# Patent

## Title
Lung-targeting nanobodies against human pulmonary surfactant protein A and a method for producing the same

## Abstract

The present invention relates to the field of biochemistry and pharmaceutical technologies. The present invention provides nanobodies that bind to human pulmonary surfactant protein A (SP-A) as well as the preparing methods and use of the same. The nanobody comprise an amino acid sequence having the formula of Q(x)2LVESGG(x)2V(x)2G(x)SL(x)LS(x)24E(x)n2 KG(x)4S(x)n3T(x)2Y(x)C(x)n4S(x)n5V(x)n6R; wherein x is amino acid; n2 ̃n6 are positive integers; 1≦n2≦21; 1≦n3≦19; 1≦n4≦50; 1≦n5≦22; 1≦n6≦8. The present invention take fresh frozen sections of lung as antigen, gene sequences with high affinity with hSP-A were obtained by constructing an SP-A antibody library and affinity selection, and nanobodies with high affinity and small molecular weight were obtained by induced expression of the gene sequences through a prokaryotic expression vector. Immunohistochemistry and in vivo imaging in nude mice showed the nanobodies have high specificity for targeting lung tissue.

## Background
FIELD OF THE INVENTION
The present invention relates to the field of biochemistry and pharmaceutical technologies, particularly to nanobodies that bind to human pulmonary surfactant protein A (SP-A) with specificity.
BACKGROUND OF THE INVENTION
In the beginning of 20th century, the Nobel Prize winner German scientist Paul Ehrlich proposed the idea of “magic bullet” for future drug development, i.e., an ideal drug that would selectively destroy diseased cells without affecting healthy cells. In the past several decades, scientists have been exploring to develop such ideal drugs.
In the 1970s, targeted drug delivery system were developed and widely used for the treatment of cancer. Meanwhile, with the advancement of research, new targeted drug delivery carriers have, emerged, the routes of administration have, been broadened, and targeted drug delivery system have been expanded to treat many diseases other than cancer.
Developing targeted drugs for respiratory diseases is one of the hotspots, and it is primarily focused on the following areas:
1. Targeted treatment of airway diseases by inhalation.
Starting in the early 1950s, inhaled corticosteroids have been used for the treatment of asthma and COPD. Since then, with the improvement in inhaled drugs and devices, inhaled corticosteroids have become the main therapeutic agents for the treatment of asthma and COPD. However, inhaled drugs are mainly suitable for topical treatment of airway diseases, and are not effective against parenchyma and interstitial lung diseases due to low bioavailability.
Passive lung-targeting drugs through drug carriers.
2. Currently, a variety of drug carriers such as liposomes, microparticles, microspheres are used in the research of lung-targeted drug delivery. However, these passive targeting drug carriers have poor tissue selectivity, and cannot avoid significant residue in the liver, spleen and other organs. Therefore, they don't achieve optimal targeting effect.
The ligand-receptor or antigen-antibody binding is a special recognition mechanism of the human body, and it has been reported that the mechanism could achieve active drug targeting to enhance drug efficacy and reduce the side effects. For example, a composite drug made of paclitaxel liposomes and a monoclonal antibodies against the epidermal growth factor has anti-tumor effect 25 times greater then that of the drug without the monoclonal antibody. Thus, to achieve ideal active lung targeting effect, it is critical to find a receptor in the lung tissue with high specificity and prepare a targeting ligand with high affinity. Studies have shown that pulmonary alveolar type II epithelial cells which account for 16% of the total cells in lung parenchyma have proliferation and secretion functions. Type II cells can synthesize and secrete pulmonary surfactant. The main components of the pulmonary surfactant are lipids (90%) and proteins (10%), and the proteins are specific pulmonary surfactant proteins (SPs). SPs have been named as SP-A, SP-B, SP-C, SP-D, based on the order of discovery, and SP-A was first discovered and has strong expression in pulmonary alveolar type II epithelial cells with abundant signals, and is rarely expressed in other tissues. Thus, SP-A is highly lung-specific, and is an ideal receptor in the lung tissue with specificity.
In addition to high affinity, an ideal targeting ligand should have a low molecular weight, high tissue penetration, and weak immunogenicity. Antigen-antibody binding is the strongest recognition mechanism, and therefore an antibody is the preferred ligand. However, although of high affinity, full antibodies are not ideal ligands due to their large molecular weight (with a relative molecular weight of 150,000), weak tissue penetration and strong immunogenicity. With the development of antibody and gene engineering technologies, antibody fragments (Fab, ScFv) now have the advantages of low molecular weight and weak immunogenicity, but they have lower stability and affinity than full antibodies.
In 1993, scientists from Belgium first reported the existence of Heavy Chain antibodies (HCAbs) without the light chain in the blood of camelids. The variable domain (VHH) of the heavy chains of HCAbs has a complete and independent antigen-binding capacity, and if cloned, a single domain antibodies in the nanometer scale which are known as Nanobodies® (Nbs) can be obtained. A nanobody has many advantages as a ligand: 1) small molecular weight, strong tissue penetration, and high affinity. It has a molecular weight of only 15,000 which is the lowest molecular weight among the known antibody molecules; its ability to penetrate tissues is significantly superior to full antibody, and its affinity with specific antigen is of nmol scale. 2) Stable structure. It can maintain stability even if stored at 37° C. for a week, under high temperature (90° C.), or under strong denaturing conditions such as being exposed to chaotropic agent, protease and strong pH value. 3) Weak immunogenicity. As its gene has high homology with human VH III family, it has weak immunogenicity and good biocompatibility. Because of these advantages, nanobodies have been studied extensively as new antibody drugs, but their use as targeted ligands for SP-A has not been reported.
SUMMARY OF THE INVENTION
The present invention provides a solution for the above-mentioned deficiencies of the prior art. The prior application CN104109207A discloses nanobodies that bind to rat's pulmonary surfactant protein A (SP-A)-, and the applicant continues to work on the nanobodies that bind to human pulmonary surfactant protein A (SP-A).
The present invention provides nanobodies that bind to human pulmonary surfactant protein A (SP-A) as well as the preparing methods and use of the same.
The present invention also provides nucleic acid encoding nanobodies that bind to pulmonary surfactant protein A.
The technical solutions are as follows:
In accordance with the first aspect of the present invention, a lung-targeting nanobody is provided. The nanobody comprises an amino acid sequence having the formula of Q(x)2LVESGG(x)2V (x)2G(x) SL(x) LS(x)24E (x)n2 KG(x)4S(x)n3T(x)2Y(x)C(x)n4S(x)n5V(x)n6R; wherein x is any amino acid; n2 ̃n6 are positive integers; 1≦n2≦21; 1≦n3≦19; 1≦n4≦50; 1≦n5≦22; 1≦n6≦8. Preferably, 17≦n2≦21; n3 is 18 or 19; 16≦n4≦50; 17≦n5≦22; n6 is 7 or 8.
In accordance with another embodiment of the present invention, the nanobody comprises an amino acid sequence having the formula of Q(X1)LVESGG(X2)V(X3)G(X4)SL(X5) LS(X6) E(X7) KG(X8) S(X9) T(X10) Y(X11) C(X12) S(X13) V(X14)R, wherein
X1 is selected from a group consisting of LQ (SEQ ID NO:16, 17, 18, 19, 20, 26, 30) and VK (SEQ ID NO:21, 22, 23, 24, 25, 27, 28, 29);
X2 is selected from a group consisting of GS (SEQ ID NO:21, 22, 23, 24, 25, 27, 28, 29), GL (SEQ ID NO:16, 17, 18, 19, 20, 26, 30) and DL (SEQ ID NO:17);
X3 is selected from a group consisting of QS (SEQ ID NO:30) and QP (SEQ ID NO:16, 18, 19, 20, 26);
X4 is G (SEQ ID NO:16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30);
X5 is selected from a group consisting of I (SEQ ID NO:28), S, R (SEQ ID NO:16, 18, 21, 22, 23, 24, 25, 26, 27, 29, 30) and T (SEQ ID NO:17);
X6 is selected from a group consisting of









