Single-chain antibody and use thereof

Abstract

The application discloses a single-chain antibody, the amino acid sequence of which is shown as SEQ ID NO:1, and the nucleotide sequence of which is shown as SEQ ID NO:2. The single-chain antibody against rabies virus is screened from an antibody library of immunized mice by using a phage display technology. Compared with a traditional cell fusion technology, the phage display technology is simple in operation, low in cost, short in time, and high in antibody discovery and screening efficiency. The single-chain antibody obtained by the application is low in molecular weight and immunogenicity, high in antibody specificity and affinity, and has the activity of neutralizing rabies virus, thereby providing a new idea for basic research of rabies virus and development of therapeutic drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a single-chain antibody specifically recognizing rabies virus and application thereof in anti-rabies virus. BACKGROUND

[0002] Rabies is a fatal zoonosis caused by rabies virus (RABV) infection, commonly known as mad dog disease. Rabies has been a serious public health problem in more than 150 countries and regions (mainly in Asia and Africa). Once the virus infects the central nervous system and clinical symptoms appear, the mortality rate is almost 100%, and about 60,000 people die worldwide each year. The incubation period of rabies is usually 2-3 months, but it can also vary from one week to one year, depending on factors such as the location of the virus entry and the viral load. The initial symptoms of rabies include fever, pain, and unusual or unexplained tingling, stinging, or burning sensations at the wound site, among other general symptoms. As the virus moves to the central nervous system, the brain and spinal cord develop progressive and fatal inflammation. There is no WHO-approved diagnostic tool for detecting rabies virus infection before clinical onset, and there is no specific treatment for rabies after onset. Human clinical rabies can be controlled, but almost cannot be cured, and death is inevitable, and it can also cause severe neurological deficits. Currently, the most effective way to prevent rabies is through post-exposure prophylaxis (PEP) to prevent the virus from entering the central nervous system, but effective rabies vaccines and immunoglobulins for humans are often difficult to obtain or unaffordable, so it is imperative to develop drugs to treat rabies.

[0003] Single-chain antibody (ScFv) is a new type of genetically engineered antibody formed by coupling the variable regions of heavy and light chains of an antibody through a flexible short peptide (Linker). Compared to traditional intact antibodies, it has no constant region fragment, low immunogenicity, simple structure, small molecular weight, easy molecular modification, and strong tissue penetration ability, so it has some unique advantages in disease treatment and research. So far, ScFv can be expressed in various expression systems such as E. coli, mammalian cells, yeast, plants, and insect cells, each host has its own advantages and disadvantages in folding and expressing ScFv. In most cases, ScFv selected from phage libraries is sufficient for use in research such as ELISA, Western blot, or immunofluorescence.

[0004] Phage display technology is a technology that uses genetic engineering principles to insert foreign genes encoding polypeptides or proteins into the appropriate position of the corresponding vector plasmid encoding phage coat genes to form a recombinant plasmid. When the phage infects E. coli for replication, the foreign genes will be expressed on the surface of the phage in the form of fusion proteins together with the phage coat protein genes. The proteins or polypeptides displayed in this way can maintain a relatively independent spatial structure and biological activity. Through biological panning, phages that can specifically bind to target molecules (such as enzymes, cell surface receptors, and chemical drugs) with affinity are screened. The unbound free phages are eluted, and the phages specifically bound to the target molecules are eluted by competing receptors or acid. The eluted phages are used to infect host cells for propagation and expansion. After 3-5 rounds of "adsorption-elution-expansion", the phages specifically bound to the target molecules are highly enriched. Phage display technology is a widely used protein and polypeptide screening technology, which is commonly used in the fields of microbial diagnosis and treatment, epitope labeling and screening, biological medicine and antibody process development, etc. SUMMARY

[0005] The present application provides a single-chain antibody capable of specifically recognizing rabies virus, the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the single-chain antibody is shown in SEQ ID NO: 2.

[0006] Another object of the present application is to provide the use of the single-chain antibody described above, i.e., its application in the preparation of anti-rabies virus drugs.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] 1. Animal immunization and acquisition of immune tissues

[0009] Mice were immunized with rabies virus standard challenge strain CVS virus liquid, and the serum titer was determined by indirect immunofluorescence experiment (IFA) and enzyme-linked immunosorbent assay (ELISA) coated with recombinant rabies virus nucleoprotein. According to the determination results, specific and high-titer immune mice were selected, and their spleens were collected, crushed, and stored for use;

