CN113512110A

CN113512110A - BVDV high-specificity nano antibody and preparation method and application thereof

Info

Publication number: CN113512110A
Application number: CN202110392556.XA
Authority: CN
Inventors: 盛金良; 杨艳; 陈创夫; 肖盛中; 李岩; 周子恒; 李玉豪; 宋雯妍; 郑婷
Original assignee: Shihezi University
Current assignee: Xinjiang Fangmu Biotechnology Co ltd; Shihezi University
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2021-10-19
Anticipated expiration: 2041-04-13
Also published as: CN116023479A; CN113512110B; CN115925895A

Abstract

The invention belongs to the field of biotechnology detection, and discloses an anti-BVDV-important functional protein nano antibody and a preparation method and application thereof, wherein an NS5A protein is purified mainly by constructing a BVDV-NS5A expression vector; immunizing alpaca with BVDV inactivated vaccine, separating alpaca peripheral blood lymphocyte, obtaining VHH gene by nested PCR, constructing nanometer antibody phage display library with target gene insertion rate of 90.8% and library capacity of 1.02 × 10⁷CFU/mL, capacity of 1.68 × 10 of nano antibody phage display library established by M13K07 phage rescue¹⁶CFU/mL; BVDV-important functional protein is used as target antigen, phage display library obtained by amplification is subjected to three rounds of affinity screening, nano antibody specifically combined with E0/E2/NS5A/NS3 protein is screened from the library, and the reactogenicity and specificity of the nano antibody are detected by ELISASequencing the good nano antibody, analyzing the sequence, further obtaining the gene of the specific nano antibody, constructing a prokaryotic expression vector, carrying out prokaryotic expression, purification and identification on the prokaryotic expression vector to obtain the required nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3, and verifying the reactogenicity of the nano antibody and the BVDV important functional protein through Western blot.

Description

BVDV high-specificity nano antibody and preparation method and application thereof

Technical Field

The invention belongs to the field of biotechnology detection, and particularly relates to a preparation method and application of a nano antibody for resisting an important functional protein of BVDV (BVDV). The method comprises the steps of firstly expressing and purifying to obtain BVDV-NS5A protein, immunizing alpaca by using a BVDV inactivated vaccine, separating lymphocytes and serum, screening a specific nano antibody by using four functional proteins of BVDV-E0, E2, NS5A and NS3 through a constructed nano antibody phage display library, detecting the reactogenicity by ELISA, expressing and purifying the nano antibody with good specificity, and verifying the specificity of the nano antibody by using Wb.

Background

Bovine Viral Diarrhea/mucosal disease (BVD-MD) is an infectious disease caused by Bovine Viral Diarrhea Virus (BVDV). Cattle of various ages are susceptible to infection, with the highest susceptibility to calves. The infection sources mainly comprise secretion, excrement, blood, spleen and the like of sick livestock, and the transmission modes are two direct contact modes or indirect contact modes. The sick cattle have acute onset of disease, the body temperature is suddenly increased to 40-42 ℃, the appetite is exhausted, the digestive tract mucosa is seriously damaged, the early stage of the disease is usually watery diarrhea, the feces have blood and mucosa at the later stage, and the death rate can reach 90 percent. BVD-MD is therefore one of the diseases of great economic significance in the cattle industry and one of the diseases to be mainly prevented in import quarantine. The disease is reported to be distributed worldwide by literature, and at present, more than 20 provinces and cities such as Xinjiang, inner Mongolia, Ningxia, Shandong, Sichuan and the like are popular in China at different degrees.

BVDV belongs to a single-stranded positive-strand RNA virus of pestivirus of flaviviridae, is a circular enveloped membrane, and is a virus of the same genus as classical swine fever virus and sheep boundary virus. BVDV mainly has four structural proteins, capsid protein (C), envelope proteins (Erns, E1 and E2) and 7 non-structural proteins (P7, NS2/NS3, NS4A, NS4B, NS5A and NS 5B).

In 1993, Hamers et al found that an antibody naturally lacking a light chain, called a Nanobody (Nb), existed in camels. The molecular size of the nanobody is only 15kDa, a single-chain variable fragment (30kDa), a Fab fragment (60kDa) and a complete antibody (150 kDa). Although different subfamilies can be distinguished in dromedaries by the length of CDR2 and the position of another cysteine in CDR1 or frame-2, all nanobodies belong to the same sequence family, closely related to the family of group III human VH 3; the antibody only has a variable region of a heavy chain, has the advantages of good water solubility, high temperature resistance, easy expression in a prokaryotic system, strong tissue penetrability, high stability, even oral absorption without degradation, high affinity of antigen combination and the like, and is an ideal research tool and can be used for developing complex nano biotechnology; after the alpaca is inoculated in the test, the VHH gene can be cloned into a phagemid vector, and then antigen-specific VHH can be selected through phage display aiming at the antigen. Due to the characteristics of small volume and natural solubility and the unique capability of targeting alternative epitopes, the nanobody is very attractive in the aspects of tumor targeting, diagnosis, even in-vivo treatment and the like.

Disclosure of Invention

The invention aims to provide a method for preparing a specific nano antibody of an important functional protein for resisting BVDV;

the current antibody obtaining method comprises a monoclonal antibody, a ribosome display library, a phage display library and the like, wherein the most widely applied technology is the phage display library;

compared with the conventional antibody, the nano antibody screened by the technology has the characteristics of small relative molecular mass, strong stability, good solubility, good antigen binding property, easy expression, low immunogenicity and the like, and has wider application range than the conventional antibody.

Aiming at the defects in the prior art, the invention aims to provide a method for preparing an anti-BVDV nano antibody and application thereof.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

the nano antibody has the characteristics which are not possessed by the traditional antibody, and has the advantages of high water solubility, strong stability, strong antigen recognition capability, low immunogenicity, strong penetrability and the like; one current focus of research on nanobodies is on their unique epitopes that conventional antibodies do not possess.