(SEQ ID NO:21,22,23,24,25,27,28,29)



CTASGSDYRWMYIARFRQCPGKER,



 



(SEQ ID NO:16,26)



CAASEFTLDYYEIGWFRQAPGKDR,



 



(SEQ ID NO:20)



CAASGFNLDDYADIGWFRQAPGKER,



 



(SEQ ID NO:19)



CAVRGRDLDYYVIGWFRQAPGKER,



 



(SEQ ID NO:18)



CTASKFHLDSYAVAWFRQTPGKER,



 



(SEQ ID NO:30)



CAASGFTFNDYRMSWVRQAPGKGL 



and 



 



(SEQ ID NO:17)



CTASGTFKIYSMGWYRRPQR;






X7 is selected from a group consisting of









(SEQ ID NO:21,22,23,24,25,27,28,29)



GVAAIYTDDTDDSSPIYATSA, 



 



(SEQ ID NO:16,26)



GLSCIGYSDRIAYYSESV,



 



(SEQ ID NO:20)



RVLCITISDGTTYYEDSG,



 



(SEQ ID NO:19)



GVSCINNSDDTTYYSDSV,



 



(SEQ ID NO:18)



AVSFINTSDDVTYFADSV, 



 



(SEQ ID NO:30)



WVSDINSGGSSTYYADSV 



and



 



(SEQ ID NO:17)



LVAEMLNGGDTQYSDSV;






X8 is RFTIRFSIRFTV;

X9 is selected from a group consisting of









(SEQ ID NO:21,22,23,24,25,27,28,29)



QDKDKNAVYLQMNSPKPED,



 



(SEQ ID NO:16,26)



RDDATSTVSLYMDMMIPED,



 



(SEQ ID NO:20)



TDIAKNTVFLQMDSLKAED,



 



(SEQ ID NO:19)



RDHAKNTVYLQMNNLKPED,



 



(SEQ ID NO:18)



RDNSKNTVYLQMNVLKPED,



 



(SEQ ID NO:30)



RDNAKNTLYLQMNSLKPED



and



 



(SEQ ID NO:17)



RTNNTMYLHMNNLKPED;



 



X10 is AMGTALSIAIAV;






X11 is any amino acid or NULL;

X12 is selected from a group consisting of









(SEQ ID NO:21,22,23,24,25,27,28,29)



AARAFGGTWSLSSPDDFSAWGQGTQVTVS,



 



(SEQ ID NO:16,26)



AGSVVEPYELLPAAEYDYWGQGTRVTVS,



 



(SEQ ID NO:20)



AGDPAPFCLYNTYVPRTWGQGTQVTVS,



 



(SEQ ID NO:19)



AADFDRLDFTVKAMCVMKFFYYWGQGTQVTVS,



 



(SEQ ID NO:18)



AAVRSPGPTGPSMQPMWSVPDLYDYWGQGTQVTVS,



 



(SEQ ID NO:30)



VALLGRGCSGLVQGAFGPWGQGTQVTVS,



 



(SEQ ID NO:17)



NLQDWYSEPAGDYWGPGTQVTVS;






X13 is selected from a group consisting of









(SEQ ID NO:23,24,25,27,28,29)



GTNEVCKWPPRPCGRRCAGA, 



 



(SEQ ID NO:16,20,26,30)



AHHSEDPGPRGLAAAGAP 



and



 



(SEQ ID NO:17,18,19)



EPKTPKPQGPRGLAAAGAP;






X14 is selected from a group consisting of (SEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28, 29) and PYPDPLEP (SEQ ID NO:16, 17, 18, 19, 20, 26, 30).

Preferably, X11 is Y, or V.
In accordance with another embodiment of the present invention, the nanobody comprises an amino acid sequence comprising any of SEQ ID NOs 16 to 30.
In accordance with the second aspect of the present invention, the present invention provides nucleic acids encoding the lung-targeting nanobody. Said nucleic acids encode the nanobody described in claim 1.
In accordance with an embodiment of the present invention, the nucleic acid comprise a polynucleotide sequence comprising any of SEQ ID NOs 1 to 15.
In accordance with the third aspect of the present invention, the present invention provides a method of preparing the antibody, comprising the steps of:

    
    
        Step 1: fresh frozen human lung tissue sections were employed as antigen to screen the constructed nanobody library;
        Step 2: selecting strains with high affinity with human pulmonary surfactant protein A and obtaining the relevant gene protein sequences;
        Step 3: inducing the expression of the obtained gene sequences in Step 2.
    
