Construction method and application of pig whole sperm membrane protein antigen specific nano antibody library

Abstract

The invention discloses a pig whole sperm membrane protein antigen specific nano antibody library construction method which comprises the following steps: S1, immunizing alpaca with pig whole sperms, and separating peripheral blood cells after immunization; s2, RNA in peripheral blood cells is extracted, cDNA is obtained through reverse transcription, and a VHH coding sequence of an antibody is amplified; s3, cloning the amplified VHH coding sequence to a bacteriophage plasmid vector to prepare a recombinant bacteriophage vector; s4, transforming the recombinant bacteriophage vector into escherichia coli and carrying out enlarged culture to obtain a bacteriophage display library; and S5, performing panning on the phage display library for a plurality of times by using the pig X / Y sperms, and constructing the pig single sex sperm specific nano antibody library. According to the method, the pig whole sperm is adopted as an immunogen for immunizing alpaca for the first time, the capacity of the constructed antibody library reaches 3.4 * 10 < 8 > pfu, the abundance of the antibody library is higher, the coverage degree of whole sperm antigens is higher, and the variety is more complete, so that the capture success rate of the pig sex control specific antibody is increased, and a foundation is laid for industrial application of the pig sperm specific antibody.

Description

Technical Field

[0001] The present invention relates to the field of antibody library preparation and animal breeding, and in particular to a method for constructing a porcine whole sperm membrane protein antigen-specific nano antibody library and its application. Background Art

[0002] In the breeding system of sows, there is a large difference in economic value between males and females, and sows create more economic benefits than boars. If a breakthrough is made in the technology of controlling the sex of sows, the number of sows per litter can be increased from 45%-50% to 65%. Each sow used to produce purebred pigs can generate an additional benefit of 20,000 yuan, and used to produce binary hybrid sows, it can generate an additional benefit of 10,000 yuan. If it is promoted nationwide, an additional output value of 36 billion yuan can be generated just by adjusting the sow ratio. Therefore, the sex control technology of sows is a breeding technology of great value. Breakthroughs in the sex control technology of sows can optimize the breeding system of sows, maximize the use of breeding resources, improve the efficiency and benefits of pig breeding, and promote high-quality development of the industry.

[0003] At present, X and Y sperm sorting technology based on flow cytometry has been applied in the cattle industry, and the accuracy is above 90%. However, its sorting efficiency is far from meeting the needs of pig breeding. For example, the current flow cytometry sperm sorting speed is 20 million / hour, and the amount of sperm required for artificial insemination of breeding pigs is 2.5-3 billion. The time required for sorting for one mating is as high as 100 hours, which cannot be applied in production practice. In addition, during the flow sorting process, sperm staining, laser irradiation and subsequent treatment will damage the structure and vitality of sperm and reduce the fertilization ability of sperm. In addition, flow cytometry sperm sorting requires expensive equipment and professional operators, which increases the barriers and costs of the application of sex control technology, and is not conducive to the establishment of an efficient livestock and poultry breeding technology system. Therefore, it is necessary to develop a new antibody library that can be used for pig X and Y sperm sorting and for the research and development of sperm immunosorting technology. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a porcine whole sperm membrane protein antigen-specific nanoantibody library and its application, by which sperm-specific antibodies can be efficiently screened and used for sorting porcine X and Y sperm to solve the above-mentioned problems.