[0010] 2. Construction of single-chain antibody phage library

[0011] Total RNA was extracted from mouse spleen tissue, and cDNA library was obtained by reverse transcription. Using mouse antibody specific amplification primer, two rounds of PCR reaction were carried out to amplify the heavy chain variable region and light chain variable region sequence of the antibody. Homologous recombination reaction was carried out to construct single-chain antibody phagemid cloning vector, which was transformed into TG1 competent cells to construct the single-chain antibody gene library. The constructed single-chain antibody gene library was rescued by helper phage, and after three rounds of enrichment screening, the phage monoclonal colonies after the third round of screening were randomly picked, and the supernatant was detected by ELISA to screen positive monoclonal with high affinity. The sequence of the single-chain antibody was analyzed by sequencing, and the single-chain antibody with correct sequence was selected. The phage monoclonal colonies were cultured, rescued by helper phage and enriched by precipitation, and then the endotoxin was removed by dialysis. The content of the precipitated phage was determined, and the phage monoclonal was mixed with rabies virus, vortexed and incubated. The mixed solution after incubation was incubated with mouse brain neuroblastoma (N2a) cells again, and the neutralization activity of the phage monoclonal was verified by Western blot and IFA. After all the experimental verifications were completed, the single-chain antibody with the best neutralization activity was selected, and the nucleotide sequence (SEQ ID NO: 2) obtained by sequencing was analyzed again. The amino acid sequence (SEQ ID NO: 1) of the single-chain antibody was obtained by using the sequence.

[0012] Advantages and technical effects of the present application:

[0013] The present application uses phage display technology to screen single-chain antibodies against rabies virus from the antibody library of immunized mice. Compared with the traditional cell fusion technology, the operation is simple, the cost is low, the time is short, and the efficiency of antibody discovery and screening is improved.

[0014] The single-chain antibody obtained by the present application has low molecular weight and low immunogenicity, high specificity and high affinity, and has the activity of neutralizing rabies virus, which provides a new idea for the basic research of rabies virus and the development of therapeutic drugs. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 IFA detection of the recognition of rabies virus by the polyclonal serum of 5 immunized mice;

[0016] Figure 2 The results of ELISA detection of the titer of the polyclonal serum of 5 immunized mice. The upper graph shows the immunized mice 2 and 5, and the lower graph shows the immunized mice 1, 3 and 4. Mock is the blank control;

[0017] Figure 3 IFA identification of the specific recognition ability of the polyclonal serum of the immunized mice 2 and 5 to the rabies virus protein.

[0018] Figure 4 Results of Western blot to identify the specific recognition ability of the polyclonal serum of the immunized mice No. 2 and No. 5 to the denatured rabies virus protein;

[0019] Figure 5 PCR amplification results of the antibody heavy chain variable region and light chain variable region fragments using the mouse spleen cDNA as the template, wherein Fig. A is the first round of amplification of the antibody heavy chain variable region gene fragments (VH), lanes 1-2 are VH1, lanes 3-4 are VH2, and lane 5 is a control; Fig. B is the first round of amplification of the antibody light chain variable region gene fragments (VL), lanes 1-2 are VL1, lanes 3-4 are VL2, and lane 5 is a control; Fig. C is the second round of amplification of the antibody heavy chain variable region gene fragments, lanes 1-2 are VH1, lanes 3-4 are VH2, and lane 5 is a control; and Fig. D is the second round of amplification of the antibody light chain variable region gene fragments, lanes 1-2 are VL1, lanes 3-4 are VL2, and lane 5 is a control;

[0020] Figure 6 Results of bacterial liquid PCR to identify the positive transformants, a total of 30 single colonies, the upper graph is the result of 15 single colonies, and the lower graph is the result of the remaining 15 single colonies, lanes 1-15 are different single colonies;

[0021] Figure 7 Results of Dot blot experiment to identify the library phages after three rounds of panning; Fig. A shows that the library phages after three rounds of panning can specifically recognize the rabies virus particles, wherein 1 and 2 are the rabies virus CVS and Flury particles cultured in the previous stage and concentrated and precipitated by PEG8000, 3 is the supernatant of normal NA cells concentrated and precipitated by PEG8000, and 4 is PBS; Fig. B shows that there are detectable phages in the enriched library, wherein 1 is the precipitated library phages, 2 is the helper phage M13K07, and 3 is PBS; 8000

[0022] Figure 8 Results of bacterial liquid PCR to identify the gene fragment deletion of the phage single colonies after three rounds of biological panning;

[0023] Figure 9 Results of the affinity of 30 single phages of the third round of screening to the rabies virus particles;

[0024] Figure 10 Results of ELISA to identify the activity of 6 single phages, wherein Fig. A is the result of binding to the rabies virus CVS strain, and Fig. B is the result of binding to the rabies virus Flury strain;

[0025] Figure 11 ​Dot blot verification of the result of the No. 3 monoclonal phage recognizing rabies virus particles, A is the result of the No. 3 monoclonal phage specifically recognizing rabies virus particles, 1 and 2 in the figure are PEG 8000 Concentrate the precipitated rabies virus CVS and Flury virus particles, 3 is PEG8000 concentrated precipitated normal N2a cell supernatant, 4 is PBS; B is the result of directly detecting precipitated No. 3 phage, 1 is precipitated No. 3 phage, 2 is helper phage M13K07, 3 is PBS;