    


In the method, preferably, the nanobody library in step 1 is pre-built anti pulmonary surfactant protein A nanobody libraries, by affinity selection.
Technical route of the method is shown in FIG. 9.
In accordance with the fourth aspect of the present invention, the present invention provides the use of nanobody as targeted ligand for SP-A.
In accordance with a preferred embodiment of the present invention, the specific target of the nanobodies is pulmonary surfactant protein A (SP-A).
SP-A was the first discovered pulmonary surfactant protein, has strong expression in pulmonary alveolar type II epithelial cells with abundant signals, and is rarely expressed in other tissues. SP-A is highly lung-specific, and is an ideal lung-specific receptor. In accordance with embodiments of the present invention, alpacas were immunized with SP-A, an antibody library was built, affinity selection was employed to screen and identify genes with lung-targeting specificity, and SP-A nanobodies with high affinity were obtained by prokaryotic expression. In vivo and in vitro experiments were conducted to verify the nanobody has high specificity for targeting lung tissue.







TABLE 1







Abbreviation of amino acid











Full name
Abbreviation
Abbreviation






alanine
Ala
A



arginine
Arg
R



asparagine
Asn
N



aspartic acid
Asp
D



cysteine
Cys
C



glutanine
Gln
Q



glutamic acid
Glu
E



Glyicine
Gly
G



histidine
His
H



isoleucine
Ile
I



leucine
Leu
L



lysine
Lys
K



methionine
Met
M



phenylalanine
Phe
F



proline
Pro
P



serine
Ser
S



threonine
Thr
T



tryptophan
Trp
W



tyrosine
Tyr
Y



valine
Val
V









Specifically, constructed anti pulmonary surfactant protein A (SP-A) nanobody library is incubated in fresh frozen sections of human lung, after several rounds of affinity selection, human lung tissue SP-A nanobody libraries is built, and 15 nanobodies strains which could bind human lung SP-A efficiently are screened out. Sequencing analysis showed they were all VHH sequences (nanobody sequences).
Nb4 had the highest affinity, and were selected as the preferred embodiments for prokaryotic expression to obtain nanobodies with a molecular weight of about 190,000 and a size of nanometer scale. In vitro Western Blot and ELISA experiments, Nb4 showed good affinity with hSP-A, immunohistochemistry and in vivo imaging results showed its lung-targeting specificity as it could bind to natural SP-A in the lung tissue.
In accordance to another embodiment of the present invention, synthetic method was used to obtain the polypeptide of the human lung tissue nanobody.
To further optimize the human lung tissue nanobody of the present invention, the active region of the polypeptide sequences of the selected clones were tested. Wherein the polypeptide of Nb4 (without the MQAQKAG part, SEQ ID NO:16) is obtained by synthetic method. Testing results showed that the functional polypeptides of Nb4 still have good lung-targeting distribution specificity after the removal of MQAQKAG.
The present invention provides human pulmonary surfactant protein A nanobody (hSPA-Nb) against the human pulmonary surfactant protein A (SP-A). And through a variety of methods are verified human lung tissue SPA-Nb prepared by the invention has a good lung-targeting distribution specificity. The operation flow of the present invention is shown in FIG. 8.
In accordance with embodiments of the present invention, the human lung tissue SPA-Nb coding sequence refers to the nucleotide sequence of the SPA-Nb polypeptide, such as the sequences from SEQ ID NO:16 to SEQ ID NO:30 and its degenerate sequence. The degenerate sequence refers to sequences from SEQ ID NO:16 to SEQ ID NO:30 wherein one or more codons were substituted.
Corresponding amino acid codon see FIG. 10.
The SPA-Nb coding sequences also include variants of SEQ ID NO:16 to SEQ ID NO:30 that encoding proteins with the same functions as SPA-Nb. Such variants include (but are not limited to): the deletion, insertion or substitution of a plurality (usually 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) of nucleotides, and the adding at the 5′ and/or 3′ end of a plurality of (typically less than 60, preferably less than 30, more preferably less than 10, the top for 5 or less) nucleotides.
Once the SPA-Nb coding sequence is obtained, large quantities of the recombinant sequences can be obtained. This is usually done by cloning the sequence into a vector, and transferring to the cells, then using conventional methods to isolate the sequences from the proliferated host cell.
In addition, the sequences can also be obtained by synthetic methods, as the length of the inhibitory factor of the nanobodies of the present invention is short. Typically, a number of small fragments can be synthesized first, and a long fragment can be formed by linking the small fragments.
In accordance with the present invention, various forms of vectors known in the art, such as those that are commercially available, can be used. For example, using a commercially available vector, the nucleotide sequence encoding the polypeptide of the invention can be operably linked to expression control sequence to form a protein expression vector.
As used herein, the term “operably linked” means the situation where part of the DNA sequence can affect the activity of other part of the DNA sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
In accordance with embodiments of the present invention, the term “host cell” includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cells, such as CHO cells, COS cells and the like.
The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, total protein extracted from fresh human lung tissue serves as an antigen which verify antibody targeting and specificity. These fragments or functional regions can be prepared using recombinant or synthesized by synthetic peptide synthesizer. Antibodies that bind unmodified human lung SPA gene product could be produced by immunizing animals with gene products of prokaryotic cells (such as E. Coli); antibodies binding to post-translationally modified forms thereof can be acquired by immunizing animals with gene products produced by eukaryotic cells (e.g., yeast or insect cells).
The technical solution of the present invention has the following technical effects compared with the prior art:
The present invention provides nanobodies that bind to human pulmonary surfactant protein A (hSP-A) with specificity. The present invention take fresh frozen sections of lung as antigen, gene sequences with high affinity with hSP-A were obtained by constructing an SP-A antibody library and affinity selection, and nanobodies with high affinity and small molecule weight were obtained by induced expression of the gene sequences through a prokaryotic expression vector. Immunohistochemistry and in vivo imaging in nude mice showed the nanobodies have high specificity for targeting lung tissue. By providing nanobodies with lung-targeting specificity, the present invention provides tools for further research on lung-targeting ligands for targeted drug delivery for human lung diseases.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows Western blot and ELISA result of human pulmonary surfactant protein A (hSP-A); 1A is the result of Western blot for hSP-A (1: Mark, 2: hSP-A); 1B is the result of ELISA (1: hSP-A, 2: negative protein).
FIG. 2A is PHAGE-ELISA of affinity selection; FIG. 2B is comparison of the coding sequences of the 15 clones.
FIG. 3 is SDS-PAGE of human lung nanobody Nb4, (1: Mark, 2: Nb4).
FIG. 4 is electron microscopy image of human lung tissue nanobodies Nb4.
FIG. 5 shows the Western blot, ELISA results of purified SPA-Nb; wherein 5A for the Western blot (positive: SP-A-mono-ant, 17: Nb4, negative: H1N1 nanobodies); 5B for the ELISA test (−1: SP-A-mono-ant, 2: Nb4, 3: irrelevant nanobody); ★ represent P≦0.001, ▴ represent P>0.05.
FIG. 6 is immunostaining result of human lung tissue nanobody Nb4 with sliced tissues of human lung, heart, liver, spleen, muscle.
FIG. 7 shows cell immunofluorescence result of human lung tissue nanobodies Nb4 with A549, L-02,293T cells.
FIG. 8 shows images of human lung tissue nanobodies Nb4 with FITC mark in the body of nude mouse at different times (respectively: after intravenous injection of 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h).
FIG. 9 shows the preparation process of human lung tissue hSPA-Nb.
FIG. 10 shows corresponding amino acid codons.