In the structural protein of the BVDV, the E0 protein belongs to a protein with high conservation, has certain immunogenicity, and plays an important role in the pathogenic process of viruses of the same genus; the C end of the E2 protein contains a hydrophobic membrane anchoring area which is positioned on the surface of the envelope and stimulates the organism to generate immune response and virus neutralizing antibody; the NS3 protein is not an essential protein for viral replication and the specific function is not known; the NS5A protein is hydrophilic. Both serine and threonine residues of NS5A are phosphorylated in members of the flaviviridae family, which contributes to the propagation of the virus and plays an important role in the virus life cycle.

The genome of a BVDV NADL strain is taken as a template, a target NS5A gene is amplified, is cloned into a prokaryotic expression vector pET-30a, is respectively transformed into escherichia coli Top10 clone bacteria and BL21(DE3) expression bacteria, and is subjected to IPTG induced expression and Ni-IDA affinity chromatography column purification to obtain recombinant protein, and the reactogenicity is detected by WB.

The nano antibody is prepared by the following steps: immunizing alpaca by using a BVDV inactivated vaccine, separating lymphocytes and serum, constructing a nano antibody library, carrying out rescue and propagation of helper phage M13K07 on the nano antibody library, and obtaining a nano antibody phage display library. And then BVDV-important functional protein is used as a target antigen, a phage display library obtained by amplification is subjected to three rounds of 'adsorption-elution-amplification' affinity screening, monoclonal antibodies of nano antibodies obtained by random screening are subjected to ELISA to detect the reactogenicity and are sent to a biotechnology company for sequencing, sequences are analyzed, and the nano antibodies with good specificity are subjected to the purification of Nb-Y1, Nb-Y2 and Nb-Y3 proteins by constructing prokaryotic expression vectors pET30a-Nb-Y1, pET30a-Nb-Y2 and pET30a-Nb-Y3 expression nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 through a His tag nickel column. The binding capacity of purified nano-antibodies Nb-Y1, Nb-Y2 and Nb-Y3 and recombinant protein BVVD-E0/E2/NS5A/NS3 is verified by Western blotting.

The invention screens out the specific and affinity nano-antibody of the BVDV-important functional protein; provides a foundation for establishing a method for detecting BVDV antigen based on nano-antibody ELISA and provides a material basis for the research and development of nano-antibody biological preparations.

The invention belongs to the field of biotechnology detection, and discloses a preparation method and application of a BVDV-important functional protein nano antibody, wherein an NS5A protein is purified mainly by constructing a BVDV-NS5A expression vector; immunizing alpaca with BVDV inactivated vaccine, separating alpaca peripheral blood lymphocyte, obtaining VHH gene by nested PCR, constructing nanometer antibody phage display library with target gene insertion rate of 90.8% and library capacity of 1.02 × 10⁷cfu/mL, capacity of 1.68 x 10 of nano antibody phage display library established by M13 phage rescue¹⁶CFU/mL; BVDV-important functional protein is used as a target antigen, three rounds of affinity screening are carried out on a phage display library obtained by propagation, a nano antibody which is specifically combined with E0/E2/NS5A/NS3 protein is screened out from the library, the reactogenicity of the nano antibody is detected by ELISA, the nano antibody with good specificity is sequenced and analyzed for sequence, then the gene of the specific nano antibody is obtained, a prokaryotic expression vector is constructed, and prokaryotic expression, purification and identification are carried out on the prokaryotic expression vector, so that the required nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 are obtained.

Drawings

FIG. 1 is a prediction of the dominant epitope of BVDV NS5A, and the curve shows the trend line of the change of the amino acid epitope threshold.

FIG. 2 is a PCR amplification electrophoresis diagram of NS5A target gene fragment using BVDV NADL standard strain genome as template, wherein M is DNA Marker; lanes 1-4 show the NS5A gene.

FIG. 3 is a double-restriction electrophoresis chart of the recombinant expression plasmid pET-30a-NS5A for identifying Nde I and Hind III, wherein: lane M is DL 10000 Marker; lane 1 is plasmid control; lanes 2-5 show the double digestion results for Nde I and Hind III.

FIG. 4 is an SDS-PAGE analysis of the expression product of pET-30a-NS 5A. In the figure: lane M is SDS-PAGE Protein marker; lane 0 is 0h control; lane 1 induction at 15 ℃ for 16 h; lane 2 was induced at 37 ℃ for 16 h.

FIG. 5 shows affinity chromatography purification of the supernatant of NS5A protein. In the figure: lane M is SDS-PAGE Protein marker; lane 1 is the supernatant of the NS5A expression strain after disruption and centrifugation; lane 2 is the effluent of the supernatant after Ni-IDA; lanes 3-4 are elution fractions of 50mM imidazole; lanes 5-7 are elution fractions of 100mM imidazole; lanes 8-10 are the elution fractions of 500mM imidazole.

FIG. 6 shows the result of SDS-PAGE analysis of the purification of NS5A protein from inclusion bodies. In the figure: lane M is SDS-PAGE Protein marker; lane 1 is the supernatant of inclusion bodies of NS5A protein after solubilization and centrifugation; lane 2 is the effluent of the inclusion body supernatant of NS5A protein after passing through Ni-IDA; lanes 3-8 are elution fractions of 50mM imidazole; lanes 9-11 are 300mM imidazole fractions eluted.

Fig. 7 is a NS5A protein purification assay. In the figure: lane M is SDS-PAGE Marker; lane 1 is BSA (1.5 μ g); lane 2 shows NS5Aprotein (1.5. mu.g).

FIG. 8 is a Western Blot analysis of purified proteins. In the figure: lane M is Western Blot Marker; lane 1 is the NS5A protein at about 57 kDa.

FIG. 9 shows the detection of the ELISA titer of BVDV-specific antibodies in alpaca serum

FIG. 10 shows the PCR results for the target gene of VHH. In the figure: lane M2 is DL700 DNA Marker; lanes 6-11 are two rounds of PCR products.

