Single-chain antibody and application thereof

Abstract

The invention discloses a single-chain antibody, the amino acid sequence of the single-chain antibody is shown as SEQ ID NO: 1, and the nucleotide sequence of the single-chain antibody is shown as SEQ ID NO: 2. Compared with the traditional cell fusion technology, the single-chain antibody for resisting rabies virus is screened from an immune mouse antibody library by utilizing a phage display technology, the operation is simple, the cost is lower, the time is short, and the discovery and screening efficiency of the antibody is improved; the obtained single-chain antibody is very low in molecular weight and immunogenicity, strong in antibody specificity and relatively high in affinity, has the activity of neutralizing the rabies virus, and provides a new thought for basic research of the rabies virus and development of therapeutic drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a single-chain antibody that specifically recognizes rabies virus and its application in resisting rabies virus. Background Art

[0002] Rabies is a fatal zoonotic infectious disease caused by infection with the rabies virus (RABV), commonly known as mad dog disease. Rabies has always 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%, causing about 60,000 deaths worldwide each year. The incubation period of rabies is usually 2-3 months, but it may also vary from one week to one year, depending on factors such as the location of virus entry and viral load. The initial symptoms of rabies include general symptoms such as fever, pain, and abnormal or unexplained tingling, stinging or burning sensations at the wound site. As the virus transfers to the central nervous system, progressive and fatal inflammation of the brain and spinal cord occurs. There is currently no WHO-approved diagnostic tool for detecting rabies virus infection before clinical onset, and there is no specific treatment drug after rabies occurs. Clinical rabies in humans can be controlled, but it is almost incurable, death is inevitable, and it will cause severe neurological deficits. Currently, the most effective treatment for rabies is post-exposure prophylaxis (PEP) to prevent the virus from entering the central nervous system, but effective rabies vaccines and immunoglobulins 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 that is formed by coupling the heavy chain variable region and light chain variable region of an antibody with a flexible short peptide (Linker). Compared with traditional complete 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 certain unique advantages in disease treatment and research. So far, ScFv can be expressed in a variety of expression systems such as Escherichia 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 screened from phage libraries is sufficient for use in studies such as ELISA, Western blot, or immunofluorescence.

[0004] Phage display technology is the application of genetic engineering principles. The exogenous gene encoding a polypeptide or protein is inserted into the appropriate position of the corresponding vector plasmid encoding the phage coat gene to form a recombinant plasmid. When it infects Escherichia coli for replication, the exogenous gene will be expressed on the phage surface in the form of a fusion protein together with the phage coat protein gene. The protein or polypeptide displayed in this way can maintain a relatively independent spatial structure and biological activity. Then, through biological selection, phages that have affinity and specific binding to target molecules (such as enzymes, cell surface receptors, chemical drugs, etc.) are screened out. Unbound free phages are eluted, and then phages that specifically bind to the target molecule are eluted by competing receptors or acid elution. The eluted phages are used to infect host cells for reproduction and amplification. After 3-5 rounds of "adsorption-elution-amplification", phages that specifically bind to the target molecule are highly enriched. Phage display technology has become a widely used protein and peptide screening technology, which is often used in the fields of microbial diagnosis and treatment, epitope tagging and screening, and the development of biomedicine and antibody processes. Summary of the invention

[0005] The present invention 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 invention is to provide the use of the above-mentioned single-chain antibody, that is, to use it in the preparation of anti-rabies virus drugs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

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

[0009] The CVS virus solution of the standard attack strain of rabies virus was used to immunize mice, and the serum titer was determined by coating the recombinant rabies virus nucleoprotein with indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). According to the test results, immunized mice with good specificity and high serum titer were selected, and their spleens were obtained, crushed, collected, and frozen for later use;

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

[0011] Total RNA was extracted from mouse spleen tissue and reverse transcribed to obtain a cDNA library. Using cDNA as a template and mouse antibody-specific amplification primers, two rounds of PCR reactions were performed to amplify the antibody heavy chain variable region and light chain variable region sequences. Homologous recombination reactions were performed to construct a single-chain antibody phagemid cloning vector, which was transformed into TG1 competent cells to construct an anti-rabies virus single-chain antibody gene library. The constructed single-chain antibody gene library was rescued by auxiliary phage. After three rounds of enrichment and panning, the phage monoclonal colonies after the third round of panning were randomly picked and cultured until During the growth period, helper phages were added for rescue, and the supernatant was taken for ELISA detection, positive monoclonal clones with higher affinity were screened, and sequencing analysis was performed on them, and monoclonal clones with complete and correct sequences were selected, and the monoclonal phage colonies were expanded and cultured, and after the helper phages were rescued and precipitated and enriched, endotoxins were removed by dialysis, and the content of the precipitated phages was determined, and the monoclonal phages were mixed with rabies virus, vortexed and incubated, and the mixed solution after incubation was incubated again with cultured mouse brain neuroma cells (N2a), and the neutralization activity of the monoclonal phages was verified by Western blot, IFA and other experiments. After all experimental verifications were completed, the monoclonal clone with the best neutralization activity was selected, and the nucleotide sequence (SEQ ID NO: 2) obtained by sequencing was compared and analyzed again, and the amino acid sequence (SEQ ID NO: 1) of the single-chain antibody was obtained by using this sequence.

