production of immune response inoculating 3 groups of cd1 mice with the corresponding antigen at regular intervals ( table 2 ) by ip administration . we also used a group of 3 cd1 with the same characteristics that received no inoculation . there has been regular blood collection ( table 2 ) for further evaluation of the presence of ig directed against the target antigen . protein bg : the immunological features on this fragment were unknown . after obtaining the construct pqehbg we proceeded to the production and analysis of the respective recombinant antigens ( fig5 a ). demonstration of the effect of the presence of the fragment h on the induction of a specific immune response against the bg has been performed by inoculation a groups of 3 cd1 mice with 20 μg of antigen hbg . the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hbg ( fig5 b ). there is the appearance of ig g anti - bg from the 3rd inoculation onwards . the antibody levels reach a plateau after the 4th ip that has been maintained even 47 days after last inoculation . to assess the specificity of produced polyclonal antibodies we performed blots using as antigen the bg . there is , using the sera from negative and hbg groups harvested post 7th ip , diluted 1 / 1000 , the appearance of precipitates corresponding to bg . the presence of significant cross - react with antigens of e . coli was not detected . to assess whether the ig produced were capable of recognizing the native protein existing in the wall of giardia we considered the realization of immunofluorescence with sera from group hbg which revealed the presence of immunofluorescence in the wall of these structures . protein fragment ent : due to the low molecular weight of this polypeptide ( 7 kda ) and since it represents only a portion of a protein , this fragment had characteristics associated with low immunogenicity . after obtaining the construct pqehent we proceeded to the production and analysis of their recombinant antigens . demonstration of the production of immune response was performed proceeding to inoculations of 3 cd1 mice with 50 μg of hent antigen . the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hent ( fig5 b ). there is the appearance of ig g anti - ent from the 5th ip onwards . the antibody levels reach a plateau after the 5th ip that was maintained even 90 days after last inoculation . to assess the specificity of polyclonal antibodies produced we performed blots using as antigen the fent . the recombinant protein fent was further identified with specific antisera anti fh8 ( fig6 b ), diluted 1 / 100 , that allows the viewing of fent polymers , whose locations are indicated by arrows . there is , using the sera of negative and hent groups , harvested post 7 th ip , and diluted 1 / 1000 , the appearance of precipitates , in hent group corresponding fent . immunoblottings performed with the same sera using antigen fh8 didn &# 39 ; t shows the appearance of precipitate . all these results shows that the antibodies developed by the group hent are specific of fragment ent since the presence of significant cross - reactions with antigens of e . coli or the presence of anti - ig fragment h was not detected . to assess whether the ig produced were capable of recognizing the native protein existing in the wall of entamoeba trophozoites we performed immunofluorescence with sera from group hent which revealed the presence of fluorescent wall structures ( fig6 c ). protein fragment pfsp : due to the low molecular weight polypeptide ( 7 kda ) and since it represents only a portion of a protein , this fragment had characteristics associated with low immunogenicity . after obtaining the construct pqehpfsp we proceeded to the production and analysis of the respective recombinant antigens under denaturing conditions . demonstration of the production of immune response was performed proceeding to the inoculation of cd1 mice with 50 μg of antigen hpfsp . the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hpfsp ( fig7 b ). there is the appearance of igg anti - pfsp from the 4 th ip onwards . the antibody levels reach a plateau after the 7th ip that has been maintained even 82 days after last inoculation . to assess the specificity of produced polyclonal antibodies we performed blot using as antigen the fpfsp . the recombinant protein was located with specific anti fh8 antisera ( fig7 b . a ), diluted to 1 / 100 , that allows the viewing of fpfsp polymers . using pool of sera from hpfsp and negative groups harvested post 6 th ip and 14 days post 7th ip , diluted 1 / 200 , we observe the appearance of precipitates corresponding to fpfsp . immunoblottings performed with the same sera using antigen fh8 didn &# 39 ; t shows the appearance of precipitate . all these results shows that the antibodies developed by the group hpfsp are specific of fragment pfsp since the presence of significant cross - reactions with antigens of e . coli or the presence of anti - ig fragment h was not detected . il5 protein fragment : due to the low molecular weight polypeptide ( 7 kda ) and since it represents only a portion of a protein with high homology with the il of 5 mice and has been described as non immunogenic , this fragment had characteristics associated with low immunogenicity . after obtaining the construct pqehil5 we proceeded to the production and analysis of the respective recombinant antigens under denaturing conditions . demonstration of the production of immune response was performed inoculating cd1 mice with about 20 μg of hil5 ( table 2 ). the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hil5 ( fig8 a ). there is the appearance of ig g anti - il5 from the 4 th ip onwards . the antibody level grows due to the inoculations throughout the study period . to assess the specificity of polyclonal antibodies produced we performed blots using as antigen the fil5 . the location of the recombinant protein fil5 was carried out with the specific anti - fh8 antisera diluted to 1 / 100 , that allows the viewing of fil5 polymers indicated with arrow . it was found ( fig8 b ), using sera from hil5 and negative groups obtained post 6th ip , diluted to 1 / 1000 , the appearance of precipitates corresponding to fil5 . immunoblottings performed with the same sera using antigen fh8 didn &# 39 ; t shows the appearance of precipitate . all these results shows that the antibodies developed by the group hil5 are specific of fragment il5 since the presence of significant cross - reactions with antigens of e . coli or the presence of anti - ig fragment h was not detected . protein toxo the immunological features on this fragment were unknown . after obtaining the construct pqehtoxo we proceeded to the production and analysis of their recombinant antigens . demonstration of the