CN115925895A - anti-BVDV E0 nano antibody and preparation method and application thereof - Google Patents
anti-BVDV E0 nano antibody and preparation method and application thereof Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Abstract
The invention belongs to the field of biotechnology detection, and discloses an anti-BVDV-important functional protein nano antibody, 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 × 10 7 CFU/mL, capacity of 1.68 x 10 of nano antibody phage display library established by M13K07 phage rescue 16 CFU/mL; BVDV-important functional protein is used as target antigen, phage display library obtained by propagation is subjected to three rounds of affinity screening, and the library is screenedSelecting a nano antibody specifically combined with the E0/E2/NS5A/NS3 protein, detecting the reactionogenicity of the nano antibody through ELISA, sequencing the nano antibody with good specificity, analyzing the sequence, further obtaining the gene of the specific nano antibody, constructing a prokaryotic expression vector, performing 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 reactionogenicity of the nano antibody and the BVDV important functional protein through Westernblot.
Description
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 BVDV important functional protein.
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 injured, 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 worldwide distributed according to literature reports, and more than 20 provinces and municipalities such as Sinkiang, inner Mongolia, ningxia, shandong and Sichuan 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 nanobody has a molecular size of only 15kDa, a single chain variable fragment (30 kDa), a Fab fragment (60 kDa), and a full antibody (150 kDa). Although different subfamilies can be distinguished in the dromedary 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 methods comprise monoclonal antibodies, ribosome display libraries, phage display libraries and the like, wherein the most widely applied technology is the phage display libraries;
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.
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 the same virus; the C end of the E2 protein contains a hydrophobic membrane anchoring area which is positioned on the surface of the cyst membrane and stimulates an organism to generate immune response and virus neutralizing antibodies; 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 an escherichia coli Top10 clone bacterium and a BL21 (DE 3) expression bacterium, is subjected to IPTG induced expression and Ni-IDA affinity chromatography column purification to obtain a recombinant protein, and the reactogenicity is detected through 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 auxiliary phage M13K07 rescue and propagation 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, a nano antibody monoclonal obtained by random screening is subjected to ELISA to detect the reactionogenicity and is sent to a biotechnology company for sequencing, the sequence is analyzed, and the nano antibodies with good specificity are subjected to the construction of prokaryotic expression vectors pET30a-Nb-Y1, pET30a-Nb-Y2 and pET30a-Nb-Y3 to express nano antibodies Nb-Y1, nb-Y2 and Nb-Y3, so that the proteins Nb-Y1, nb-Y2 and Nb-Y3 are purified by a His label nickel column. The binding capacity of purified nano-antibodies Nb-Y1, nb-Y2 and Nb-Y3 and recombinant protein BVVD-E0/E2/NS5A/NS3 protein 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 × 10 7 cfu/mL, capacity of 1.68 x 10 of nano antibody phage display library established by M13 phage rescue 16 CFU/mL; the BVDV-important functional protein is taken as a target antigen to carry out the amplification on the obtained phage display libraryThree rounds of affinity screening are carried out, a nano antibody specifically combined with the E0/E2/NS5A/NS3 protein is screened out from the library, the reactogenicity of the nano antibody is detected through 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 are NS5A genes.
FIG. 3 is a double-restriction enzyme electrophoresis diagram of pET-30a-NS5A recombinant expression plasmid identification Nde I and Hind III, wherein: lane M is DL10000 Marker; lane 1 is a plasmid control; lanes 2-5 show the double digestion results for Nde I and Hind III.
FIG. 4 is an SDS-PAGE analysis of pET-30a-NS5A expression products. In the figure: lane M is SDS-PAGE Protein marker; lane 0 is 0h control; lane 1 induction at 15 ℃ for 16h; lane 2 was induced at 37 ℃ for 16h.
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 expressing 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 NS5A protein inclusion body supernatant after Ni-IDA; lanes 3-8 are elution fractions of 50mM imidazole; lanes 9-11 are 300mM imidazole fractions eluted.
Fig. 7 is an 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 57kDa.
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 enzyme cutting identification 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 a 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 16h; 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 SDS-PAGE analysis for purification of Nb-Y1 protein from inclusion bodies. 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 eluted fractions of 50mM Imidazole; lane 5 is the elution fraction of 300mM Imidazole.
FIG. 16 shows Nb-Y1 protein concentration measurement. 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.
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 16h; 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 SDS-PAGE analysis for purification of Nb-Y2protein from inclusion bodies. 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 is Nb-Y2protein concentration determination. In the figure: lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y2protein (2.00. Mu.g); lane M1 is SDS-PAGE 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 16h; lane 2 is the supernatant after the whole strain is disrupted; lane 3 is the pellet after the whole strain is disrupted.