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

[0013] The present invention utilizes phage display technology to screen single-chain antibodies against rabies virus from an immune mouse antibody library. Compared with traditional cell fusion technology, the present invention is simple to operate, low in cost, and short in time, thereby improving the efficiency of antibody discovery and screening.

[0014] The single-chain antibody obtained by the present invention has very low molecular weight and immunogenicity, strong antibody specificity, high affinity, and activity in neutralizing rabies virus, thus providing new ideas for basic research on rabies virus and development of therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of indirect immunofluorescence (IFA) detection of rabies virus by polyantisera from 5 immunized mice;

[0016] Figure 2 The results of ELISA test of the titer of polyclonal antibody sera from 5 immunized mice are shown in the upper figure. The upper figure shows mice immunized with No. 2 and No. 5, and the lower figure shows mice immunized with No. 1, No. 3, and No. 4. Mock in the figure is a blank control.

[0017] Figure 3 The results of IFA identification of the specific recognition ability of polysera from mice immunized with No. 2 and No. 5 against rabies virus proteins;

[0018] Figure 4 Western blot analysis of the specific recognition of polysera from mice immunized with No. 2 and No. 5 against denatured rabies virus proteins;

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

[0020] Figure 6 The positive transformants were identified by bacterial liquid PCR. There were 30 monoclonal colonies in total. The upper figure shows the result of 15 monoclonal colonies, and the lower figure shows the result of the remaining 15 monoclonal colonies. Lanes 1-15 are different monoclonal colonies respectively.

[0021] Figure 7 The results of the Dot blot experiment to identify the library phage after three rounds of panning; Figure A shows that the library phage after three rounds of screening can specifically recognize rabies virus particles. In the figure, 1 and 2 are rabies virus CVS and Flury virus particles that were cultured in the early stage and concentrated and precipitated with PEG8000, respectively, and 3 is PEG 8000 The supernatant of normal NA cells was concentrated and precipitated, and 4 is PBS; the results in Figure B show that there are detectable phages in the enriched library, in which 1 is the precipitated library phage, 2 is the helper phage M13K07, and 3 is PBS;

[0022] Figure 8 The results of bacterial liquid PCR identification of gene fragment deletion in phage monoclonal colonies after three rounds of bio-panning;

[0023] Fig. 9 The results of the third round of screening of 30 monoclonal phages for the affinity of rabies virus particles;

[0024] Fig.10 The results of ELISA for identifying the activity of 6 monoclonal phages, of which Figure A shows the results of binding to rabies virus CVS strain, and Figure B shows the results of binding to rabies virus Flury strain;

[0025] Fig.11This is the result of Dot blot verification of the recognition of rabies virus particles by monoclonal phage No. 3. Figure A shows the result of the specific recognition of rabies virus particles by monoclonal phage No. 3. In the figure, 1 and 2 are PEG 8000 Figure 1 shows the concentrated and precipitated rabies virus CVS and Flury virus particles, Figure 3 shows the supernatant of normal N2a cells concentrated and precipitated by PEG8000, and Figure 4 shows PBS; Figure B shows the result of directly detecting the precipitated phage No. 3, in which Figure 1 shows the precipitated phage No. 3, 2 shows the helper phage M13K07, and 3 shows PBS;

[0026] Fig.12 The neutralization activity results of 6 monoclonal phages were identified by Western blot. The upper figure is the result of Western blot verification, and the lower figure is the statistical result of protein band quantification.

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

[0028] To make the purpose, technical scheme and benefit of the embodiments of the present invention clearer, the technical scheme of the present invention will be described in detail below in conjunction with the accompanying drawings and specific examples. The following examples are only used to understand the present invention and should not be used as limiting the scope of the present invention. They should be understood as detailed descriptions of certain aspects, characteristics and embodiments of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used in the examples are not specified by the manufacturer, and are all commercially available conventional products. The technical methods used are carried out according to the kit instructions or conventional methods without special instructions.