[0026] Figure 12 The result of Western blot identification of the neutralizing activity of 6 strains of monoclonal phage, the upper figure is the result of Western blot verification, and the lower figure is the quantitative statistical result of the protein band;

[0027] Figure 13 The result of IFA identification of the neutralizing activity of No. 3 monoclonal phage, 1N1 in the figure is the anti-rabies virus nucleoprotein monoclonal antibody 1N1 (Anti-RABV-N) screened in the laboratory in the early stage, and the normal infected virus and uninfected virus (Mock) incubated N2a cells are controls. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and benefits of the embodiments of the present application clearer, the technical scheme of the present application will be described in detail below in combination with the drawings and specific embodiments. The following embodiments are only used to understand the present application, and should not be regarded as limiting the scope of the present application, and it should be understood as a detailed description of certain aspects, characteristics and embodiments of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments used in the embodiments are not marked by the manufacturer, which are conventional products that can be purchased in the market, and the technical methods used are not specially described, which are carried out according to the kit instruction method or the conventional method.

[0029] The rabies virus recombinant nucleoprotein (pET-32a-RABV-N) plasmid in the example was constructed by the laboratory in the early stage. The strain was cultured and induced to express the recombinant protein. The protein was purified according to the Ni-NTA affinity chromatography column operation method, and was used as the coating antigen for ELISA detection. The rabies virus glycoprotein plasmid pLVX-RABV-G and the nucleoprotein plasmid Flag-RABV-N were constructed by the laboratory in the early stage. The preparation method was referred to “Jia T. Functional research on AAV-mediated anti-rabies virus intracellular antibody and its PROTAC molecule [D]. Kunming University of Science and Technology, 2023. DOI:10.27200 / d”, and the plasmid extraction was performed according to the plasmid extraction kit instruction book purchased from Kang Weishijie Century Biotechnology Co., Ltd. The pCANTAB-5E vector was purchased from Wuhan Moliang Biological Technology Co., Ltd. After the transformation of DH5α competent cells, the strain was preserved.

[0030] Example 1: Animal immunization and acquisition of immune tissues

[0031] Take 250 μL of the rabies virus standard challenge strain CVS virus liquid prepared from the brain tissue of the laboratory in the early stage, irradiate it under ultraviolet light for 15 min for inactivation treatment. Mix the inactivated virus with Freund's incomplete adjuvant at a volume ratio of 1:1. After complete emulsification, use the abdominal subcutaneous multi-point injection method to immunize 5 mice for the first time, with an immunization dose of 100 μL per mouse. Then, immunize every 2 weeks, and replace the Freund's incomplete adjuvant with Freund's complete adjuvant. After the third immunization, collect the mouse tail vein blood one week after each immunization, and determine the serum titer:

[0032] 1. Collection and storage of mouse immune serum: take the immunized mouse, fix it with a fixer, cut off 2-3 mm of the mouse tail, and collect the flowing blood into a 0.5 mL centrifuge tube with a pipettor, taking care to avoid hemolysis. Place it at room temperature for 2 h, and then store it overnight in a 4°C refrigerator. Centrifuge the overnight-treated blood at 4°C and 3000 rpm for 15 min, then aspirate the supernatant into a new centrifuge tube. Add glycerol at a volume ratio of 1:1, mix well, and store it at -40°C or -80°C for standby use.

[0033] 2. Detection of immune mouse polyclonal serum titer by indirect immunofluorescence assay (IFA)

[0034] 96-well cell plate culture mouse brain tumor cells (N2a), 37°C, 5% CO2 incubator culture, until the cells adhere to the growth density of about 80%, each hole according to the multiplicity of infection MOI = 0.01 to the N2a cells into the rabies virus, placed in 37°C incubator continue to culture 48h, so that the virus as far as possible completely infected the cells in the hole. Virus infected N2a cell supernatant harmless treatment, fixed liquid fixation of cells, after the completion of the liquid, until the plate completely dry, add 200 μL / hole blocking fluid, placed in 4°C refrigerator blocking overnight. Discard the blocking fluid, PBST buffer washing 5 times, each hole 100 μL diluted serum (serum and blocking fluid according to 1:200, 1:400, 1:800, 1:1600 dilution), while setting up no virus infected N2a cells as blank control (Control); placed in 37°C incubation 2h, discard the primary antibody dilution, PBST buffer washing 5 times, add 50 μL / hole (or 100 μL / hole) dilution of FITC labeled goat anti mouse IgG antibody (antibody: blocking fluid = 1:1500, volume ratio), 37°C avoid light incubation 1h; discard the secondary antibody dilution, PBST washing 5 times, retain the last washing liquid, fluorescence microscope observation; results see Figure 1 As can be seen from the figure, the immune effect of the 2nd, 3rd and 5th mice is better, and the serum titer is at least 1:1600;