DETAILED DESCRIPTION
The present invention is further illustrated using the following embodiments, but any of the embodiments or its combinations thereof should not be construed as a limitation to the scope of the present invention.
Example 1. The Preparation of Human Pulmonary Surfactant Protein A (hSP-A)
1.1 the Preparation of Human Pulmonary Surfactant Protein A (hSP-A)
Grind 5 mg fresh human lung tissue with the mixture of protein lysate and PMSF in a tissue grinder for 3 min (60 HZ, 90S), centrifuged supernatant, measuring protein content (BCA).
1.2 hSP-A Identification:
1.2.1 Western Blot:
Purified hSP-A was isolated by SDS-PAGE and transferred onto nitrocellulose membrane. It was sealed in 20% goat serum and incubated for 2 hours, then immune serum containing mouse polyclonal antibody against hSP-A (at room temperature for 2 hours, and washed 3 times with PBS) and serum containing anti-mouse IgG-HRP (at room temperature for 1 hours, washed 3 times with PBS) were added sequentially. Scanning of fluorescence scanner and photographs of the camera displays the target bands are around 35 Kd, 70 Kd, 120 Kd, multiple bands (FIG. 1A).
1.2.2 ELISA Test:
ELISA test was performed to measure the immunological activity of the purified protein. An ELISA plate with 96 wells were coated with purified hSP-A and an irrelevant protein, and incubated overnight at 4° C. The next day, it was sealed in 3% skim milk and incubated at 37° C. for an hour, then immune serum containing hSP-A monoclonal antibody (at room temperature for 2 hours, and washed 3 times with PBS) and serum containing goat anti-mouse IgG-HRP (at room temperature for 1 hours, washed 3 times with PBS) were added sequentially. TMB was added last to develop the image, and sulfuric acid was added to stop the reaction. The OD value of each well was measured using the chromogenic microplate, which showed that, compared with the control group, both purified hSP-A and SP-A monoclonal antibodies had obvious binding activity (FIG. 1B).
Example 2. Screening of hSPA-Specific Nanobody (rSPA-Nb)
Affinity selection technique was employed to screen the VHH antibody library with acetone fixed fresh frozen sections of human lung.
2.1 Simplified Procedure of Affinity Selection:


    
    
        (1) fix fresh-frozen human lung slice with cold acetone for 30 min.
        (2) wash the tubes 10 times using PBS, and dried by shaking.
        (3) The tubes were blocked using 20% goat serum (1 ml serum was added in 4 ml PBS) and incubated for 2 hours at 37° C. The blocking solution was discarded, and the tubes were washed 3 times using PBS and dried.
        (4) 200 μl of the prepared phage library was added to each fresh human lung slice, and incubated overnight at 4° C.
        (5) The phage library on the slices was disposed, and the slices were washed three times with PBS, and dried.
        (6) coat host strain TG1 which OD600 is 0.8200 μl on each slice, 37° C. 1 h, to wash away the bound phage library; wash with PBS 10 times, drying, scraping the tissue on the slide into 2YTAG, 3° C. until turbidity. This completed the first round of selection, and the first antibody library was obtained. The output of the antibody library was calculated.
        (7) The selection steps were repeated for 3 times to obtain the third antibody library.

2.2 Preliminary Selection of Positive Nanobodies Using Indirect Phage ELISA.

        (1) Single colonies obtained from the three rounds of selections and grown on 2YTAG plates were inoculated into the 96-well culture plate at 30° C., and cultured with shaking overnight.
        (2) 300 ul of M13K07 helper phage was put in each well of another 96-well culture plate (labeled P1 Plate) the next day.
        (3) 40 ul of cultured medium were taken from each well of the Master Plate, which was cultured overnight, and put in each well of the P1 Plate, and incubated at 37° C. with shaking overnight. The culture supernatant was prepared by centrifugation at 150 rpm for 20 minutes set aside, and the recombinant antibody was obtained.
        (4) A 96-well microtiter plate was coated with hSP-A and incubated overnight at 4° C.
        (5) 160 ul of recombinant antibody was mixed with 40 μL of MPBS, incubated for 20 minutes at room temperature. It was then added to blocked microtiter wells and incubated overnight at 4° C.
    