[0029] The rabies virus recombinant nucleoprotein (pET-32a-RABV-N) plasmid in the embodiment was constructed by this laboratory in the early stage, and 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 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 Ting. Functional study of AAV-mediated anti-rabies virus intracellular antibodies and their PROTAC molecules [D]. Kunming University of Science and Technology, 2023. DOI: 10.27200 / d", and the plasmid was extracted according to the instructions of the plasmid extraction kit purchased from Kangwei Century Biotechnology Co., Ltd. The pCANTAB-5E vector was purchased from Wuhan Miaoling Biotechnology Co., Ltd. and the strain was preserved after transformation of DH5α competent cells.

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

[0031] Take 250 μL of the rabies virus standard attack strain CVS virus liquid prepared in the early stage of the laboratory from the brain tissue of suckling mice, place it under ultraviolet light for 15 minutes for inactivation, and mix the inactivated virus with Freund's incomplete adjuvant in a volume ratio of 1:1. After complete emulsification, use the abdominal subcutaneous multi-point injection method to perform the first immunization on 5 mice, with an immunization dose of 100 μL per mouse. After that, immunization is performed every 2 weeks, and Freund's incomplete adjuvant is replaced with Freund's complete adjuvant. Starting from the third immunization, the tail vein blood of the mice is collected one week after each immunization, and the serum titer is measured:

[0032] 1. Collection and storage of mouse immune serum: Take the immune mouse, fix it with a fixator, cut off 2-3 mm of the mouse tail, and collect the outflowing blood with a pipette into a 0.5 mL centrifuge tube. Be careful to avoid hemolysis. Leave it at room temperature for 2 hours, and then place it in a 4°C refrigerator overnight. After the overnight treated blood is centrifuged at 4°C and 3000 rpm for 15 minutes, the supernatant is aspirated and placed in a new centrifuge tube, and glycerol is added at a volume ratio of 1:1. After mixing, it is stored in a -40°C or -80°C refrigerator for later use.

[0033] 2. Indirect immunofluorescence assay (IFA) to detect the titer of polyclonal antiserum of immunized mice

[0034] Mouse brain neuroma cells (N2a) were cultured in a 96-well cell plate at 37°C and 5% CO2 incubator. When the cells adhered to the wall and grew to a density of about 80%, rabies virus was added to the N2a cells in each well at a multiplicity of infection MOI = 0.01, and the cells were placed in a 37°C incubator for 48 hours to allow the virus to infect the cells in the well as completely as possible. The supernatant of the virus-infected N2a cells was detoxified, and the cells were fixed with a fixative. After the fixation was completed, the liquid was discarded, and after the plate was completely air-dried, 200 μL / well of blocking solution was added, and the plate was placed in a 4°C refrigerator for overnight blocking. The blocking solution was discarded, and the cells were washed 5 times with PBST buffer. 100 μL of diluted serum was added to each well (serum and blocking solution were diluted at 1:200, 1:400, 1:800, and 1:1600), and N2a cells not infected with the virus were set as blank controls (Control); incubated at 37°C for 2 h, the primary antibody diluent was discarded, the cells were washed 5 times with PBST buffer, 50 μL / well (or 100 μL / well) of diluted FITC-labeled goat anti-mouse IgG antibody (antibody: blocking solution = 1:1500, volume ratio) was added, and the cells were incubated at 37°C in the dark for 1 h; the secondary antibody diluent was discarded, the cells were washed 5 times with PBST, the last washing solution was retained, and the cells were observed under a fluorescence microscope; the results are shown in the figure. Figure 1 ,It can be seen from the figure that the immune effect of mice No. 2, No. 3 and No. 5 is better, and the serum titer is at least 1:1600;

[0035] 3. Detection of polyclonal antibody titer of immunized mice by enzyme-linked immunosorbent assay (ELISA)