[0005] According to the first aspect of the present invention, a method for constructing a porcine whole sperm membrane protein antigen-specific nanoantibody library is provided, the method comprising the following steps: S1: immunizing alpacas with porcine whole sperm, and isolating peripheral blood cells of the alpacas after immunization; S2: extracting RNA from peripheral blood cells, reversely transcribing to obtain cDNA, and amplifying the VHH coding sequence of the antibody; S3: cloning the amplified VHH coding sequence into a phage plasmid vector to prepare a recombinant phage vector carrying the porcine sperm antibody gene; S4: transforming the recombinant phage vector into coli and expand the culture to obtain a pig whole sperm antibody phage display library; S5: Use pig X / Y sperm to perform several pannings on the phage display library obtained in step S4 to construct a pig single sex sperm membrane protein antigen specific nano antibody library: use X sperm for negative screening, use Y sperm for positive screening, and perform panning to construct a Y sperm membrane protein antigen specific nano antibody library; use Y sperm for negative screening, use X sperm for positive screening, and perform panning to construct an X sperm membrane protein antigen specific nano antibody library. The present invention innovatively uses pig whole sperm to immunize alpacas, and uses phage display technology to construct a sperm immune nano antibody library, and the library capacity reaches 3.4×10 8 pfu, the antibody library has a higher abundance, a higher coverage of all sperm antigens, and a more complete range of antibodies, thereby improving the success rate of capturing pig sex-controlled specific antibodies. In addition, a nano-antibody library targeting a single sex can be obtained by positive / negative screening using X / Y sperm, and this method only requires 4-7 rounds of panning to enrich a large number of positive antibodies. Combined with high-throughput amplicon sequencing technology and biological big data analysis technology, it can achieve rapid development and screening of antibodies, laying the foundation for the industrial application of pig sperm-specific antibodies.

[0006] In some embodiments, in step S1, the pig sperm is injected subcutaneously into the alpaca for at least two consecutive immunizations with an interval of one week, thereby achieving the best immunization effect and the highest antibody titer.

[0007] In certain embodiments, the phage plasmid used in step S3 is pCom3XSS or a variant thereof.

[0008] In certain embodiments, the expanded culture in step S4 is obtained by adding helper phage for rescue.

[0009] In certain embodiments, the several rounds of panning in step S5 refers to at least 2 rounds of panning.

[0010] In certain embodiments, X sperm is used for negative screening, and Y sperm is used for positive screening, and at least one round of panning is required when constructing a Y sperm-specific nanoantibody library for panning.

[0011] In certain embodiments, Y sperm is used for negative screening and X sperm is used for positive screening. It is recommended that at least one round of panning be performed when the X sperm-specific nanobody library is panned.

[0012] According to the second aspect of the present invention, a porcine whole sperm membrane protein antigen-specific nanoantibody library prepared by the above method is provided. The antibody library of the library has a higher abundance, a higher coverage of whole sperm antigens, and a more complete variety, thereby improving the success rate of capturing porcine sex control-specific antibodies.

[0013] According to a third aspect of the present invention, there is provided a pig whole sperm membrane protein antigen-specific nanoantibody library for use in screening pig sperm-specific nanoantibodies, wherein the pig sperm-specific nanoantibody library is prepared by the above method.

[0014] According to a fourth aspect of the present invention, there is provided a porcine whole sperm membrane protein antigen-specific nanoantibody library for use in pig X and Y sperm sorting, wherein the porcine whole sperm membrane protein antigen-specific nanoantibody library is prepared by the above method.

[0015] Beneficial effects of the present invention: The present invention innovatively uses whole pig sperm to immunize alpacas, and uses phage display technology to construct a whole pig sperm membrane protein antigen-specific nanoantibody library, which can be used for screening of pig sperm-specific antibodies and further for sorting pig sperm to achieve sex control of pig semen. This nanoantibody library uses whole pig sperm to immunize alpacas for the first time. Since pig sperm is used as an immunogen, the antibody library capacity constructed by it reaches 3.4×10 8 pfu, the antibody library has a higher abundance, a higher coverage of all sperm antigens and a more complete variety, thereby increasing the success rate of capturing pig sex-controlled specific antibodies. In addition, nanoantibody libraries targeting a single sex can be obtained by positive / negative screening using X / Y sperm, laying the foundation for the industrial application of pig sperm-specific antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the monitoring result of serum antibody titer of alpaca immunized with pig sperm;

[0017] Figure 2 This is the result of monoclonal antibody abundance analysis of pig sperm panning library;

[0018] Figure 3 This is the result of monoclonal antibody binding specificity analysis of the pig sperm panning library;

[0019] Figure 4 Figure 1 is the result of differential antibody flow cytometry analysis;

[0020] Figure 5 This is the result map of differential antibody binding position location;

[0021] Figure 6 This is a clustering result diagram of the pig sperm panning nanoantibody library;

[0022] Figure 7 This is a graph showing the results of the analysis of the binding characteristics of X / Y sperm-specific antibodies on the sperm membrane surface. DETAILED DESCRIPTION

[0023] The invention will be further described in detail below in conjunction with the accompanying drawings.