    


(6) Washing and adding HRP secondary antibody: HRP-labeled antibody against M13K07 was diluted 1:1000 in PBS, 200 ul of that was added to each well, and incubated and reacted for 1 hour at 37° C.
(7) 200 ul TMB substrate solution was added to each well, incubated at 37° C. for about 45 minutes to develop the image, 100 ul of stop solution was added to each well to stop the development process, and measurements were taken at 450 nm. Preliminary screening was conducted to select positive clones binding to hSP-A with specificity. If a clone has affinity value greater than 3 times the affinity value for the negative control great, then it is considered to be a positive clone.
Preliminary screening by indirect Phage ELISA showed that 15 sequences had affinity value greater three times the affinity value for the negative control group, and these 15 sequences were positive clones (FIG. 2).
Example 3. Expression and Purification of hSPA-Nb with Specificity
3.1 Construction of hSPA-Nb Prokaryotic Expression Vector
The 15 clones selected by Phage ELISA were sent for sequencing (FIG. 3). No. 17 (Nb17) and No. 4 (Nb4) which had high affinity were PCR amplified using clone plasmid carrying BamH I and Xho I restriction sites. After the restriction digest, it was cloned to PET-26b (+) plasmid, and sent for sequencing.
3.2 Expression and Purification of Nanobodies
Recombinant plasmid with correct sequence was transformed into E. coli BL21 (DE3), the expression conditions were optimized, and protein expression was induced at 25° C., 0.8 mmol/L IPTG. The expressed product was purified with nickel affinity chromatography and molecular sieve. SDS-PAGE electrophoresis showed that the expressed nanobody had a molecular weight of 19 kDa (FIG. 4). As measured by BCA, the purified proteins had concentration levels of 10 mg/L and 12 mg/L, respectively. Observed under the electron microscope, the size of the antibodies was in the nanometer scale. (FIG. 5).
The 15 clones obtained by the present invention are effective lung-targeting ligands as their nucleotide sequences and amino acid sequences specifically bind to SP-A, which are listed below:
1) Nucleotide sequence listing:








1) Nucleotide sequence listing:



NO. 1, Nb4 (SEQ ID NO 1):


TTGCAGGCCCAGCTGGCCGGTCAGTTGCAGCTCGTGGAGTCGGGGGGAGG


 


CTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAAT


 


TCACTTTGGATTATTATGAAATAGGCTGGTTCCGGCAGGCCCCGGGGAAG


 


GACCGTGAGGGGCTCTCATGTATTGGTTATAGTGACAGAATCGCGTATTA


 


TTCAGAGTCCGTGAAGGGCCGATTCACCACCGTCAGAGACGACGCCACGA


 


GCACGGTCTCTCTTTATATGGATATGATGATTCCAGAGGACACAGGCACT


 


TATTATTGTGCGGGGTCGGTTGTGGAGCCTTACGAGTTACTGCCAGCGGC


 


TGAATATGACTACTGGGGACAGGGGACCCGGGTCACTGTCTCCTCAGCGC


 


ACCACAGCGAAGACCCCGGCCCCCGAGGCCTTGCGGCCGCAGGTGCGCCG


 


GTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGCA;


 


NO. 2, Nb6 (SEQ ID NO 2):


TGGCAGGCCCAGCTGGCCGTTCAGTTGCAGCTCGTGGAGTCTGGGGGAGA


 


CTTGGCGCAGCCTGGGGGGTCTCTGACACTCTCCTGTACAGCCTCTGGAA


 


CGTTCAAGATCTATTCCATGGGCTGGTACCGCCGCCCTCAGCGCGAGTTG


 


GTCGCGGAAATGCTTAATGGTGGTGACACACAATATTCAGACTCCGTGAA


 


GGGCCGATTCACCATCTCCAGAACCAACAACACGATGTATCTCCACATGA


 


ACAACCTGAAACCTGAGGACACGGCCGTCTATTATTGTAATCTACAGGAT


 


TGGTATAGCGAACCTGCGGGCGACTATTGGGGCCCGGGGACCCAGGTCAC


 


CGTCTCCTCAGCGCACCACAGCGAAGACCCCGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


A;


 


NO. 3, Nb11 (SEQ ID NO 3):


ATGCAGGCCCAGCTGGCCGGTCAGTTGCAGCTCGTGGAGTCTGGGGGAGG


 


CTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTAAAT


 


TCCATTTGGATTCTTATGCCGTAGCCTGGTTCCGCCAGACCCCAGGGAAG


 


GAGCGTGAGGCGGTCTCATTTATAAATACTAGTGATGATGTCACATACTT


 


TGCTGACTCCGTAAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGA


 


ACACGGTATATCTGCAAATGAACGTCCTGAAACCAGAAGACACTTCTATT


 


TATGTGTGTGCAGCGGTAAGAAGTCCCGGCCCTACCGGCCCTAGTATGCA


 


GCCTATGTGGTCGGTGCCTGACCTGTATGACTACTGGGGCCAGGGGACCC


 


AGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCGGCCCCCGAGGC


 


CTTGCGGCCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCG


 


TGCCGCA;


 


NO. 4, Nb15 (SEQ ID NO 4):


ATGCAGGCCCAGCTGGCCGGTCAGTTGCAGCTCGTGGAGTCTGGGGGAGG


 


CTTGGTGCAGCCTGGGGGGTCTCTGAGCGTCTCCTGCGCAGTCCGAGGAC


 


GCGATTTGGATTATTATGTCATCGGTTGGTTCCGCCAGGCCCCAGGGAAG


 


GAGCGTGAGGGTGTTTCATGCATTAATAATAGTGATGATACCACATACTA


 


TTCAGACTCCGTGAAGGGCCGATTTACCATCTCGAGAGATCACGCCAAGA


 


ACACGGTATATCTCCAAATGAACAACCTGAAACCTGAGGACACCGCCCTT


 


TATTACTGTGCAGCGGATTTCGATCGCCTCGATTTTACTGTTAAGGCTAT


 


GTGTGTTATGAAGTTCTTTTACTACTGGGGCCAGGGGACGCAGGTCACCG


 


TCTCCTCAGAACCCAAGACACCAAAACCACAAGGCCCCCGAGGCCTTGCG


 


GCCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


A;


 


NO. 5, Nb17 (SEQ ID NO 5):


ATGCAGGCCCAGCTGGCCGTTCAGTTGCAGCTCGTGGAGTCAGGTGGAGG


 