[0035] 3, enzyme linked immunosorbent assay (ELISA) detection of immune mouse polyclonal serum titer

[0036] The antigen (pET-32a-RABV-N recombinant protein) is diluted with coating solution to 10 μg / mL, 100 μL per well is added to the microplate, and incubated at 37°C for 2h (or 4°C overnight). Discard the liquid in the microplate, wash with PBST for 3 times, add 200 μL blocking solution to each well, and incubate at 37°C for 2h. Discard the blocking solution, wash with PBST for 5 times, then add 200 μL of gradient diluted serum (serum dilution with serum: 5% skim milk = 1:200, 1:400, two-fold gradient dilution, a total of 12 gradients) to the microplate, and incubate at 37°C for 2h (or 4°C overnight). Discard the serum dilution, wash with PBST for 5 times, then add 50 μL of diluted HRP labeled goat anti mouse IgG antibody (antibody: 5% skim milk = 1:10000) to each well, incubate at 37°C for 1h, then discard the secondary antibody dilution, wash with PBST for 5 times, add 100 μL TMB color developing liquid to each well, and develop color at 37°C for 15 min in the dark. Add 50 μL of 2M H2SO4 to terminate the reaction, and measure the OD450nm absorbance value by enzyme marker. Figure 2The results showed that the serum titers of the mice reached 1:25600, but the immune effects of the No. 2 and No. 5 mice were better.

[0037] 4. The specific recognition ability of the polyclonal serum of the No. 2 and No. 5 immunized mice to the rabies virus protein was further detected by IFA method

[0038] The rabies virus glycoprotein plasmid pLVX-RABV-G and the nucleoprotein plasmid Flag-RABV-N were transfected into 293T cells according to the instructions of the PEI 40K Transfection Reagent transfection reagent, and the cells were cultured at 37°C for 48h. The cell supernatant was discarded, the cells were fixed with fixing solution, and the results were observed after blocking, serum incubation, and fluorescence secondary antibody incubation (the wells of cells without transfection of plasmid were also incubated with serum as control (Control)). The dilution ratios of the serum of the immunized mice were 1:200, 1:400, 1:800, 1:1600, and 1:3200. The results are shown in Figure 3 The results showed that the polyclonal serum of the No. 2 and No. 5 immunized mice could specifically recognize the rabies virus glycoprotein and nucleoprotein, and the serum titers could reach 1:3200.

[0039] 5. Western blot was used to detect the specific recognition ability of the serum of the No. 2 and No. 5 immunized mice to the denatured rabies virus protein

[0040] The rabies virus glycoprotein plasmid pLVX-RABV-G and nucleoprotein plasmid Flag-RABV-N were transfected into 293T cells cultured in a 6-well cell plate according to the transfection reagent instructions of PEI 40K Transfection Reagent. After 48 h of culture, the cell supernatant was discarded, 200 μL RIPA cell lysis solution was added to each well, and the cells were lysed at low temperature for 15-20 min. 293T cells transfected with no plasmid were used as a control. The cell lysate was collected, centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant was collected as the total cell protein sample. 5x SDS loading buffer was added, mixed, heated at 95°C for 10 min in a metal bath, and then cooled. SDS-PAGE electrophoresis was performed. After electrophoresis, the concentrated gel was cut off, and the "sandwich" method was used to place the sponge pad, filter paper, NC membrane, protein gel, filter paper, and sponge pad. The protein was transferred from the negative electrode to the positive electrode horizontally to the NC membrane in a transmembrane instrument at a constant current of 160 mA for 120 min. After transmembrane, the NC membrane was taken out and placed in a suitable size incubation box. 5% skim milk was added and blocked at 37°C for 2 h. The blocking solution was discarded, and the membrane was washed with PBST 5 times, each for 5 min. The immune mouse polyclonal serum was diluted at a ratio of 1:1000, and then incubated with the membrane at 4°C overnight (or 37°C for 2 h). The primary antibody diluent was discarded, the membrane was washed with PBST 5 times, each for 5 min, the diluted HRP-labeled goat anti-mouse IgG antibody (antibody: 5% skim milk = 1:10000, volume ratio) was added, and incubated at 37°C for 1 h. The secondary antibody diluent was discarded, the membrane was washed with PBST several times, ECL developing solution was prepared, and chemiluminescence imaging was performed. The results are shown in Figure 4 The results show that the immune mouse polyclonal serum of No. 2 and No. 5 can specifically recognize denatured rabies virus glycoprotein and nucleoprotein.