[0024] Example 1: Construction of Nanoantibody Library of Alpaca Immunized with Pig Whole Sperm

[0025] 5 mL of blood was collected from healthy alpacas before immunization, and serum was separated as negative serum. In the first week, the first immunization was carried out, 10 million fresh pig sperm were taken, centrifuged at 800 g for 10 minutes at room temperature, the supernatant was removed, and 1 mL of PBS was added for washing, and repeated twice. After washing, it was resuspended with 1 mL of PBS, and complete Freund's adjuvant was added to the cells (10 8 ) 1:1 mixed emulsified, and injected subcutaneously at multiple points. Following the first immunization, the second, third, and fourth immunizations were performed in the same way in the third, fifth, and seventh weeks to strengthen the immunization. During the immunization process, the alpaca serum was separated and the titer of pig sperm-specific antibodies in the serum was monitored using immunofluorescence technology. The results are as follows Figure 1 As shown in: It shows that the first immunization can induce alpacas to secrete pig sperm-specific antibodies, the antibody titer continues to increase after the first booster immunization, the antibody titer reaches the highest after the second booster immunization, and is maintained until the final immunization, when the antibody titer reaches the requirements for antibody library construction.

[0026] 7-10 days after the fourth immunization, 5 ml of peripheral blood was collected, serum was separated, and the total IgG titer against the antigen was detected. The immune response was monitored by ELISA. After the animal serum antibody titer reached the standard (>100000), 50 mL of peripheral blood was collected for subsequent nanoantibody library construction. Peripheral blood cells (PBMC) were separated from the peripheral blood, and total RNA was extracted twice, obtaining a total of 247.84 μg of RNA. After RNA was reverse transcribed into cDNA, the cDNA was used as a template to amplify the coding sequence of the variable region (VHH) of the heavy chain of the alpaca antibody in two rounds using nested PCR technology to obtain 1238.8 μg of DNA, which was cloned into the phage plasmid pCom3XSS (Apark Bio, P001). The recombinant phage plasmid carrying the pig sperm antibody gene was electrotransformed into TG1 Escherichia coli (Apark Bio, P021), and the antibody bacterial library was obtained by expansion culture. The library capacity was determined to be 3.4×10 8pfu, the positive rate was 96%, and the diversity was 100% (Table 1). The bacterial library was added to 100 mL of 2×TY medium until OD600 = 0.1, and cultured at 37°C 250rpm until OD600 = about 0.5, and helper phage M13KO7 (New England Biolabs (NEB, N0315S) was added at a ratio of 1:10-20 (bacterial number: helper phage number) to rescue, and cultured overnight, centrifuged at 4°C 4000rmp for 10min, and the supernatant was taken to purify the phage using PEG / NaCl solution to obtain the pig whole sperm antibody phage display library. The library titer was determined to be 1.2×10 14 pfu / mL (Table 1).

[0027] Table 1 Results of the construction of the nanoantibody library of pig sperm immunized alpaca

[0028]

[0029] The above results show that by using pig whole sperm to immunize alpacas and using phage display technology to construct a sperm immune nanoantibody library, the library capacity reached 3.4×10 8 pfu, the antibody library has a higher abundance, a higher coverage of sperm antigens, and a more complete variety, thereby increasing the success rate of capturing pig sex control-specific antibodies. Moreover, after rescue with helper phage and expanded culture, the library titer can reach 1.2×10 14 pfu / mL.