[0036] The antigen (pET-32a-RABV-N recombinant protein) was diluted to 10 μg / mL with the coating solution, and 100 μL was added to each well of the microplate, incubated at 37°C for 2 h (or incubated at 4°C overnight), the liquid in the microplate was discarded, washed 3 times with PBST, 200 μL of blocking solution was added to each well, and the wells were blocked at 37°C for 2 h; the blocking solution was discarded, and the wells were washed 5 times with PBST, and 200 μL of serum diluted in a gradient manner was added to the microwells in sequence (serum was diluted in a two-fold gradient of serum: 5% skim milk = 1:200, 1:400, for a total of 12 gradients), and non-immunized clean mouse serum was diluted as a control, and incubated at 37°C for 2 h (or incubated at 4°C overnight). The serum diluent was discarded, and the plates were washed with PBST for 5 times. Then, 50 μL of diluted HRP-labeled goat anti-mouse IgG antibody (antibody: 5% skim milk = 1:10000) was added to each well. After incubation at 37°C for 1 hour, the secondary antibody diluent was discarded. After washing with PBST for 5 times, 100 μL of TMB colorimetric solution was added to each well. The plates were colorimetric for 15 minutes at 37°C in the dark. Then, 50 μL of 2MH2SO4 was added to the microplate to terminate the reaction. The absorbance at OD450nm was measured by an enzyme reader. The results are shown in the table. Figure 2,The results showed that the titer of mouse serum reached 1:25600, but the immune effect of mice No. 2 and No. 5 was better;

[0037] 4. The IFA method was further used to detect the specific recognition ability of polysera from mice immunized with No. 2 and No. 5 against rabies virus proteins.

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

[0039] 5. Western blot detection of the specific recognition ability of sera from mice immunized with No. 2 and No. 5 for denatured rabies virus protein

[0040] Similarly, according to the instructions of PEI 40K Transfection Reagent, the rabies virus glycoprotein plasmid pLVX-RABV-G and the nucleoprotein plasmid Flag-RABV-N were transfected into 293T cells cultured in 6-well cell plates. After 48 h of culture, the cell supernatant was discarded, 200 μL RIPA cell lysis buffer was added to each well, and the cells were lysed at low temperature for 15-20 min. At the same time, the 293T cells without plasmid transfection were lysed as a control, and the cell lysate was collected and centrifuged at 4°C and 12000 rpm for 20 min. The supernatant was collected as the total cell protein sample, 5×SDS loading buffer was added, mixed, and heated in a 95°C metal bath for 10 min. After the sample cooled, SDS-PAGE electrophoresis was performed. After the electrophoresis, the concentrated gel was cut off, and the sponge pad, filter paper, NC membrane, protein gel, filter paper, and sponge pad were placed in a "sandwich" method and placed in a transfer instrument. The protein was horizontally transferred from the negative electrode to the positive electrode on the NC membrane, and the constant current was 160mA for 120min. After the transfer, the NC membrane was removed and placed in an incubation box of appropriate size, 5% skim milk powder was added, and the membrane was blocked at 37℃ for 2h. The blocking solution was discarded, and the membrane was washed 5 times with PBST, each time for 5min. The immune mouse polyclonal antibody serum was diluted at a ratio of 1:1000 and incubated with the membrane at 4℃ overnight (or blocked at 37℃ for 2h). The primary antibody diluent was discarded, and the membrane was washed 5 times with PBST, each time for 5min. The diluted HRP-labeled goat anti-mouse IgG antibody (antibody: 5% skim milk = 1:10000, volume ratio) was added and incubated at 37℃ for 1h. The secondary antibody diluent was discarded, and the membrane was washed several times with PBST. The ECL colorimetric solution was prepared for chemiluminescence imaging. Results are shown in Figure 4 The results showed that the polyantibodies from mice immunized with No. 2 and No. 5 could specifically recognize the 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 tissue materials for the subsequent construction of single-chain antibody gene library; they were killed by bleeding, and the spleens were removed. After mixing, they were fully ground and crushed in a glass tissue grinder, collected in EP tubes, and frozen at -80°C for later use.

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

[0043] 1. RNA extraction and reverse transcription

[0044] The spleen tissue fluid was taken out at -80℃, thawed at low temperature, and RZ lysis solution was added. Total RNA was extracted using RNAsimple Total RNA Kit from Tiangen Biotechnology Co., Ltd. The RNA concentration was determined using a UV spectrophotometer, and reverse transcription was performed according to the instructions of the Green Tag Mix and HiScriptⅡ1st Strand cDNA Synthesis Kit (+gDNA wiper) purchased from Novizan Biopharmaceutical Technology Co., Ltd. to obtain cDNA;

[0045] 2. Amplification of target fragment

[0046] Mouse antibody specific amplification primers were designed and synthesized. The primer sequences are shown in Table 1. The cDNA obtained by reverse transcription was used as a template for the first round of PCR reaction. The reaction system was shown in Table 2 and Table 3. The amplification conditions were shown in Table 4. The gene fragments of the heavy chain variable region and light chain variable region of the mouse antibody were amplified respectively. After the reaction, 1.5% agarose gel electrophoresis was performed. The results were as follows Figure 5 A, Figure 5B, the target band size is consistent with the sequence size of the antibody heavy chain and light chain variable region, 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 genes (VH1 and VH2)