CTTGGTGCAGCCTGGGGGGTCTCTGAGACTCGCCTGTGCAGCTTCTGGAT


 


TCAATTTGGATGATTATGCAGACATAGGCTGGTTCCGCCAGGCCCCAGGG


 


AAGGAGCGTGAACGAGTCCTTTGTATTACTATTAGTGATGGTACCACATA


 


CTATGAAGACTCCGGGAAGGGCCGATTCTCCATCTCCACAGACATCGCCA


 


AGAACACGGTGTTTCTTCAAATGGACAGCCTGAAAGCTGAGGACACAGCC


 


GTTTATTATTGTGCAGGAGATCCCGCCCCTTTTTGTCTCTATAACACCTA


 


TGTACCGCGAACCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGC


 


ACCACAGCGAAGACCCCGGCCCCCGAGGCCTTGCGGCCGCAGGTGCGCCG


 


GTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGCA;


 


NO. 6, Nb22 (SEQ ID NO 6):


CTCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGA


 


GGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGG


 


ATCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGA


 


AGGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGT


 


AGTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGA


 


CAAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGG


 


ACACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGC


 


TTGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCAC


 


CGTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCG


 


GCCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


ATAGACTGT;


 


NO. 7, Nb23 (SEQ ID NO 7):


TCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGAG


 


GCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGA


 


TCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGAA


 


GGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGTA


 


GTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGAC


 


AAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGGA


 


CACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGCT


 


TGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCACC


 


GTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


A;


 


NO. 8, Nb25 (SEQ ID NO 8):


TGCTCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGG


 


GAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCT


 


GGATCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGG


 


GAAGGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATA


 


GTAGTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAA


 


GACAAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGA


 


GGACACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGA


 


GCTTGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTC


 


ACCGTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTG


 


CGGCCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCC


 


GCA;


 


NO. 9, Nb26 (SEQ ID NO 9):


TCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGAG


 


GCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGA


 


TCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGAA


 


GGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGTA


 


GTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGAC


 


AAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGGA


 


CACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGCT


 


TGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCACC


 


GTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGCA


 


TAGACTGT;


 


NO. 10, Nb27 (SEQ ID NO 10):


TCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGAG


 


GCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGA


 


TCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGAA


 


GGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGTA


 


GTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGAC


 


AAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGGA


 


CACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGCT


 


TGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCACC


 


GTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGCA


 


TAGACTGT;


 


NO. 11, Nb28 (SEQ ID NO 11):


ATGCAGGCCCAGCTGGCCGGTCAGTTGCAGCTCGTGGAGTCGGGGGGAGG


 


CTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAAT


 


TCACTTTGGATTATTATGAAATAGGCTGGTTCCGGCAGGCCCCGGGGAAG


 


GACCGTGAGGGGCTCTCATGTATTGGTTATAGTGACAGAATCGCGTATTA


 


TTCAGAGTCCGTGAAGGGCCGATTCACCACCGTCAGAGACGACGCCACGA


 


GCACGGTCTCTCTTTATATGGATATGATGATTCCAGAGGACACAGGCACT


 


TATTATTGTGCGGGGTCGGTTGTGGAGCCTTACGAGTTACTGCCAGCGGC


 


TGAATATGACTACTGGGGACAGGGGACCCGGGTCACTGTCTCCTCAGCGC


 


ACCACAGCGAAGACCCCGGCCCCCGAGGCCTTGCGGCCGCAGGTGCGCCG


 


GTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGCA;


 


NO. 12, Nb29 (SEQ ID NO 12):


TCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGAG


 


GCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGA


 


TCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGAA


 


GGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGTA


 


GTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGAC


 


AAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGGA


 


CACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGCT


 


TGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCACC


 


GTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


A;


 


NO. 13, Nb38 (SEQ ID NO 13):


TCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGAG


 


GCTCGGTGCAGGCTGGAGGGTCTCTGATACTCTCCTGTACAGCCTCTGGA


 


TCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGAA


 


GGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGTA


 


GTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGAC


 


AAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGGA


 


CACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGCT


 


TGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCACC


 


GTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


A;


 


NO. 14, Nb39 (SEQ ID NO 14):


TCTTCTACAAGGTGTCCAGGCTCAGGTGAAGCTGGTGGAGTCTGGGGGAG


 


GCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGA


 


TCAGACTACAGATGGATGTACATCGCCCGGTTTCGCCAATGTCCAGGGAA


 


GGAGCGCGAGGGGGTCGCAGCAATTTATACTGATGATACTGATGATAGTA


 


GTCCGATCTATGCCACCTCCGCCAAGGGCCGATTCACCATCTCCCAAGAC


 


AAGGACAAGAACGCGGTATATCTGCAAATGAACAGCCCGAAACCTGAGGA


 


CACTGCCATGTACTACTGTGCGGCAAGAGCGTTCGGTGGTACCTGGAGCT


 


TGAGCTCCCCGGACGACTTTAGTGCCTGGGGCCAGGGGACCCAGGTCACC


 


GTCTCCTCAGGAACGAATGAAGTATGCAAGTGGCCCCCGAGGCCTTGCGG


 


CCGCAGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGC


 


A;


 


NO. 15, Nb43 (SEQ ID NO 15):


ATGCAGGCCCAGCTGGCCGTTCAGTTGCAGCTCGTGGAGTCGGGGGGAGG


 


CTTGGTGCAATCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAT


 


TCACTTTCAATGACTATCGCATGAGCTGGGTCCGCCAGGCTCCAGGAAAG


 


GGGCTCGAGTGGGTCTCAGATATTAACAGTGGTGGTAGTAGTACATACTA


 


TGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGA


 


ACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCATT


 


TACTACTGTGTGGCCCTACTTGGGCGCGGTTGTTCAGGCTTGGTTCAGGG


 


GGCCTTTGGACCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGC


 


ACCACAGCGAAGACCCCGGCCCCCGAGGCCTTGCGGCCGCAGGTGCGCCG


 


GTGCCGTATCCGGATCCGCTGGAACCGCGTGCCGCA;


 


2) Amino acid sequence listing:


NO. 16, Nb4 (SEQ ID NO 16):