FIG. 11 shows the restriction of pCANTAB5E + VHH. In the figure: lane M is DL5000 DNA Marker; lanes 1-5 are pCANTAB5E + VHH ligated plasmids.

FIG. 12 is a graph of the capacity characterization titer assay of the Nanobody library.

FIG. 13 is a colony PCR identification of phage antibody display library insertion rates. In the figure: lane M is DL700 DNA Marker; lanes 1-12 are two rounds of PCR products.

FIG. 14 is an SDS-PAGE analysis of the expression of Nb-Y1 protein in BL21(DE 3). In the figure: lane M is SDS-PAGE Protein Marker; lane 0 is control (no IPTG added); lane 1 was induced at 37 ℃ for 16 h; lane 2 is the supernatant of the whole cell after disruption; lane 3 is the pellet after the disruption of the whole strain.

FIG. 15 shows the result of protein purification from Nb-Y1 in inclusion bodies analyzed by SDS-PAGE. The figure is as follows: lane 1 is the supernatant after solubilization and centrifugation of inclusion bodies; lane 2 is the effluent of the supernatant incubated with Ni-IDA; lanes 3-4 are the elution fractions of 50mM Imidazole; lane 5 is the elution fraction of 300mM Imidazole.

FIG. 16 shows the measurement of the Nb-Y1 protein concentration. In the figure: lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y1, protein (2.00. mu.g); lane M1 is SDS-PAGE Marker M2 Western Blot Marker

FIG. 17 shows the Nb-Y1 protein WB assay. In the figure: lane M2 is Western Blot Marker; lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y1 protein (2.00. mu.g).

FIG. 18 is an SDS-PAGE analysis of the expression of Nb-Y2protein in BL21(DE 3). In the figure: lane M is SDS-PAGE Protein Marker; lane 0 is control (no IPTG added); lane 1 was induced at 37 ℃ for 16 h; lane 2 is the supernatant of the whole cell after disruption; lane 3 is the pellet after the disruption of the whole strain.

FIG. 19 shows the result of protein purification from Nb-Y2 in inclusion bodies analyzed by SDS-PAGE. The figure is as follows: lane 1 is the supernatant after solubilization and centrifugation of inclusion bodies; lane 2 is the effluent of the supernatant incubated with Ni-IDA; lanes 3-4 are the elution fractions of 50mM Imidazole; lane 5 is the elution fraction of 300mM Imidazole.

FIG. 20 shows the Nb-Y2protein concentration measurement. In the figure: lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y2protein (2.00. mu.g); lane M1 is an SDS-PAGE Marker M2 Western Blot Marker.

FIG. 21 shows the Nb-Y2protein WB assay. In the figure: lane M2 is Western Blot Marker; lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y2protein (2.00. mu.g).

FIG. 22 is an SDS-PAGE analysis of the expression of Nb-Y3 protein in BL21(DE 3). In the figure: lane M is SDS-PAGE Protein Marker; lane 0 is control (no IPTG added); lane 1 was induced at 37 ℃ for 16 h; lane 2 is the supernatant of the whole cell after disruption; lane 3 is the pellet after the disruption of the whole strain.

FIG. 23 shows the result of protein purification from Nb-Y3 in inclusion bodies analyzed by SDS-PAGE. The figure is as follows: lane 1 is the supernatant after solubilization and centrifugation of inclusion bodies; lane 2 is the effluent of the supernatant incubated with Ni-IDA; lanes 3-4 are the elution fractions of 50mM Imidazole; lane 5 is the elution fraction of 300mM Imidazole.

FIG. 24 shows the Nb-Y3 protein concentration measurement. In the figure: lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y3 protein (2.00. mu.g); lane M1 is an SDS-PAGE Marker M2 Western Blot Marker.

FIG. 25 shows the Nb-Y3 protein WB assay. In the figure: lane M2 is Western Blot Marker; lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y3 protein (2.00. mu.g).

FIG. 26 is a double-restriction electrophoresis chart of pET-30a-Nb-Y1 recombinant expression plasmid identification Nde I and Hind III, in which: lane M is DL 4500 Marker; lane 1 is plasmid control; lane 2 shows the double digestion results with Nde I and Hind III.

FIG. 27 is a double-restriction electrophoresis chart of recombinant expression plasmid pET-30a-Nb-Y2 for identification of Nde I and Hind III, in which: lane M is DL 4500 Marker; lane 1 is plasmid control; lane 2 shows the double digestion results with Nde I and Hind III.

FIG. 28 is a double-restriction electrophoresis chart of pET-30a-Nb-Y3 recombinant expression plasmid identification Nde I and Hind III, in which: lane M is DL 4500 Marker; lane 1 is plasmid control; lane 2 shows the double digestion results with Nde I and Hind III.

Fig. 29 is WB verification of the specificity of nanobody Nb-Y1. In the figure: lane M is 100kDa Western Blot Marker.

Fig. 30 is WB validation of the specificity of nanobody Nb-Y2. In the figure: lane M is 100kDa Western Blot Marker.

Fig. 31 is WB verification of the specificity of nanobody Nb-Y3. In the figure: lane M is 100kDa Western Blot Marker.

Detailed Description

1. Dominant epitope prediction

According to the BVDV NADL standard strain (NC-001461), an online software Predicting Anti-genetic Peptides server is used for Predicting the dominant epitope of the target protein NS5A of the NADL strain. (FIG. 1)

Gene amplification of NS5A protein and construction of prokaryotic expression vector

And (3) amplifying an NS5A target gene fragment by using a BVDV NADL standard strain genome as a template. (FIG. 2) the NS5A protein amino acid sequence was optimized using codon Optimization software MaxCodon (TM) Optimization Program (V13), NS5A gene was inserted into expression vector pET-30a via restriction enzyme sites Nde I/Hind III, and the accuracy of the final expression vector was confirmed by digestion and sequencing to obtain prokaryotic expression plasmid pET-30a-NS5A (FIG. 3).