[0041] According to the above results, the spleens of immune mice No. 2 and No. 5 with good specificity and high serum titer were selected as the tissue material for constructing a single-chain antibody gene library in the future. They were killed by exsanguination, their spleens were removed and mixed, and then ground in a glass tissue grinder. The product was collected in an EP tube and stored at -80°C for standby.

[0042] Example 2: Construction of a single-chain antibody phage library and evaluation of the library capacity

[0043] 1. RNA extraction and reverse transcription

[0044] The spleen tissue fluid was taken out at -80℃, and after low-temperature thawing, RZ lysis solution was added. The total RNA was extracted using RNAsimple Total RNA Kit from Tiangen Biosciences Co., Ltd., and the RNA concentration was determined by ultraviolet spectrophotometry. Then, the reverse transcription was performed according to the instructions of the Green Tag Mix and HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) purchased from Nuozhan Biomedicals Co., Ltd., to obtain cDNA.

[0045] 2. Amplification of the target fragment

[0046] The specific amplification primers for the murine antibody were designed and synthesized. The primer sequences are shown in Table 1. The cDNA obtained by reverse transcription was used as the template for the first round of PCR reaction. The reaction system is shown in Table 2 and Table 3, and the amplification conditions are shown in Table 4. The murine antibody heavy chain variable region and light chain variable region gene fragments were amplified, respectively. After the reaction, 1.5% agarose gel electrophoresis was performed, and the results are shown in Figure 5 A, 5B, the size of the target band is consistent with the size of the antibody heavy chain and light chain variable region sequence, and DNA gel recovery was performed;

[0047] Table 1

[0048]

[0049]

[0050] Table 2: Reaction system for the first round of PCR amplification of antibody heavy chain variable region gene (VH1 and VH2)

[0051]

[0052] Table 3: Reaction system for the first round of PCR amplification of light chain variable region gene (VL1 and VL2)

[0053]

[0054] The recovered target fragment was used as the template for the second round of PCR reaction. The reaction system is shown in Table 5 and Table 6, and the amplification conditions are the same as the first round of reaction program. This round of amplification reaction adds a Linker sequence between the heavy chain variable region and the light chain variable region fragments, and adds SfiI and NotI enzyme cutting sites at the 5' end and 3' end of ScFv, respectively. The amplification results are shown in Figure 5C, 5D, the size of the target band also conforms to the antibody heavy and light chain variable region sequence size, and the DNA gel is recovered, the concentration is measured, and the sample is stored at -40°C for standby. In order to ensure the integrity of the sequence diversity of the library constructed subsequently, in this step, as many times of two-round PCR amplification as possible are carried out to recover the antibody heavy chain variable region (VH1 and VH2) and light chain variable region gene fragments (VL1 and VL2);

[0055] Table 4: PCR reaction program

[0056]

[0057] Table 5: Reaction system of second-round PCR amplification of antibody heavy chain variable region gene (VH1 and VH2)

[0058]

[0059] Table 6: Reaction system of second-round PCR amplification of antibody light chain variable region gene (VH1 and VH2)

[0060]

[0061] 3. Single-chain antibody gene library construction and library capacity evaluation

[0062] (1) Preparation of linearized pCANTAB-5E vector

[0063] The pCANTAB-5E strain preserved in the early stage of the experiment was used, and after low-temperature thawing, it was inoculated on an LB plate containing 100 μg / mL ampicillin by streaking method, and was incubated at 37°C for overnight. After the single colony was picked and expanded, the plasmid was extracted using the EndoFree Plasmid Midi Kit of Kangweishijie Bioengineering, the concentration was measured, and then the reaction system of Tables 7 and 8 was prepared according to the instructions of the restriction enzymes Sfi I and Not I of Takara. Since the reaction temperatures of the two restriction enzymes are different, the reaction system shown in Table 7 is first prepared, and the plasmid is digested at 50°C for 3 h. Then, the components shown in Table 8 are added to the same reaction system, and the plasmid is digested at 37°C for 3-4 h. After the reaction is completed, 1 / 10 volume of 10×DNA Loading Buffer is added to terminate the reaction, and 1% agarose gel electrophoresis is performed to observe the digestion results, and the linearized pCANTAB-5E vector is recovered;

[0064] Table 7

[0065]

[0066] Table 8

[0067]

[0068] (2) Use Beijing Qianke Biotechnology Co., Ltd. Treief Seamless Cloning Kit to perform multi-fragment homologous recombination reaction on the second round of amplified 2 groups of heavy chain variable region fragments (VH1 and VH2), 2 groups of light chain variable region fragments (VL1 and VL2), and linearized pCANTAB-5E vector for homologous recombination reaction. The reaction system is shown in Table 9;

[0069] Table 9

[0070]