  1
LQAQLAGQLQ LVESGGGLVQ PGGSLRLSCA ASEFTLDYYE IGWFRQAPGK DREGLSCIGY



 61
SDRIAYYSES VKGRFTTVRD DATSTVSLYM DMMIPEDTGT YYCAGSVVEP YELLPAAEYD


121
YWGQGTRVTV SSAHHSEDPG PRGLAAAGAP VPYPDPLEPR AA;


 








NO. 17, Nb6 (SEQ ID NO 17):










  1
WQAQLAVQLQ LVESGGDLAQ PGGSLTLSCT ASGTFKIYSM GWYRRPQREL VAEMLNGGDT



 61
QYSDSVKGRF TISRTNNTMY LHMNNLKPED TAVYYCNLQD WYSEPAGDYW GPGTQVTVSS


121
AHHSEDPGPR GLAAAGAPVP YPDPLEPRAA;


 








NO. 18, Nb11 (SEQ ID NO 18):










  1
MQAQLAGQLQ LVESGGGLVQ PGGSLRLSCT ASKFHLDSYA VAWFRQTPGK EREAVSFINT



 61
SDDVTYFADS VKGRFTISRD NSKNTVYLQM NVLKPEDTSI YVCAAVRSPG PTGPSMQPMW


121
VPDLYDYWGQ GTQVTVSSAH HSEDPGPRGL AAAGAPVPYP DPLEPRAA


 








NO. 19, Nb15 (SEQ ID NO 19):










  1
MQAQLAGQLQ LVESGGGLVQ PGGSLSVSCA VRGRDLDYYV IGWFRQAPGK EREGVSCINN



 61
SDDTTYYSDS VKGRFTISRD HAKNTVYLQM NNLKPEDTAL YYCAADFDRL DFTVKAMCVM


121
KFFYYWGQGT QVTVSSEP KTPKPQGPRG LAAAGAPVPY PDLEPRAA;


 








NO. 20, Nb17 (SEQ ID NO 20):










  1
MQAQLAVQLQ LVESGGGLVQ PGGSLRLACA ASGFNLDDYA DIGWFRQAPG KERERVLCIT



 61
ISDGTTYYED SGKGRFSIST DIAKNTVFLQ MDSLKAEDTA VYYCAGDPAP FCLYNTYVPR


121
TWGQGTQVTV SSAHHSEDPG PRGLAAAGAP VPYPDPLEPRAA;


 








NO. 21, Nb22 (SEQ ID NO 21):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLRLSCT ASGSDYRWMY IARFRQCPGK EREGVAAIY



 61
TDDTDDSSPI YATSAKGRFT ISQDKDKNAV YLQMNSPKPE DTAMYYCAAR AFGGTWSLSS


121
PDDFSAWGQG TQVTVSSGTN EVCKWPPRPC GRRCAGAVSG SAGTACRIDC 


 








NO. 22, Nb23 (SEQ ID NO 22):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLRLSCT ASGSDYRWMY IARFRQCPGK EREGVAAIYT



 61
DDTDDSSPIY ATSAKGRFTI SQDKDKNAVY LQMNSPKPED TAMYYCAARA FGGTWSLSSP


121
DDFSAWGQGT QVTVSSGTNE VCKWPPRPCG RRCAGAVSGS AGTACRIDC


 








NO. 23, Nb25 (SEQ ID NO 23):










  1
ALLQGVQAQV KLVESGGGSV QAGGSLRLSC TASGSDYRWM YIARFRQCPG KEREGVAAIY



 61
TDDTDDSSPI YATSAKGRFT ISQDKDKNAV YLQMNSPKPE DTAMYYCAAR AFGGTWSLSS


121
PDDFSAWGQG TQVTVSSGTN EVCKWPPRPC GRRCAGAVSG SAGTACRIDC


 








NO. 24, Nb26 (SEQ ID NO 24):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLRLSCT ASGSDYRWMY IARFRQCPGK EREGVAAIYT



 61
DDTDDSSPIY ATSAKGRFTI SQDKDKNAVY LQMNSPKPED TAMYYCAARA FGGTWSLSSP


121
DDFSAWGQGT QVTVSSGTNE VCKWPPRPCG RRCAGAVSGS AGTACRIDC 


 








NO. 25, Nb27 (SEQ ID NO 25):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLRLSCT ASGSDYRWMY IARFRQCPGK EREGVAAIYT



 61
DDTDDSSPIY ATSAKGRFTI SQDKDKNAVY LQMNSPKPED TAMYYCAARA FGGTWSLSSP


121
DDFSAWGQGT QVTVSSGTNE VCKWPPRPCG RRCAGAVSGS AGTACRIDC


 








NO. 26, Nb28 (SEQ ID NO 26):










  1
MQAQLAGQLQ LVESGGGLVQ PGGSLRLSCA ASEFTLDYYE IGWFRQAPGK DREGLSCIGY



 61
SDRIAYYSES VKGRFTTVRD DATSTVSLYM DMMIPEDTGT YYCAGSVVEP YELLPAAEYD


121
YWGQGTRVTV SSAHHSEDPG PRGLAAAGAP VPYPDPLEPR AA;


 








NO. 27, Nb29 (SEQ ID NO 27):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLRLSCT ASGSDYRWMY IARFRQCPGK EREGVAAIYT



 61
DDTDDSSPIY ATSAKGRFTI SQDKDKNAVY LQMNSPKPED TAMYYCAARA FGGTWSLSSP


121
DDFSAWGQGT QVTVSSGTNE VCKWPPRPCG RRCAGAVSGS AGTACRIDC


 








NO. 28, Nb38 (SEQ ID NO 28):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLILSCT ASGSDYRWMY IARFRQCPGK EREGVAAIYT



 61
DDTDDSSPIY ATSAKGRFTI SQDKDKNAVY LQMNSPKPED TAMYYCAARA FGGTWSLSSP


121
DDFSAWGQGT QVTVSSGTNE VCKWPPRPCG RRCAGAVSGS AGTACRIDC


 








NO. 29, Nb39 (SEQ ID NO 29):