TABLE 1 double digestion reaction System

Table1-5 Reaction system of double enzyme digestion

2. Expression vector transformation and inducible expression

The constructed plasmid containing the BVDV NS5A gene was transformed into BL21(DE3) competent cells, which were then spread evenly onto LB plates (containing 50. mu.g/mL kanamycin sulfate), and then placed upside down in a 37 ℃ incubator overnight. From the transformed plate, a single clone was selected, inoculated into 4mL of LB medium (containing 50. mu.g/mL kanamycin sulfate), cultured to OD600nm of 0.5-0.8, added to the test tube culture medium at a final concentration of 0.1mM IPTG, and then placed at 15 ℃ for induction expression at 37 ℃ for 0h, 16 h. (FIG. 4)

Affinity chromatography purification of the supernatant of the NS5A protein

After induction of the expression bacteria, the supernatant and the precipitate are separated by ultrasonic crushing and centrifugation. And (3) sucking the supernatant, adding the supernatant into a Ni-IDA column balanced by Buffer A, eluting proteins by Buffer B with different imidazole concentrations, collecting eluted components at various concentrations, and analyzing the protein expression form by SDS-PAGE. The supernatant after the crushing and centrifugation of the expression bacteria has no target protein expression. (FIG. 5)

Affinity chromatography purification of inclusion bodies of NS5A protein

After the inclusion bodies were washed with 50mM Tris (pH8.5),150mM NaCl containing 1% Triton X-100, 5mM EDTA, and 2mM DTT, the inclusion bodies were solubilized with 50mM Tris (pH8.5),150mM NaCl, and 8M Urea buffer while equilibrating the Ni-IDA column, and finally the target protein was eluted with equilibration buffer of different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE assay (FIG. 6), and the protein was diluted to 0.1mg/mL, and dialyzed to buffer [50mM Tris (pH8.5),150mM NaCl, 2mM EDTA, 4mM GSH, 0.4mM GSSG, and 0.4M Arginine ] at 4 ℃ to allow a small amount of protein to precipitate during renaturation, and after renaturation, the Nb 2-synthesized protein was finally dialyzed to storage solution 50mM Tris (pH8.5),150mM NaCl, and 10% Glycerol solution for about 6-8 h. After dialysis renaturation is finished, concentration is increased, the mixture is filtered by a 0.22um filter and then subpackaged, and the mixture is frozen to be at-80 ℃.

Purification and concentration determination of NS5A protein

After the protein was purified, the concentration of the protein was measured by the Bradford method, and the concentration of the protein was 0.331 mg/mL. SDS-PAGE analysis was performed using BSA as a control and SDS-PAGE analysis. As can be seen in FIGS. 1-7, the desired protein was of the expected size, indicating that the NS5A proteins were all purified successfully. (FIG. 7)

Western Blot analysis of NS5A protein

Western-blot analysis of the antigenicity of the NS5A protein after renaturation, obvious bands (57KDa) can be seen in the NS5A protein at expected sizes, and the result shows that the purified recombinant protein NS5A has good antigenicity (figure 8)

7. Immunization of alpaca

The alpaca was immunized with BVDV inactivated vaccine, whole blood was collected using anticoagulation tubes (EDTA) at 0d, 21d, 49d, and 70d, and peripheral blood lymphocytes and serum were isolated. The result of the measurement of the antibody titer shows that the alpaca serum antibody titer can reach 1: 51200. (FIG. 9)

Obtaining the VHH Gene

Extracting RNA of peripheral blood lymphocytes, performing reverse transcription to form cDNA, synthesizing nested primers through a reference document to perform nested PCR amplification on a target gene of a nano antibody, and identifying through agarose gel electrophoresis, wherein the target fragment is about 400 bp. (FIG. 10)

TABLE 2 primers for amplification of target genes

Table1 The primers for target gene PCR amplification

9. Construction of recombinant vector pCANTAB5E + VHH

The purified target fragment and the cryopreserved plasmid pCANTAB5E were recovered after agarose gel electrophoresis, and digested with restriction enzymes Sfi I and Not I. The cleaved VHH target fragment was ligated to pCANTAB5E in a 200. mu.L sterile EP tube. The prepared connection system is placed in a low-temperature connector and connected for 16 hours at the low temperature of 16 ℃. The recombinant vector pCANTAB5E + VHH is subjected to enzyme digestion identification. (FIG. 11)

10. Transformation of ligation products and library identification

The connected recombinant vector pCANTAB5E + VHH is transferred into TG1 competent cells, the transformed bacteria are spread on LB-AMP solid culture medium, and the strain is placed in an incubator at 37 ℃ for overnight culture. The next day, 1mL LB-AMP liquid medium was added to the plates, colonies were collected using a sterile cell scraper, 20. mu.L of the collected colonies were inoculated into 20mL of 2 XYT/AMP medium, the plates were placed in a shaker at 37 ℃ and 200rpm/min, and cultured until logarithmic growth phase, with an OD600nm value of 0.8-1.0. Add 100. mu. L M13K07 helper phage, mix gently, and let stand at 37 ℃ for 30 min. 2800g was centrifuged at room temperature for 10min, the supernatant was discarded, and the cells were resuspended in 200mL of 2 XYT/AMP medium and cultured in a shaker at 37 ℃ and 200rpm/min for 12 h. Placing in a centrifuge, centrifuging at 12000g for 15min at 4 deg.C, collecting supernatant, adding 0.1mL precooled PEG/NaCl, mixing by turning upside down, and standing on ice for 2 h. Placing the mixture into a centrifuge, centrifuging the mixture for 10min at 4 ℃ and 10000 g, discarding the supernatant, resuspending the phage precipitate by using 1mL of PBS (phosphate buffer solution), and incubating the precipitate overnight in a shaking table at 4 ℃ to fully dissolve the phage.