FIG. 23 shows the result of SDS-PAGE analysis for purification of Nb-Y3 protein from inclusion bodies. 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 is Nb-Y3 protein concentration determination. In the figure: lane 1 is BSA (1.50 μ g); lane 2 is Nb-Y3 protein (2.00. Mu.g); lane M1 is SDS-PAGE 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 DL4500 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 pET-30a-Nb-Y2 recombinant expression plasmid identification Nde I and Hind III, in which: lane M is DL4500 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 DL4500 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 verification 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 the 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 through 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 (DE 3) competent cells, which were then spread evenly onto LB plates (containing 50. Mu.g/mL kanamycin sulfate), followed by inverting 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, 16h. (FIG. 4)
Affinity chromatography purification of supernatant of 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 NS5A protein inclusion bodies
After the inclusion bodies were washed with 50mM Tris (pH 8.5), 150mM NaCl containing 1% Triton X-100, 5mM EDTA, 2mM DTT, the inclusion bodies were solubilized with 50mM Tris (pH 8.5), 150mM NaCl,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, dialyzed to buffer [50mM Tris (pH 8.5), 150mM NaCl,2mM EDTA,4mM GSH,0.4mM GSSG,0.4M Arginine ] at 4 ℃ with a small amount of protein precipitated during renaturation, and after renaturation the Nb2 synthetic protein was finally dialyzed to a stock solution of 50mM Tris (pH 8.5), 150mM NaCl,10 mM Glycerol for about 6-8h. 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 0.331mg/mL. SDS-PAGE analysis was performed using BSA as a control and SDS-PAGE analysis. As can be seen in FIGS. 1-7, the protein of interest was of the expected size, indicating that both NS5A proteins were successfully purified. (FIG. 7)
Western Blot analysis of NS5A protein
Western-blot analysis is carried out after NS5A renaturation, antigenicity of the NS5A protein is analyzed, obvious bands (57 KDa) can be seen at expected sizes of the NS5A protein, 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 antibody titer is measured, and the result shows that the alpaca serum antibody titer can reach 1. (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 target genes of the nano antibodies, and identifying through agarose gel electrophoresis, wherein target fragments are about 400 bp. (FIG. 10)
TABLE 2 primers for amplification of genes of interest
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 ℃. And carrying out enzyme digestion identification on the recombinant vector pCANTAB5E + VHH. (FIG. 11)
10. Transformation of ligation products and library identification
The connected recombinant vector pCANTAB5E + VHH is transferred into TG1 competent cells, and the transformed bacteria are spread on LB-AMP solid culture medium and 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 with 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 to logarithmic 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 30min.2800g was centrifuged at room temperature for 10min, the supernatant was discarded, and the cells were resuspended in 200mL 2 XYT/AMP medium, and cultured in a shaker at 37 ℃ and 200rpm/min for 12h. 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 2h. Placing the mixture into a centrifuge, centrifuging the mixture for 10min at 4 ℃ and 10000g, 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.
Taking the stored rescue phage for 2 XYT according to 10-1, 10-2, 10-3, 10-4 \8230 \8230and10-16 gradient dilution, adding TG1 in logarithmic phase according to the proportion of 1. 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. The final storage capacity is 1.02X 10 7 VHH 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 (3) carrying out specific nano antibody panning by taking four proteins of E0, E2, NS5A and NS3 as coating antigens. 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 times 5 Doubling, 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 gently, incubating at 37 deg.C for 30min, adding 1695l 2 XYT-AMP liquid culture medium, culturing at 37 deg.C and 200rpm/min in a shaker to logarithmic growth phase, and collecting D 600nm The value is between 0.6 and 0.8; mu.L of M13K07 helper phage was added to the medium at logarithmic phase, gently mixed, incubated at 37 ℃ for 10min, and centrifuged at 2800g for 10min, and the supernatant was discarded. Suspending thallus with 2 XYT-AMP liquid medium, placing in shaker at 37 deg.C and 220rpCulturing for 14h at m/min. 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 obtained by elution after the third round of screening by E0, E2, NS5A and NS3, respectively taking 100 mu L of a sample with the dilution of 2 XYT of 108, adding TG1 in an isometric logarithmic phase, uniformly mixing, and standing for 15min at 37 ℃; coating the infected TG1 on an LB-AMP solid culture medium, putting the LB-AMP solid culture medium into an incubator, and culturing for 8h at 37 ℃; randomly picking 96 monoclonal colonies of each protein, inoculating the colonies into 200 mu L LB-AMP liquid culture medium, and culturing for 10h; uniformly mixing the bacterial liquid and a TB culture medium according to the proportion of 1; IPTG was added to a final concentration of 0.1mM and induced overnight; centrifuging at 4 deg.C and 3200g for 10min, discarding supernatant, and freezing thallus precipitate in refrigerator at-20 deg.C for 30min. 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 at 225rpm/min for 30min on a shaker at 37 ℃. Centrifuging at 4 deg.C for 15min at 3500g, 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 was added to a 96-well microplate (each protein coated on 2 plates) 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, taking the crude soluble recombinant nano-antibody extract, diluting with 5% skimmed milk powder 1, adding 100 μ L per well, placing in a 37 ℃ incubator, and incubating for 45min. Wash 3 times with PBS' T, same protein, different plates, add respectively 1. Washing with PBS' T for 3 times, adding ELISA developing solution, placing in 37 deg.C incubator, and developing for 15min. Add 50. Mu.L of ELISA stop solution to each well and measure D using microplate reader 450nm The value of OD analyzed was more than 3 times greater than that of PBS control, and the test was positive.