production of immune response was performed by inoculating cd1 mice with 20 μg of antigen htoxo ( table 2 ). the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen htoxo ( fig9 a ). there is the appearance of igg anti - toxo from the 4th inoculation onwards . to assess the specificity of produced polyclonal antibodies we performed blots using as antigen toxo . there is , using sera harvested post 4th ip , diluted at 1 / 1000 , the appearance of precipitates corresponding to recombinant toxo in the group htoxo . the presence of significant cross - reactions with antigens of e . coli was not observed . fragment cd4 : this fragment was shown to be poorly immunogenic . after obtaining the construct pqehcd4 we proceeded to the production and analysis of the respective recombinant antigens under denaturing conditions . demonstration of the production of immune response was performed by inoculating cd1 mice with 30 μg of antigen hcd4 ( table 2 ). the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hcd4 ( fig1 a ). there is the appearance of anti - cd4 from the 4th inoculation onwards . the antibody levels reach a plateau after the 4th ip that remains 82 days after last inoculation . to assess the specificity of produced polyclonal antibodies we performed blots using as antigen the recombinant cd4 . using a pool of sera harvested 14 days after the 7th ip and diluted 1 / 500 , the appearance of precipitates corresponding to cd4 is observed in the group hcd4 , ( fig1 b ). pal protein : this protein was shown to be very immunogenic . after obtaining the construct pqehpal we proceeded to the production and analysis of their recombinant antigens . demonstration of the production of immune response was performed by inoculating cd1 mice with 30 μg of antigen hpal ( table 2 ). the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hpal ( fig1 a ). there is the appearance of ig g anti - pal from the 2nd inoculation onwards . to assess the specificity of produced polyclonal antibodies we performed blots using as antigen the hpal . there is , using the sera of harvest post 4th ip , diluted 1 / 4000 , the appearance of precipitates corresponding to recombinant pal ( fig1 b ) in the group hpal . lec protein : this protein was considered moderately immunogenic but due to its hemagglutinating activity , when in native form , we have developed specific antibodies against the antigen in denatured conditions . we proceeded to the production and analysis of recombinant antigen hlec under denaturing conditions . demonstration of the production of immune response was performed by inoculating cd1 mice with 12 . 5 μg of antigen hlec . the evaluation of the presence of specific antibody response was performed by elisa with plates containing the antigen hlec ( fig1 a ). there is the appearance of igg anti - lec from the 4th inoculation onwards . the antibody levels reach a plateau after the 4th ip that was maintained during the period under review . to assess the specificity of produced polyclonal antibodies we performed blots using as antigen the hlec . there is , using sera harvested post 6th ip , diluted 1 / 1000 , the appearance of precipitates corresponding to hlec ( fig1 b ) for the group hlec . the demonstrations described for the fragments cwg and cp12 showed that the presence of the fragment h in the recombinant protein can significantly increase the specific immune response developed by the mice . thus the increase in immunogenicity is a characteristic associated with the recombinant antigen which allows the production of specific polyclonal antibodies , even though that in some of the extractions , including hent , hil5 , hpfsp hlec the presence of e . coli contaminants was significant . so despite the contamination with proteins from e . coli , antibodies produced are essentially specific for the target fragment . the development of polyclonal antibodies against a recombinant antigen may be associated with protection of host where they develop antibodies against the infectious organism that contains the corresponding antigen . this depends on a number of factors ; especially the role or importance of this antigen has the mechanism of infection of infectious organism . in the case of mice inoculated with the protein hcp12 , hcwg and hbg , these antigens represent , in the mechanism of infection by cryptosporidium ( cp12 ) and giardia ( cwg and bg ), a crucial role in the invasion or cell adhesion to the host organism , and therefore , as described in the literature , are target candidates for vaccine development . the development of antibodies against these antigens has been described in the literature as protecting from infection by infectious agents ( tellez et al ., 2003 , jenkins et al ., 1998 , abdul - wahid et al . 2007 ). in a very similar way to that described in previous literature , and to assess the protection of mice injected with hcp12 , hcwg hbg in face of infection by the parasites cryptosporidium and giardia , we have indications that there is an effect of protection against infection by of mice pre - inoculated with these antigens . the use of inocula consisting of soluble proteins eliminates much of the undesirable effects caused by adjuvants . in none of the mice used in described experiments were detected side effects that may result from inoculation of antigens . we also performed the production of polyclonal sera against some of the fragments described above ( hcwg , hcp12 , htoxo , hil5 , hent and hbg ) in the rabbit model using subcutaneous inoculations in the inner thigh and , as in mice , we didn &# 39 ; t observe any side effects from the administration of antigens . as in model mice , rabbits produced evidence of very significant polyclonal antibodies against the antigen of interest . in all targets evaluated we could demonstrate the existence of a significant immune response by demonstrating the production of specific ig . another very important feature , observed particularly in the case of the response against hcwg , hcd4 , hpfsp , hent and hbg , is that it was possible to detect specific response up to 3 months after last immunization , suggesting the development of memory cells , a characteristic essential for the development of vaccines . although the examples given are based on models for production of recombinant proteins that produce antigen fusion containing the fragment h , this fragment can be added to the targets through other processes . the activity shown should be included in the field of adjuvants and as they can be used in various formulations in order to improve or increase the intensity and specificity of the immune response resulting from the application of a target antigen . abdul - wahid , a & amp ; 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