The stored rescue phage is taken and diluted by 2 XYT according to the gradient of 10-1, 10-2, 10-3 and 10-4 … … 10-16, TG1 in the logarithmic phase is added according to the proportion of 1:1, and the incubation is carried out for 5-10min at room temperature. The incubated mixture was aspirated by 100. mu.L, and the resulting solution was spread on LB-AMP solid medium by plate-spreading. The culture was carried out overnight at 37 ℃. The next day, the single clones on the plate were counted and the rescue phage titer was calculated. And randomly picking 96 monoclonal colonies from the transformed culture plate to perform PCR of bacterial liquid, identifying the positive rate of target fragment insertion of a PCR product, and calculating library capacity. Finally the obtained library capacity is 1.0210⁷VHH library in CFU/mL (FIG. 12). 90.8% of the clones contained the insertion of the gene of interest. (FIG. 13)

11. Specific nano antibody panning

And E0, E2, NS5A and NS3 are used as coating antigens to carry out specific nano antibody panning. Proteins E0, E2, NS5A, NS3 were diluted to 100 μ g/mL with coating solution, each protein coating 16 wells (PBS for negative control instead of antigen protein); discarding the coating solution, adding 200 μ L of 5% skimmed milk powder, placing into incubator, and sealing at 37 deg.C for 2 hr. Washing with PBS' T for 4 times, collecting rescued phage solution, diluting with 2% skimmed milk powder 10 times⁵Doubling, adding 100 mu L of the extract into each hole, and incubating for 2h at 37 ℃; phage samples were discarded, washed 5 times with PBS' T and PBS, and 100. mu.L of freshly prepared 0.1M triethylamine was added to each well, incubated at room temperature for 10min, and rapidly neutralized with an equal volume of 1M Tris-HCl, pH 7.4. Measuring the concentration of the eluted phage, collecting 400 μ L eluate, infecting 4mL TG1 in logarithmic growth phase, mixing, incubating at 37 deg.C for 30min, adding 16mL 2 XYT-AMP liquid culture medium, culturing in shaker at 37 deg.C and 200rpm/min to logarithmic growth phase, and D_600nmThe value is between 0.6 and 0.8; after 20. mu. L M13K07 helper phage was added to the medium at logarithmic phase, gently mixed, incubated at 37 ℃ for 1h, centrifuged at 2800g for 10min, and the supernatant was discarded. The mycelia were suspended in 2 XYT-AMP liquid medium, and cultured on a shaker at 37 ℃ and 220rpm/min for 14 hours. Then, the concentration and purification of the phage particles are carried out. Three rounds of panning were performed in triplicate.

12. Induced expression of recombinant nano antibody and crude extract acquisition

Respectively taking phages eluted after the third round of screening of E0, E2, NS5A and NS3, respectively adding 100 mu L of the phages into TG1 in an isometric logarithmic growth phase by using a sample with the dilution of 2 XYT of 108, uniformly mixing, and standing for 15min at 37 ℃; coating infected TG1 on LB-AMP solid culture medium, placing in incubator, and culturing at 37 deg.C for 8 hr; randomly picking 96 monoclonal colonies of each protein, inoculating the colonies into 200 mu L LB-AMP liquid culture medium, and culturing for 10 h; uniformly mixing the bacterial liquid and the TB culture medium in a ratio of 1:100, correspondingly numbering, putting into a shaking table at 37 ℃, and culturing at 200rpm/min until logarithmic phase; IPTG was added to a final concentration of 0.1mM and induction was carried out overnight; centrifuging at 4 deg.C and 3200 g for 10min, discarding supernatant, and freezing thallus precipitate in refrigerator at-20 deg.C for 30 min. The mixture was allowed to stand at room temperature until it was melted, 500. mu.L of sterile PBS was added to each tube, and the mixture was incubated on a shaker at 37 ℃ and 225rpm/min for 30 min. Centrifuging at 4 deg.C for 15min at 3500 g, and collecting supernatant as crude extract of soluble recombinant nanometer antibody.

13. ELISA detection of soluble recombinant nanobody

The four proteins E0, E2, NS5A and NS3 were diluted to 10. mu.g/mL with the coating solution, and 100. mu.L of each protein-coated 2-plate was added to each well of a 96-well microplate and coated overnight at 4 ℃. Blank wells were coated with no protein using PBS as a no antigen control. Discarding the coating solution, patting to dry, adding 200 μ L5% skimmed milk powder into each well, placing into 37 deg.C incubator, and sealing for 2 hr. Washing with PBS' T for 3 times, diluting the crude extract with 5% skimmed milk powder 1:1, adding 100 μ L into each well, placing in 37 deg.C incubator, and incubating for 45 min. Washing with PBS' T for 3 times, adding enzyme-labeled E-tag labeled with HRP and mouse-resistant M13 labeled with HRP diluted at a ratio of 1: 2000 times to different plates, placing in a 37 deg.C incubator, and incubating for 45 min. Washing with PBS' T for 3 times, adding ELISA developing solution, placing in 37 deg.C incubator, and developing for 15 min. Add 50. mu.L of ELISA stop solution to each well and measure D using microplate reader_450nmThe value of the OD value of the assay was more than 3 times greater than that of the PBS control, and the assay was positive.

14. Specific nanobody sequencing analysis

Two groups of ELISA are selected to be positive, and positive clone preserving fluid with higher OD value is transferred to a fresh 20mL2 XYT-Amp liquid culture medium for overnight culture. The next day, bacterial liquid PCR was performed using primer VHH-F, VHH-R, the PCR product was sent to Huada Gene (Beijing) GmbH for sequencing, and after Blast comparison, DNAMAN software was used to analyze and compare amino acid sequence homology. The result obtained 7 nanobodies of different amino acid sequences.