14. Specific nanobody sequencing analysis
Two groups of ELISA positive clone preservation solution with higher OD value are selected and transferred into a fresh 20mL 2 XYT-Amp liquid culture medium for overnight culture. The next day, bacterial liquid PCR was performed using primers VHH-F, VHH-R, and the PCR product was sent to Huada Gene (Beijing) GmbH for sequencing, and after Blast comparison, amino acid sequence homology was analyzed and compared using DNAMAN software. 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 adopting codon Optimization software MaxCodon TM Optimization Program (V13), the Nb-Y1, nb-Y2 and Nb-Y3 genes are inserted into an expression vector pET30a by adopting whole-gene synthesis and restriction enzyme 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 clone strain and a BL21 (DE 3) expression strain. (pET 30a-Nb-Y1 FIG. 26) (pET 30a-Nb-Y2 FIG. 27) (pET 30a-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 (DE 3) competent cells, and then spread evenly onto LB plates (containing 50. Mu.g/mL kanamycin sulfate), followed by inverting in a 37 ℃ incubator overnight. A single clone was selected from the transformed plate, inoculated into 4mL of LB medium (containing 50. Mu.g/mL of kanamycin sulfate), and cultured to D 600nm 0.5 to 0.8, IPTG was added to the test tube culture medium at a final concentration of 0.5mM, followed by induction of expression at 37 ℃. Expanding and culturing to D 600nm If the concentration is not more than 0.8, the cells are induced at 37 ℃ for 16 hours by adding IPTG 0.5mM in final concentration, and then the cells are collected.
17. SDS-PAGE analysis identification expression result of nano antibodies Nb-Y1, nb-Y2 and Nb-Y3
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 in 20mM Tris (pH 8.0), 300mM NaCl,2mM Imidazole 1% Triton X-100,1mM DTT,1mM PMSF, and the supernatant and pellet were subjected to SDS-PAGE analysis. (E2-1 FIG. 14) (E2-3 FIG. 18) (NS 2-3 FIG. 22)
18. The inclusion body purifies the proteins of nano antibodies Nb-Y1, nb-Y2 and Nb-Y3 through affinity chromatography
After inclusion bodies were washed with 20mM Tris (pH 8.0), 300mM NaCl containing 1% Triton X-100,2mM EDTA, and 5mM DTT, the inclusion bodies were dissolved in 20mM Tris (pH 8.0), 300mM NaCl,8M Urea, and 2mM Imidazole buffer while equilibrating the Ni-IDA column, and finally the target protein was eluted with equilibration buffers of different Imidazole concentrations, and each eluted fraction was collected for SDS-PAGE analysis. (Nb-Y1 FIG. 15) (Nb-Y2 FIG. 19) (Nb-Y3 FIG. 23)
Relatively high purity Lane 3-5 was collected by Ni-IDA affinity chromatography, added to the treated dialysis bag, and dialyzed into buffer solution [1 XPBS (pH 7.4), 4mM GSH,0.4mM GSSG,0.4M L-Arginine,1M Urea,5% Glycerol ] at 4 ℃ for renaturation, after which the Nb-Y1, nb-Y2, nb-Y3 proteins were finally dialyzed into stock solution 1 XPBS (pH 7.4), 5 Glycerol solution for about 6-8h. After the renaturation by dialysis, the supernatant was filtered through a 0.22 μm filter and dispensed, and was frozen to-80 ℃.
19. Determination of protein concentration 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-antibody 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 combined Horse Radish 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, heated to boil for 10min, and subjected to 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 transferring to a plate, sealing with 5% skimmed milk powder at room temperature for 2h, and then incubating and combining horseradish peroxidase (HRP) nano antibody Nb-Y1, nb-Y2 and Nb-Y3 proteins by using an incubation box, wherein the dilution is 1:5000, and incubating for 1h at room temperature by 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 showed that Nb-Y1 and Nb-Y2 were capable of specifically binding to E0, and Nb-Y3 was capable of specifically binding to E2. (FIG. 29), (FIG. 30), and (FIG. 31).
Claims (1)
1. A nanobody against BVDV E0, characterized by: the amino acid sequence is SEQ ID NO.3.
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