  1
LLQGVQAQVK LVESGGGSVQ AGGSLRLSCT ASGSDYRWMY IARFRQCPGK EREGVAAIYT



 61
DDTDDSSPIY ATSAKGRFTI SQDKDKNAVY LQMNSPKPED TAMYYCAARA FGGTWSLSSP


121
DDFSAWGQGT QVTVSSGTNE VCKWPPRPCG RRCAGAVSGS AGTACRIDC


 








NO. 30, Nb43 (SEQ ID NO 30):










  1
MQAQLAVQLQ LVESGGGLVQ SGGSLRLSCA ASGFTFNDYR MSWVRQAPGK GLEWVSDINS



 61
GGSSTYYADS VKGRFTVSRD NAKNTLYLQM NSLKPEDTAI YYCVALLGRG CSGLVQGAFG


121
PWGQGTQVTV SSAHHSEDPG PRGLAAAGAP VPYPDPLEPR AA.









Example 4. Testing of hSPA-Nb's Lung-Specificity
To further verify the affinity between hSPA-Nb and human pulmonary surfactant protein A, and whether hSPA-Nb has lung-specificity, Western blot and ELISA were used to preliminarily measure the antigen specificity of hSPA-Nb, and immunohistochemistry and in vivo imaging were used to verify its lung-specificity in vivo.
4.1 Western Blot and ELISA
Purified human lung tissue SPA-Nb4, irrelevant nanobody (H1N1 nanobodies) and commercial anti-human SP-A monoclonal antibody were selected as the primary antibody to test the affinity between SPA-Nb4 and hSPA using Western blot and ELISA (using the same method described in section 1.2). The results showed that Nb4 had significant binding specificity with hSPA (FIG. 6A, 6B).
4.2 Cell Immunofluorescence
When A549 (lung), L-02 (liver), 293T (kidney) cells were grown and cover the cell plates to 95%-100%, PBS washed 3 times, incubated in fixative 30 min, PBS washed 3 times, 0.2% Triton X-100 permeabilization 5 min, blocked for 1 h by 20% goat serum, diluted primary antibody (human lung tissues Nb4-Fitc) for the experimental group, anti-human SP-A monoclonal antibody as a positive control group, and H1N1-Fitc nanobodies as a negative control group) was dropped on. The secondary antibody was anti-mouse-IgG-APC. The results showed that Nb4 and SPA monoclonal antibody (SPA-monopoly-ant) had significant binding effect with human lung tissue (shown as green/red), wherein the human lung tissue Nb4 binding ability is similar with SPA-monopoly-ant. All three antibodies had no obvious binding effect with human heart, liver, spleen, kidney, muscle tissues, nor had the negative control group (FIG. 7).
4.3 Immunohistochemistry
The fresh human lung, liver, spleen, kidney and other tissue sections were fixed, diluted primary antibody (human lung tissues Nb4 for the experimental group, SP-A monoclonal antibody as a positive control group, and H1N1 nanobodies as a negative control group) was dropped on. The secondary antibody was His-IgG-HRP or anti-mouse-IgG-HRP. The results showed that human lung tissues Nb4 and SPA monoclonal antibody (SPA-monopoly-ant) had significant binding effect with human lung tissue (shown as brown), wherein Nb4 binding ability is similar with SPA-monopoly-ant. All three antibodies had no obvious binding effect with human heart, liver, spleen, kidney, muscle tissues, nor had the negative control group (FIG. 8).
4.4 In Vivo Lung-Specificity Testing Using FITC-Marked Nanobody in Mice
Sequence homology analysis showed that there is a high degree of homology between the amino acid sequence of human and mouse rSPA. Since it is easier to obtain in vivo imaging using nude mice, nude mice were chosen for testing specificity in vivo. Five-week-old nude mice were chosen, and after continuous inhalation anesthesia, 200 ul FITC-labeled nanobody was injected intravenously at the tail, and the dose was 1 mg/kg of the animal body weight. The nude mice were imaged at 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours after the injection, respectively. At the same time, 200 ul H1N1-Fitc nanobody was injected intravenously at the tail as the negative control group (FIG. 9). The results showed that 15 minutes after intravenous injection, the FITC-labeled nanobody began to clearly cluster in the lung. 2 hours after the injection, the clustering in the lung was still obvious, and the lung-targeting effect was similar to that of the nasal inhalation.
The above experiment was repeated using the functional region of the polypeptides of synthetic human lung tissues Nb4 (SEQ ID NO:16 and Nb17 (SEQ ID NO:20) (without the MQAQKAG portion). It was found that the synthetic polypeptides also bound to hSPA with specificity, and clustered around the lung in vivo testing.
Example 5. Clone Protein Expression and Targeting Detection
Sequence homology comparative analysis was conducted on the selected 15 sequences, and it was found that human lung tissues Nb23, Nb25, Nb27, Nb29 and Nb39 had the same polypeptide sequence, human lung tissues Nb28 and Nb4 had high sequence similarity; while the rest of the sequences were quite different.
To further verify that the 15 nanobody sequences exhibits lung-targeting affinity with SP-A, 8 clones (excluding those with the same sequence as Nb4) were expressed and purified in accordance with the method described in Examples 5 and 6. Soluble expressions of these nanobodies were obtained, where Nb1 has the least protein expression concentration of 3 mg/L, while the rest of nanobodies have an average protein expression concentration of 8 mg/L.
In Western blot and ELISA, affinity was clearly shown in all 6 proteins, and the OD450 value in ELISA for 5 nanobodies, namely human lung tissues Nb11, Nb15, Nb17, Nb6 and Nb43 were 2 times greater than that of the negative control group. Immunohistochemical staining showed that these clones had strong affinity. All clones showed significant differences with the negative control group.
In vivo specificity testing in mice showed that five nanobodies, namely Nb11, NB15, NB17, NB6 and Nb43 had specificity similar to that of Nb17; while there were variations in the clustering effect, all the images exhibited obvious clustering in the lung.
Above mentioned specific embodiments of the present invention are presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Thus, equality of changes and modifications without departing from the spirit and scope of the invention shall fall within the scope of the invention.

## Claims

1. A human lung-targeting nanobody, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

2. A preparation comprising the nanobody of claim 1.

3. The preparation of claim 2, wherein the nanobody binds to human pulmonary surfactant protein A.