15. Synthesis of nano-antibody Nb-Y1, Nb-Y2 and Nb-Y3 genes and construction of prokaryotic expression vector

The provided Nb-Y1, Nb-Y2 and Nb-Y3 protein amino acid sequences are optimized by using codon Optimization software MaxCodon TM Optimization Program (V13), Nb-Y1, Nb-Y2 and Nb-Y3 genes are inserted into an expression vector pET30a by using whole-gene synthesis and restriction enzyme cutting sites NdeI and HindIII to obtain prokaryotic expression plasmids pET30a-Nb-Y1, pET30a-Nb-Y2 and pET30a-Nb-Y3, the accuracy of the final expression vector is confirmed by an enzyme cutting method and sequencing, and finally the prokaryotic expression plasmids are respectively transferred into a Top10 cloning strain and a BL21(DE3) expression strain. (pET30a-Nb-Y1 FIG. 26) (pET30a-Nb-Y2 FIG. 27) (pET30a-Nb-Y3 FIG. 28)

16. Expression vector transformation and inducible expression

The constructed plasmids containing Nb-Y1, Nb-Y2, and Nb-Y3 genes were transformed into BL21(DE3) competent cells, and then uniformly spread on LB plates (containing 50. mu.g/mL kanamycin sulfate), followed by inverting in a 37 ℃ incubator overnight. Single colonies were picked from the transformed plates, inoculated into 4mL of LB medium (containing 50. mu.g/mL kanamycin sulfate), and allowed to grow to D_600nm0.5 to 0.8, IPTG was added to the test tube culture solution at a final concentration of 0.5mM, followed by induction of expression at 37 ℃. Expanding culture and growing to D_600nmWhen the concentration was 0.8, the cells were induced at 37 ℃ for 16 hours with the addition of 0.5mM IPTG.

17. The expression results of the nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 SDS-PAGE analysis and identification

Centrifuging induced culture solution at 12000rpm for 5min, removing supernatant, adding PBS solution to resuspend and precipitate, adding SDS-PAGE sample buffer, heating the sample at 100 deg.C for 10min, centrifuging, and collecting supernatant for electrophoresis. The whole strain was sonicated with 20mM Tris (pH8.0), 300mM NaCl, 20mM Imidazole containing 1% Triton X-100, 1mM DTT, 1mM PMSF, and the supernatant and pellet were analyzed by SDS-PAGE. (E2-1 FIG. 14) (E2-3 FIG. 18) (NS2-3 FIG. 22)

18. The inclusion bodies purify the proteins of nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 through affinity chromatography

After the inclusion bodies were washed with 20mM Tris (pH8.0), 300mM NaCl containing 1% Triton X-100, 2mM EDTA, and 5mM DTT, the inclusion bodies were solubilized with 20mM Tris (pH8.0), 300mM NaCl,8M Urea, and 20mM Imidazole buffer while equilibrating the Ni-IDA column, and finally the target protein was eluted with equilibration buffer containing different concentrations of Imidazole, and each eluted fraction was collected for SDS-PAGE analysis. (Nb-Y1 FIG. 15) (Nb-Y2 FIG. 19) (Nb-Y3 FIG. 23)

Purifying and analyzing by Ni-IDA affinity chromatography, collecting Lane 3-5 with relatively high purity, adding into treated dialysis bag, dialyzing into buffer solution [1 XPBS (pH7.4), 4mM GSH, 0.4mM GSSG, 0.4M L-Arginine, 1M Urea, 5% Glycerol ] at 4 deg.C for renaturation, and dialyzing Nb-Y1, Nb-Y2, Nb-Y3 proteins into stock solution 1 XPBS (pH7.4), 5% Glycerol solution for about 6-8 h. After the renaturation by dialysis, the supernatant was filtered through a 0.22 μm filter and dispensed, and was frozen to-80 ℃.

19. Protein concentration determination of nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3

Protein concentration was determined using the Bradford protein concentration assay kit. (Nb-Y1 FIG. 16) (Nb-Y2 FIG. 20(Nb-Y3 FIG. 24)

20. Detection of nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 protein WB

The WB experimental operation flow refers to Yao Jun treatise of protein electrophoresis Experimental technology. (Nb-Y1 FIG. 17) (Nb-Y2 FIG. 21) (Nb-Y3 FIG. 25)

21. Nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 protein-conjugated horseradish peroxidase (HRP)

WB verification of the specificity of the Nanobodies Nb-Y1, Nb-Y2, Nb-Y3

80. mu.L of BVVD-E0/E2/NS5A/NS3 protein was mixed with 20. mu.L of the protein sample solution, and the mixture was boiled for 10min for SDS-PAGE. Cutting off the position of the target protein, putting the cut target protein into a membrane transfer solution for later use, respectively putting the filter paper, the glue and the PVDF membrane on a semi-wet transfer membrane instrument (3 layers of filter paper, glue, PVDF membrane and 3 layers of filter paper in sequence), and transferring for about 50 min. Then the cells are transferred to a plate and blocked by 5 percent of skimmed milk powder for 2h at room temperature, and then an incubation box is used for incubating and combining horseradish peroxidase (HRP) nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 proteins, and the cells are diluted to be 1: 5000, and incubating for 1h at room temperature by using a shaking table. After TBST cleaning for 3 times, the chemiluminescent solution was uniformly dropped on the PVDF membrane surface and placed in a chemical exposure instrument for exposure. The results show that Nb-Y1 and Nb-Y2 can be specifically combined with E0, and Nb-Y3 can be specifically combined with E2. (FIG. 29), (FIG. 30), and (FIG. 31).