[0071] After the system is prepared, gently mix the components, centrifuge briefly, and quickly place in an ice water bath for 5 min after 50℃ reaction for 35 min. Then directly transform TG1 competent cells and coat on 2xYT plates containing 0.4% glucose and ampicillin resistance, and incubate at 37℃ overnight. Add 2 mL of non-resistant 2xYT liquid medium to the surface of each plate with several single colony colonies. After the surface is wet, gently scrape the colonies with a coating rod and collect the bacterial solution in the same sterile 50 mL centrifuge tube. Mix well; this connection and transformation step is performed a total of 5 times, and 200 plates are coated. Add the mixed bacterial solution to 800 mL of ampicillin-resistant 2xYT liquid medium, and incubate at 37℃, 180 rpm for 4-6 h. Centrifuge at 5000g for 10 min at room temperature, discard the supernatant, resuspend the precipitate with 20 mL of 2xYT medium, mix well, take out 2 mL of bacterial solution and store at 4℃, add the same volume of 50% glycerol to the remaining bacterial solution, and store at -80℃ to obtain the primary single-chain antibody gene library. At the same time, perform bacterial liquid PCR identification of positive transformants, and the results are shown in Figure 6 The results show that the correct rate of library insertion fragments is 73%, and the library capacity size is 2.22x10 8 cfu / mL, and part of the single colonies with correct size of target fragments are randomly selected for sequencing by Qianke Biotechnology Co., Ltd. Comparison and analysis of the sequencing results show that each sequence is different, and the library diversity is relatively complete.

[0072] Example 3: Cell culture and concentration and precipitation of rabies virus particles

[0073] N2a cells were cultured in T75 cell culture flasks. When the cells were about 80% confluent, they were inoculated with rabies virus strain CVS and Flury at 37°C in a 5% CO2 incubator for 3-4 hours. The medium was then replaced with 20 mL of DMEM containing 2% FBS, and the cells were incubated for another 72 hours. The cell supernatant was collected and centrifuged at 3000 rpm for 10 minutes at 4°C to remove cell debris. The supernatant was collected and mixed with β-propiolactone at a volume ratio of 1:4000. The mixture was incubated overnight at 4°C to inactivate the virus. The same volume of sterile PEG8000 / NaCl solution was then added to the mixture, which was then incubated overnight at 4°C. The mixture was then centrifuged at 7000 rpm for 30 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 3-4 mL of PBS. The purified rabies virus strain CVS and Flury were obtained and stored at -80°C for future use. 8000 / NaCl solution, and incubated overnight at 4°C. The mixture was then centrifuged at 7000 rpm for 30 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 3-4 mL of PBS. The purified rabies virus strain CVS and Flury were obtained and stored at -80°C for future use.

[0074] Example 4: Rescue and titer detection of single-chain antibody phage library

[0075] The single-chain antibody gene library cells obtained in Example 2 were resuscitated and activated, and the culture medium was added to 400 mL of ampicillin-resistant 2xYT medium containing 1% glucose. The mixture was incubated at 37°C and 200 rpm until the cells entered the logarithmic growth phase. Then, 200 μL of helper phage M13K07 (titer: 3.9x10 12 pfu / mL) was added, and the mixture was incubated at 37°C for 30 minutes without shaking. Then, the mixture was incubated at 37°C and 200 rpm for another 30 minutes, followed by centrifugation at 3300g for 10 minutes at room temperature. The supernatant was discarded, and the cell precipitate was resuspended in 400 mL of ampicillin- and kanamycin-resistant 2xYT medium. The mixture was incubated at 37°C and 200 rpm overnight. The next day, the mixture was centrifuged at 4°C, and the supernatant was collected. One-fourth the volume of sterile PEG8000 / NaCl solution was added to the supernatant, and the phage was precipitated on ice. The mixture was centrifuged at 10800g for 30 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in sterile water. One-fifth the volume of PEG8000 / NaCl solution was added to the mixture, and the phage was precipitated on ice for at least 30 minutes. The mixture was centrifuged at 10800g for 10 minutes at 4°C. The supernatant was discarded, and the phage precipitate was resuspended in PBS. The mixture was centrifuged at 11600g for 10 minutes at room temperature to remove residual cell debris. The supernatant was collected and stored at 4°C for subsequent bio-panning.

[0076] Ten to twenty microliters of the supernatant were taken and used for phage titer detection using gradient dilution and Amp-resistant plate screening of phage monoclonal colonies. The measured result (3x10 10 pfu) was the input amount of phage for the first round of bio-panning.