[0030] Example 2: Selection of Nanoantibodies Specific to Pig Sperm

[0031] Take 10 million fresh pig sperm, wash 3 times with PBS, add 2 mL of PBS to resuspend, add 10 million sperm / well to 6-well cell culture, centrifuge at 1200g for 10 minutes, aspirate the supernatant, dry at 50°C, fix with 4% paraformaldehyde for 15 minutes, block with 3% BSA solution for 1 hour, wash 3 times with PBS, use the fixed well without sperm as blank control, add pig sperm immunized alpaca nanoantibody library for the first round of screening, recover and amplify the positive phages in the first round of screening, and use the obtained screening antibody library for the next round of screening. Using the same method, the flow sorted pig X sperm and Y sperm were fixed to 12-well cell culture plates, and X sperm was used for negative screening to remove antibodies binding to X sperm, and Y sperm was used for positive screening to enrich antibodies binding to Y sperm, and construct a Y sperm-specific antibody library. Alternatively, Y sperm was used for negative screening to remove antibodies binding to Y sperm, and X sperm was used for positive screening to enrich antibodies binding to X sperm, and construct an X-specific antibody library. 1000 times the number of binding phages in the previous round was used as the input library amount, and 4-7 rounds of panning were continued. The positive rate was calculated by dividing the number of binding phages by the number of input phages to evaluate the enrichment effect of each round of antibodies. The results are shown in Tables 2 and 3: The library was effectively enriched by using pig Y sperm for positive screening to pan the nanoantibody library. The 7th round of panning was 587 times richer than the 1st round of panning antibody library (Table 2); the library was also effectively enriched by using pig X sperm for positive screening to pan the nanoantibody. The library was enriched 1221 times in the 4th round compared with the 1st round (Table 3). In order to further reduce the library capacity of X sperm-specific antibodies and reduce the difficulty of antibody identification, the 5th round of screening was performed with 100 times the number of phages bound in the 4th round of panning as the library input. Thus, a highly efficient pig sperm membrane protein antigen-specific nanoantibody library was obtained after panning.

[0032] Table 2 Results of panning of pig Y sperm-specific nanoantibodies

[0033] Selection rounds Input library dosage / pfu Number of binding phages / pfu Positive ratio Round 1 <![CDATA[3.40×10 11 ]]> <![CDATA[5.06×10 5 ]]> <![CDATA[1.49×10 -6 ]]> Round 2 <![CDATA[5.06×10 8 ]]> <![CDATA[1.83×10 5 ]]> <![CDATA[3.62×10 -4 ]]> Round 3 <![CDATA[1.83×10 8 ]]> <![CDATA[3.78×10 5 ]]> <![CDATA[2.07×10 -3 ]]> Round 4 <![CDATA[3.78×10 8 ]]> <![CDATA[3.50×10 5 ]]> <![CDATA[9.26×10 -4 ]]> Round 5 <![CDATA[3.50×10 8 ]]> <![CDATA[8.64×10 4 ]]> <![CDATA[2.47×10 -4 ]]> Round 6 <![CDATA[8.64×10 7 ]]> <![CDATA[5.76×10 4 ]]> <![CDATA[6.67×10 -4 ]]> Round 7 <![CDATA[5.76×10 7 ]]> <![CDATA[5.04×10 4 ]]> <![CDATA[8.75×10 -4 ]]>

[0034] Note: Positive ratio = number of binding phages in the Nth round of screening / amount of library input.

[0035] Table 3 Results of panning of pig X sperm-specific nanoantibodies

[0036] Selection rounds Input library dosage / pfu Number of binding phages / pfu Positive ratio Round 1 <![CDATA[3.40×10 11 ]]> <![CDATA[5.06×10 5 ]]> <![CDATA[1.49×10 -6 ]]> Round 2 <![CDATA[5.06×10 8 ]]> <![CDATA[4.08×10 5 ]]> <![CDATA[8.06×10 -4 ]]> Round 3 <![CDATA[4.08×10 8 ]]> <![CDATA[5.76×10 5 ]]> <![CDATA[1.41×10 -3 ]]> Round 4 <![CDATA[5.76×10 8 ]]> <![CDATA[1.05×10 6 ]]> <![CDATA[1.82×10 -3 ]]> Round 5 <![CDATA[1.05×10 8 ]]> <![CDATA[2.60×10 4 ]]> <![CDATA[2.48×10 -4 ]]>

[0037] Note: Positive ratio = Nth round of screening, number of combined phages / amount of library input Example 3 Functional verification of pig whole sperm membrane protein antigen-specific nanoantibody library and its application in screening specific antibodies

[0038] 3.1 Analysis of the pig sperm nanoantibody library using first-generation sequencing and ELISA technology

[0039] The porcine sperm membrane protein antigen-specific nanoantibody library obtained in Example 2 was used to infect TG1 Escherichia coli, and then coated on a 2×TY solid culture plate containing ampicillin, cultured overnight, and single clone colonies were selected for sequencing to analyze the antibody coding sequence information and abundance. The results of single clone colony sequencing showed (as shown in Figure 2 As shown): After pig sperm selection, positive antibodies were effectively enriched, with the highest abundance being 13.19%, indicating that the constructed pig whole sperm membrane protein antigen-specific antibody nanolibrary is of excellent quality.