<110> river university

<120> BVDV high-specificity nano antibody and preparation method and application thereof

<141>

<160> 3

<210> 1

<211> 114

<212> PRT

<213> Nanobody gene

<220>

<221> misc_feature

<223> Nb-Y1 nano antibody gene amino acid sequence

<400> 1

HVQLQESGGGLVQPG

GSLRLSCIVSGRETV

AIGWFRQAPGKEREE

ISCIRRSGSTTNYLD

SVKGRFTISRDNAKN

TVYLQMNDLKAEDTA

RYYCAADKTCLSSWT

QAFWGQGTQVTVSS

<210> 2

<211> 122

<212> PRT

<213> Nanobody gene

<220>

<221> misc_feature

<223> Nb-Y2 nano antibody gene amino acid sequence

<400> 2

HVQLQESGGGLVQPG

GSLRLSCTASEFTLD

YYAIGWFRQAPGKER

EGVSCISSSGDTIKY

ADSVKGRFTISRDNA

KNTVYLQMNSLKPED

TAVYYCAADRADPWN

VQHMCIPRGDYWGQG

TQVTVSS

<210> 3

<211> 120

<212> PRT

<213> Nanobody gene

<220>

<221> misc_feature

<223> Nb-Y3 nano antibody gene amino acid sequence

<400> 3

HVQLQESGGGLVQPG

GSLRLSCAPSGLDYT

VIGWFRQAPGKEREG

VACIFRSGGDTAYAD

SVQGRFTASRDDTMN

TAYLQMNSLTPEDTA

VYYCAAKKYGSCLPT

TIWSSHYPYWGQGTQ

VTVSS

Claims

1. A high-specificity nano antibody for resisting BVDV is characterized in that: the amino acid sequence is one of <210>1, <210>2 or <210> 3.

2. A preparation method of a high-specificity nano antibody for resisting BVDV is characterized by comprising the following steps: at least comprises the following steps:

immune of alpaca and obtaining VHH gene

Immunizing alpaca with BVDV inactivated vaccine, collecting whole blood at 0d, 21d, 49d, and 70d with anticoagulation tube (EDTA), and separating peripheral blood lymphocyte and serum; extracting RNA of peripheral blood lymphocytes, performing reverse transcription to form cDNA, synthesizing nested primers through a reference document to perform nested PCR amplification on a target gene of a nano antibody, and identifying through agarose gel electrophoresis, wherein the target fragment is 400 bp;

② construction of recombinant vector pCANTAB5E + VHH

Taking agarose gel electrophoresis, recovering a purified target fragment and a frozen plasmid pCANTAB5E, and carrying out enzyme digestion by using restriction enzymes Sfi I and Not I; connecting the cut VHH target fragment with pCANTAB5E in a 200 mu L sterile EP tube to prepare a recombinant vector pCANTAB5E + VHH;

(iii) transformation of ligation products and library identification

Transferring the connected recombinant vector pCANTAB5E + VHH into TG1 competent cells, coating the transformed bacteria on an LB-AMP solid culture medium, and putting the LB-AMP solid culture medium into an incubator at 37 ℃ for overnight culture; from transformed platesRandomly selecting 96 monoclonal colonies as bacteria liquid PCR, identifying the positive rate of target fragment insertion of PCR products, and calculating library capacity to obtain the final library capacity of 1.02 × 10⁷A VHH library of CFU/mL; adding 1mL LB-AMP liquid medium to the plate, collecting colonies with a sterile cell scraper, inoculating 20. mu.L of the collected colonies into 20mL2 XYT/AMP medium, placing the mixture in a shaker at 37 ℃ and 200rpm/min, culturing to logarithmic growth phase, D_600nmThe value is between 0.8 and 1.0; adding 100 mu L M13K07 helper phage, mixing gently, standing at 37 deg.C for 30 min; 2800g, centrifugating at room temperature for 10min, discarding the supernatant, resuspending the mycelia in 200mL 2 XYT/AMP medium, culturing in a shaker at 37 deg.C and 200rpm/min for 12 h; placing in a centrifuge, centrifuging at 4 deg.C and 12000g for 15min, collecting supernatant, adding 0.1mL precooled PEG/NaCl, turning upside down, mixing, and standing on ice for 2 h; placing the mixture into a centrifuge, centrifuging the mixture for 10min at 4 ℃ and 10000 g, discarding supernatant, resuspending phage precipitate by using 1mL PBS, and incubating the precipitate overnight in a shaking table at 4 ℃ to fully dissolve the phage; taking the stored rescue phage with 2 XYT according to 10^-1、10^-2、10^-3、10^-4……10^-16Gradient dilution, adding TG1 in logarithmic phase at a ratio of 1:1, and incubating at room temperature for 5-10 min; sucking 100 mu L of the incubated mixed solution, and uniformly coating the mixed solution on an LB-AMP solid culture medium by using a plate coating method; culturing at 37 deg.C overnight; counting the monoclonals on the plate the next day, and calculating the titer of the rescue phage; the capacity of the phage display library of the nano antibody established by the rescue of M13K07 phage is 1.68 multiplied by 10¹⁶CFU/mL；

Specific nano antibody panning

Performing specific nano-antibody panning by taking four proteins of E0, E2, NS5A and NS3 as coating antigens, diluting the proteins of E0, E2, NS5A and NS3 to 100 mu g/mL by using coating solution, and coating 16 holes on each protein (PBS is used as a negative control to replace the antigen protein); discarding the coating solution, adding 200 μ L of 5% skimmed milk powder, placing into incubator, sealing at 37 deg.C for 2 hr, washing with PBS' T for 4 times, collecting the rescued phage solution, diluting with 2% skimmed milk powder 10 times⁵Doubling, adding 100 mu L of the extract into each hole, and incubating for 2h at 37 ℃; phage samples were discarded, washed 5 times with PBS' T and PBS, 100. mu.L of freshly prepared 0.1M triethylamine was added to each well, and the chamberIncubating for 10min, rapidly neutralizing with 1M Tris-HCl with equal volume and pH of 7.4, measuring the concentration of eluted phage, infecting TG1 with 4mL of eluate in logarithmic growth phase, mixing, incubating at 37 deg.C for 30min, adding 16mL of 2 XYT-AMP liquid culture medium, culturing in shaker at 37 deg.C and 200rpm/min to logarithmic growth phase, and D_600nmThe value is between 0.6 and 0.8; adding 20 mu L M13K07 helper phage into the culture solution reaching the logarithmic phase, gently mixing, incubating at 37 ℃ for 1h, centrifuging at 2800g for 10min, and discarding the supernatant; suspending thallus with 2 XYT-AMP liquid culture medium, culturing in shaker at 37 deg.C and 220rpm/min for 14 hr, and concentrating and purifying phage particles; repeating the steps for three times to finish three rounds of elutriation;

fifthly, induction expression of the recombinant nano antibody and crude extract acquisition