[0077] Example 5: Enrichment panning and affinity identification of single-chain antibody phage library

[0078] The purified CVS virions of Example 3 were diluted to 20 μg / mL with coating solution and added to the enzyme-linked immunosorbent plate, which was coated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed 3 times with PBST. 200 μL of 5% skim milk was added to each well, and the plate was blocked at 37°C for 2 h. After the blocking solution was discarded and the plate was washed, the remaining liquid was blotted with sterile paper. 100 μL of the enriched ScFv phage library of Example 4 (i.e., the input of the first round was 3 x 10 10 pfu) was added to each well, and the plate was incubated gently at room temperature for 1-2 h. The antibody library solution was discarded, and the plate was washed 10 times with PBST. The remaining liquid was blotted, and 50 μL of 2M Gly-HCl (pH = 2.2, containing 1 mg / mL BSA) was added to each well. The plate was shaken at room temperature for 10 min, and 150 μL of neutralization solution was added to each well for rapid neutralization of the eluent. The neutralized solution was collected, which was the product of the first round of screening. 10-15 μL of the product was taken for titer detection, and the output of the phage of this round of panning was calculated, i.e., 4 x 10 6 pfu. The remaining screening product was used to infect TG1 bacteria in the logarithmic growth phase for 30 min, and the supernatant was discarded after centrifugation at 3300 g for 5 min. The cell pellet was resuspended in 4-5 mL of antibiotic-free 2 x YT medium, and the entire resuspension was spread on a YTAG plate and incubated at 37°C in an inverted incubator overnight (12-16 h). The next day, 2-3 mL of antibiotic-free 2 x YT medium was added to the surface of the plate, and the single colonies on the surface were gently scraped off with a spreader and collected in a 50 mL centrifuge tube. The supernatant was discarded after centrifugation at 3300 g for 5 min. The pellet was resuspended in 5 mL of antibiotic-free 2 x YT medium, and 2 mL of the bacterial solution was used for expansion culture. The rescue and titer detection of the phage library were performed according to the procedures of Example 4. The enriched phage was used for the next round of biological panning. The enrichment panning was repeated for a total of 3 rounds of “adsorption-washing-elution-enrichment” biological panning procedures. The antigen coating concentration was reduced to 15 μg / mL and 10 μg / mL for the subsequent rounds, and the number of plate washing was increased to 15 and 20 for the subsequent rounds. The preliminary screening of the anti-RABV ScFv phage library was completed, and the results of the screening are shown in Table 10. The recovery ratio of the eluted phage after each round of panning was 1.33 x 10 -4 to 5.11 x 10 -1 The enrichment fold also increased from 1.0 to 68.18, indicating that the ScFv phage library was effectively and specifically enriched. At the same time, Dot blot experiments were used to preliminarily identify the library phage after 3 rounds of biological panning.

[0079] Dot blot experiment operation steps: according to the experimental requirements, cut two NC membranes of appropriate size, and press three circular holes of appropriate size on the membrane, then cut the upper left corner of the membrane and mark it. Take the purified -80℃ frozen CVS and Flury virus particles and uninfected virus N2a cell supernatant, thaw and take 20 μL of each to be spotted in the center of one of the circular holes of the NC membrane, and take 20 μL of the above-mentioned enriched library phage and 15 μL of helper phage to be spotted on the other NC membrane, spot multiple times, set up PBS control wells, and let the membrane dry completely. Place the membrane in an incubation box, add 5% skim milk, and block at 37℃ overnight. Discard the blocking solution, wash the membrane with PBST 5 times, 5 min each time. Take 2-3 mL of the above-mentioned precipitated enriched library phage and add it to the NC membrane, incubate at 37℃ for 2 h. Discard the phage solution, wash the membrane with PBST 5 times, 5 min each time. After diluting the HRP-labeled Anti-M13 monoclonal antibody (1:5000, volume ratio) with 5% skim milk, add it to the NC membrane, and incubate at 37℃ for 1 h. Discard the antibody diluent, wash the membrane with PBST 5 times, 5 min each time, prepare ECL developing solution, and perform chemiluminescence imaging. The results are shown in Figure 7 Figure 2, which shows that the screened library phage can specifically recognize rabies virus particles, and there are detectable phages in the enriched library.

[0080] Table 10: Selective enrichment and screening results of ScFv phage library

[0081]

[0082] At the same time, 20 phage monoclonal colonies after the third round of screening were randomly picked, and the ScFv library gene fragment deletion after screening was determined by PCR of the bacterial solution. The results are shown in Figure 8 Figure 3, which shows that the 20 monoclonal strains have completely correct insert sizes without insert deletion, indicating that the three rounds of specific screening did not cause insert loss, but rather screened and enriched the positive strains in the primary library that were successfully connected to the vector and had correctly inserted fragments.