[0040] Further, the flow sorted pig X and Y sperm were taken, and then centrifuged at 800g for 10 minutes, the supernatant was aspirated, and resuspended with CBS coating solution (pH9.6), and 300,000 sperm were added to each well of a 96-well ELISA plate, and coated at 4°C overnight. The supernatant was gently aspirated, dried at 50°C, fixed with 4% paraformaldehyde for 15 minutes, blocked with 3% BSA for 1 hour, washed 3 times with PBST, and the phage wells without display antibodies were added as negative controls, and the wells without antibody addition were blank controls. The same amount of the same strain of antibody was added to the X sperm and Y sperm coating wells, respectively, incubated at room temperature for 2 hours, washed 3 times with PBST, added with secondary antibody anti-M13 Bacteriophage (HRP) (11973-MM05T-H, Sino Biological Inc.), incubated at room temperature for 1 hour, washed 6 times with PBST, developed with TMB colorimetric solution, and measured OD370 absorbance to screen antibodies that differentially bind to X and Y sperm. The binding specificity of 47 highly abundant antibodies was detected by ELISA technology, and the ratio of the OD370 value of sperm X to the OD370 value of sperm Y (OD370-X / Y) greater than or equal to 1.3 was used as the specificity screening standard. Figure 3 The results showed that 11 strains of antibodies with strong binding ability to X sperm could be screened out, indicating that the nanolibrary has a high screening efficiency.

[0041] Immunofluorescence technology and flow cytometry were used to analyze the binding position and specificity of differential antibodies on the surface of sperm membrane. 10 million fresh pig sperm were taken, washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, blocked with 3% BSA for 1 hour, washed 3 times with PBST, and the phage that did not display the antibody was used as a control. The screened differential antibodies were added, incubated at room temperature for 2 hours, washed 3 times with PBST, and the secondary antibody anti-M13 Bacteriophage (FITC) (11973-MM05T-P, SinoBiological Inc.) was added, incubated at room temperature for 1 hour, washed 10 times with PBST, and resuspended with 1 mL of PBS. 700 μL of antibody-incubated sperm were taken to detect the fluorescence ratio using a flow cytometer to analyze the antibody specificity. Another 300 μL of antibody-incubated sperm was placed in a 48-well cell culture plate, centrifuged at 1200 g for 10 minutes, the supernatant was removed, DAPI staining solution was added, and incubated at room temperature for 20 minutes. The binding position of the antibody on the sperm membrane surface was observed under a fluorescence microscope. The results of flow cytometry analysis showed (eg Figure 4 The results of fluorescence observation showed that the binding sites of the 11 antibodies were all located on the mitochondrial sheath of the sperm tail (as shown in Figure 2). Figure 5 As shown). This indicates that the nanolibrary can screen out positive antibodies that are highly efficient and specifically bind to sperm.

[0042] 3.2 Analysis of the porcine sperm selection library using amplicon sequencing and biological big data analysis technology

[0043] The 6th and 7th rounds of panning eluates with positive screening of Y sperm and the 5th round of panning eluates with positive screening of X sperm were selected to infect TG1 Escherichia coli, and the phage plasmids were extracted after expansion culture to obtain antibody plasmid libraries, which were named Y5 library, Y6 library and X5 library respectively. The nanoantibody coding sequence on the plasmid was amplified using primers VHH-F (SEQ ID No: 1): 5'-AAGACA GCTATC GCGATTGCA G-3' and VHH-R (SEQ ID No: 2): 5'-GCC CCC TTATTA GCG TTT GCC ATC-3', and purified and recovered. The purified amplification product was used to construct an amplicon sequencing library, and PE250 sequencing was performed using the Illuminate high-throughput sequencing platform. After splicing and quality control of the sequencing data, the abundance of the antibody sequence was calculated. Redundant sequences were removed using CD-hit and MEGA11 software, and multiple sequences of the de-redundant sequences were aligned using Muscle software. The alignment results were AI clustered using Iq-tree2 software to obtain Y sperm-specific sequences, X sperm-specific sequences, and XY sperm common sequences. A total of 22 X sperm and Y sperm-specific sequences were randomly selected, and the sequences were codon-optimized according to the codon usage preference of the CHO cell expression system. The optimized sequences were fused with the Fc fragment of the rabbit IGg antibody to construct a fusion expression plasmid, which was transfected into CHO cells for fusion expression, and the recombinant expressed antibodies were purified. The binding characteristics of the recombinant antibodies on the surface of the pig sperm membrane were detected by immunofluorescence technology.