The phages eluted after the third round of screening of E0, E2, NS5A and NS3 were diluted to 10 with 2 XYT⁸Respectively taking 100 mu L of the sample, adding TG1 in an isometric logarithmic phase, uniformly mixing, and standing for 15min at 37 ℃; coating infected TG1 on LB-AMP solid culture medium, placing in incubator, and culturing at 37 deg.C for 8 hr; randomly picking 96 monoclonal colonies of each protein, inoculating the colonies into 200 mu L LB-AMP liquid culture medium, and culturing for 10 h; uniformly mixing the bacterial liquid and the TB culture medium in a ratio of 1:100, correspondingly numbering, putting into a shaking table at 37 ℃, and culturing at 200rpm/min until logarithmic phase; IPTG was added to a final concentration of 0.1mM and induction was carried out overnight; centrifuging at 4 deg.C for 10min at 3200 g, removing supernatant, freezing thallus precipitate in refrigerator at-20 deg.C for 30min, thawing at room temperature, adding 500 μ L sterile PBS into each tube, and culturing in shaker at 37 deg.C and 225rpm/min for 30 min; centrifuging at 4 deg.C for 15min at 3500 g, and collecting supernatant as crude extract of soluble recombinant nanometer antibody;

sixth, ELISA detection of soluble recombinant nano antibody and sequencing analysis of specific nano antibody

Diluting the four proteins of E0, E2, NS5A and NS3 to 10 mug/mL by using coating solution, taking a 96-well enzyme label plate, adding 100 mug L (each protein is coated by 2 plates) into each well, and coating overnight at 4 ℃; blank wells used PBS as no antigen control without coating protein; discarding the coating solution, patting to dry, adding 200 μ L5% skimmed milk powder into each hole, placing into 37 deg.C incubator, and sealing for 2 hr; by usingWashing with PBS' T for 3 times, diluting the crude extract with 5% skimmed milk powder 1:1, adding 100 μ L into each well, placing in 37 deg.C incubator, and incubating for 45 min; washing with PBS' T for 3 times, adding enzyme-labeled E-tag labeled with HRP and HRP mouse antibody M13 diluted by 1: 2000 times into different plates, placing into 37 deg.C incubator, and incubating for 45 min; washing with PBS' T for 3 times, adding ELISA developing solution, placing in 37 deg.C incubator, and developing for 15 min; add 50. mu.L of ELISA stop solution to each well and measure OD using microplate reader_450nmValue, based on OD value, more than 3 times greater than PBS control was analyzed as positive;

selecting two groups of ELISA positive clone preserving fluid with higher OD value to transfer the positive clone preserving fluid into a fresh 20mL2 XYT-Amp liquid culture medium for overnight culture; the next day, the primers VHH-F, VHH-R are used for carrying out bacteria liquid PCR, PCR products are sent to Huada gene (Beijing) GmbH for sequencing, and DNAMAN software is used for analyzing and comparing amino acid sequence homology after Blast comparison; the result obtains 7 nanometer antibodies with different amino acid sequences;

seventhly, synthesizing nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 genes and constructing prokaryotic expression vector

Optimizing the provided Nb-Y1, Nb-Y2 and Nb-Y3 protein amino acid sequences by adopting codon Optimization software MaxCodon TM Optimization Program (V13), inserting Nb-Y1, Nb-Y2 and Nb-Y3 genes into an expression vector pET30a by adopting whole-gene synthesis and restriction enzyme cutting sites NdeI and HindIII to obtain prokaryotic expression plasmids pET30a-Nb-Y1, pET30a-Nb-Y2 and pET30a-Nb-Y3, confirming the accuracy of the final expression vector by adopting an enzyme cutting method and sequencing, and finally respectively transferring the prokaryotic expression plasmids into a Top10 cloning strain and a BL21(DE3) expression strain;

(viii) expression vector transformation and inducible expression

The constructed plasmids containing Nb-Y1, Nb-Y2 and Nb-Y3 genes are transformed into BL21(DE3) competent cells, then are evenly coated on LB plates (containing 50 mu g/mL kanamycin sulfate), and then are placed in an incubator at 37 ℃ for overnight in an inverted mode; single colonies were picked from the transformed plates, inoculated into 4mL of LB medium (containing 50. mu.g/mL kanamycin sulfate), and allowed to grow to D_600nm0.5-0.8, adding final concentration of 0 to the test tube culture solution5mM IPTG followed by induction of expression at 37 ℃; expanding culture and growing to D_600nmWhen the concentration is 0.8, adding 0.5mM IPTG, inducing at 37 ℃ for 16h, and collecting thalli;

ninthly, the expression results of the nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 SDS-PAGE analysis and identification

Centrifuging induced culture solution at 12000rpm for 5min, removing supernatant, adding PBS solution, resuspending, adding SDS-PAGE sample buffer, heating at 100 deg.C for 10min, centrifuging, and collecting supernatant for electrophoresis; carrying out ultrasonic lysis on the whole bacteria by using 20mM Tris (pH8.0), 300mM NaCl, 20mM Imidazole containing 1% Triton X-100, 1mM DTT and 1mM PMSF, and taking supernatant and sediment for carrying out SDS-PAGE analysis and detection; the inclusion bodies purify the proteins of the nano antibodies Nb-Y1, Nb-Y2 and Nb-Y3 through affinity chromatography.