[0083] Example 6: Preparation of phage monoclonal and identification of neutralizing activity

[0084] After the third round of screening products to infect TG1 cells, coated on YTAG plate, 37°C incubator inverted culture overnight; or is taken in the third round of frozen at -80°C screening phage library, low temperature thawed after plating line method inoculated to ampicillin resistant 2 x YT plate, 37°C incubator inverted culture overnight. In the clean bench, respectively, randomly picked 100 single colonies in 1 mL ampicillin resistant 2 x YT medium, 37°C, 200 rpm culture 3-4 h to be cloudy, then add 10 μL titer about 1.5 x 10 12 pfu / mL helper phage M13K07, mixed 37°C, 30 min, then 37°C, 180 rpm culture 30 min, room temperature, 3300 g centrifugation 5 min, discard the supernatant, resuspended in 1 mL ampicillin resistant and kanamycin resistant 2 x YT medium, 37°C, 180 rpm culture overnight (10-12 h); 3300 g centrifugation 5 min, take the supernatant for ELISA detection, the operation steps refer to example 1, secondary antibody incubation 5% skim milk diluted anti-M13 (volume ratio 1:5000) monoclonal antibody, enzyme label instrument measured OD 450nm absorbance value, the results are shown in Figure 9 ; The results show that most of the screened single phage has strong affinity to rabies virus particles, and 15 single phage with strong affinity are selected for sequencing in Beijing Genki Biotechnology Co., Ltd. After comparing and analyzing the sequencing results, 6 single phage with correct and complete single-chain antibody sequences are screened, which are named No. 1 phage, No. 3 phage, No. 6 phage, No. 7 phage, No. 9 phage and 1-9 phage, respectively. The affinity and neutralizing activity of the 6 single phage are identified again.

[0085] 1. ELISA and Dot blot are used to identify the affinity of the single phage

[0086] Similarly, the pre-purified rabies virus particles CVS and Flury are coated on the enzyme-linked immunosorbent plate for ELISA identification, and the operation steps refer to example 1. The secondary antibody is also incubated with 5% skim milk diluted anti-M13 (volume ratio 1:5000) monoclonal antibody. The results are shown in Figure 10 ; No. 3 phage has the highest binding activity to rabies virus particles. Dot blot experiment is also performed, and the results are shown in Figure 11 ; No. 3 single phage can also specifically recognize rabies virus particles, and the results of figure 11B show that the phage precipitation amount of No. 3 phage is moderate, but the recognition ability to rabies virus particles is higher, which is consistent with the results of ELISA.

[0087] 2. Western blot to verify the neutralizing activity of the monoclonal phage

[0088] Six phage monoclonal colonies were expanded, and PEG 8000 / NaCl precipitation was used to enrich the phage, which was then dialyzed to remove endotoxins, and the phage content was determined. 50 μg of the monoclonal phage was mixed with 10 μL of rabies virus (calculated according to the multiplicity of infection MOI = 0.01), and DMEM medium was added to make up to 500 μL. DMEM-diluted rabies virus (RABV) and normal mouse brain neuroblastoma cells (N2a) were used as controls. After vortex mixing, the mixture was incubated at 37°C for 2 h. After incubation, the mixture was added to the mouse brain neuroblastoma cells (N2a) cultured in a 12-well cell culture plate. After 4-6 h of culture, the corresponding monoclonal phage and DMEM medium mixture 1 mL was replaced, and the serum concentration was adjusted to 2%. The same conditions were continued to culture, and the cell lysate was collected 48 h after viral infection. Western blot was used to detect the rabies virus (RABV) nucleoprotein in the cells (the primary antibody was monoclonal antibody 1N1: Anti-RABV-N). The results are shown in Figure 12 The results show that the viral protein content of phage No. 3 is the lowest, and the corresponding quantitative statistical results are also the same, indicating that phage No. 3 has good neutralizing activity against rabies virus.

[0089] 3. IFA to identify the neutralizing activity of the monoclonal phage

[0090] The No. 3 monoclonal phage was incubated with rabies virus and infected N2a cells as described above. After 48 h of cell culture, the cell supernatant was inactivated, 4% paraformaldehyde fixing solution was added, and the cells were fixed at room temperature for 15-30 min. IFA was used to detect the neutralizing ability of the monoclonal phage against rabies virus. Monoclonal antibody 1N1 (Anti-RABV-N) was used to incubate the N2a cells infected with the virus and the N2a cells not infected with the virus as controls.

[0091] The results are shown in Figure 13 Compared with the controls, phage No. 3 also has good neutralizing activity, and the neutralizing ability increases with increasing concentration.

[0092] 4. The No. 3 monoclonal phage was sent to Beijing Qianke Biological Technology Co., Ltd. for sequencing to obtain the nucleotide sequence, which was analyzed again. The sequence is shown in SEQ ID NO: 2. The single-chain antibody sequence is complete and correct, and the amino acid sequence of the single-chain antibody obtained from this sequence is shown in SEQ ID NO: 1.

Claims

1. A single-chain antibody, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The use of the single-chain antibody according to claim 1 in the preparation of anti-rabies virus drugs.

Images