[0044] A total of 3 million antibody coding sequences were obtained by sequencing the amplicons of the Y5, Y6, and X5 libraries. Using MEGA11 software to remove redundancy, 4469 sequences were obtained. Using CD-hit software to remove redundancy with a homology of 0.98, 4072 sequences were obtained. Using Iq-tree2 software, clustering diagrams were constructed for the redundant sequences removed by the two softwares, and Y sperm-specific sequences, X sperm-specific sequences, and X and Y sperm common sequences (such as Figure 6 22 X and Y sperm-specific sequences were selected for recombinant expression, and the binding characteristics of the antibodies were detected by immunofluorescence technology. The results showed that all 22 antibodies could bind to the surface of the pig sperm membrane, of which 18 bound to the mitochondrial sheath of the sperm tail, 2 bound to the sperm acrosome, 1 bound to the sperm head and tail junction, and 1 bound to the entire sperm tail (as shown in Figure 1). Figure 7 As shown). This indicates that the prepared pig sperm-specific antibody nano-library can screen out positive antibodies that specifically bind to the sperm membrane surface, and can be used to further screen out nano-antibodies for separating X and Y sperm. This also indicates that the pig sperm-specific nano-antibody library was successfully prepared with good specificity.

[0045] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all belong to the protection scope of the invention.

Claims

1. A method for constructing a porcine whole sperm membrane protein antigen-specific nanoantibody library, wherein: The method comprises the following steps: S1: Immunize alpacas with pig whole sperm, and separate alpaca peripheral blood cells after immunization; S2: Extract RNA from peripheral blood cells, reverse transcribe to obtain cDNA, and amplify the VHH coding sequence of the antibody; S3: cloning the amplified VHH coding sequence into a phage plasmid vector to prepare a recombinant phage vector carrying the pig sperm antibody gene; S4: Transform the recombinant phage vector into Escherichia coli and expand the culture to obtain the porcine whole sperm antibody phage display library; S5: Using pig X / Y sperm to perform several pannings on the phage display library obtained in step S4 to construct a pig single-sex sperm membrane protein antigen-specific nanoantibody library: using X sperm for negative screening and Y sperm for positive screening, panning to construct a Y sperm membrane protein antigen-specific nanoantibody library; using Y sperm for negative screening and X sperm for positive screening, panning to construct an X sperm membrane protein antigen-specific nanoantibody library.

2. The method according to claim 1, wherein: In the step S1, whole pig sperm is injected subcutaneously into the alpaca for immunization at least twice with an interval of 1 week.

3. The method according to claim 2, wherein: The phage plasmid used in step S3 is pCom3XSS or its similar variants.

4. The method according to claim 3, wherein: The expanded culture in step S4 is obtained by adding helper phage for rescue.

5. The method according to claim 4, wherein: The several rounds of panning in step S5 refers to at least 2 rounds of panning.

6. The method according to claim 5, wherein: X sperm is used for negative screening and Y sperm is used for positive screening. When constructing a Y sperm-specific nanoantibody library for panning, at least one round of panning is required.

7. The method according to claim 5, wherein: Using Y sperm for negative screening and X sperm for positive screening, at least one round of panning is required when constructing an X sperm-specific nanoantibody library for panning.

8. A porcine whole sperm membrane protein antigen-specific nanobody library prepared by the method described in any one of claims 1 to 7.

9. Use of the pig whole sperm membrane protein antigen-specific nanobody library described in claim 8 in screening pig sperm-specific nanobodies.

10. Use of the porcine whole sperm membrane protein antigen-specific nanoantibody library as claimed in claim 8 in porcine X and Y sperm sorting.

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