[0051]

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

[0053]

[0054] The second round of PCR reaction was carried out with the recovered target fragment as template. The reaction system is shown in Table 5 and Table 6. The amplification conditions were the same as the first round of reaction procedures. In this round of amplification reaction, a linker sequence was added between the heavy chain variable region and the light chain variable region fragments. At the same time, SfiI and NotI restriction sites were added to the 5' and 3' ends of ScFv, respectively. The amplification results are shown in Figure 5C, 5D, the target band size also conforms to the size of the antibody heavy and light chain variable region sequence, and the DNA gel is also recovered, and after the concentration is determined, it is stored at -40°C for use. In order to ensure the integrity of the sequence diversity of the library constructed later, in this step, two rounds of PCR amplification will be performed as many times as possible 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 for the second round of PCR amplification of antibody heavy chain variable region genes (VH1 and VH2)

[0058]

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

[0060]

[0061] 3. Construction of single-chain antibody gene library and evaluation of library capacity

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

[0063] Take the pCANTAB-5E strain stored in the early stage of this experiment, thaw at low temperature, inoculate it on the LB plate containing 100μɡ / mL ampicillin resistance by streaking method, and culture it upside down at 37℃ overnight. Pick the monoclonal colony and expand the culture. Use the EndoFree Plasmid Midi Kit of Kangwei Century Biology to extract the plasmid. After measuring the concentration, prepare the reaction systems in Tables 7 and 8 according to the instructions of Takara's restriction enzymes SfiⅠ and NotⅠ. Because the reaction temperatures of the two restriction enzymes are different, first prepare the reaction system shown in Table 7, perform plasmid enzyme digestion at 50℃ for 3h, then add the components shown in Table 8 to the same reaction system, perform plasmid enzyme digestion at 37℃ for 3-4h, and after the reaction is completed, add 1 / 10 volume of 10×DNA Loading Buffer to terminate the reaction, and use 1% agarose gel for electrophoresis to observe the enzyme digestion results and recover the linearized pCANTAB-5E vector;

[0064] Table 7

[0065]

[0066] Table 8

[0067]

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

[0069] Table 9

[0070]

[0071] After the system is prepared, gently mix the components, centrifuge briefly, react at 50℃ for 35min, and then quickly place in an ice water bath for 5min, then directly transform TG1 competent cells, spread on 2×YT plates containing 0.4% glucose and ampicillin resistance, and invert and culture overnight at 37℃. Add 2mL of non-resistant 2×YT liquid culture medium to the surface of each plate with several monoclonal colonies. After the surface of the plate is wet, gently scrape the colonies with a coating rod, collect all the bacterial liquid in the same sterile 50mL centrifuge tube, and mix evenly; this connection and transformation step is performed 5 times in total, and 200 plates are coated. Add all the mixed bacterial solution to 800 mL of ampicillin-resistant 2×YT liquid medium, shake and culture at 37°C and 180 rpm for 4-6 hours, centrifuge at 5000g for 10 minutes at room temperature, discard the supernatant, resuspend the precipitate with 20 mL of 2×YT medium, mix evenly, take out 2 mL of bacterial solution and set aside at 4°C, add the same volume of 50% glycerol to the remaining bacterial solution, and store at -80°C to obtain the primary single-chain antibody gene library; at the same time, perform bacterial solution PCR to identify positive transformants, and the results are shown in Figure 6 The results showed that the accuracy of the library insertion fragments reached 73%, and the library capacity was 2.22×10 8 cfu / mL, and randomly selected some monoclonal colonies with the correct target fragment size and sent them to Qingke Biotechnology for sequencing. The sequencing results were compared and analyzed, and it was found that the sequences were different and the library diversity was relatively complete.

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

[0073] N2a cells were cultured in T75 cell culture flasks. After the cells adhered to the wall and the cell density was about 80%, rabies virus CVS strain and Flury strain were inoculated and cultured in a 37°C, 5% CO2 incubator for 3-4 hours. 20 mL of DMEM medium containing 2% FBS was replaced and cultured for 72 hours under the same conditions. The cell supernatant was collected and centrifuged at 4°C, 3000 rpm for 10 minutes to remove cell debris. The supernatant was collected and β-propiolactone was added to the supernatant at a volume ratio of 1:4000. The virus was inactivated at 4°C overnight, and the same volume of sterile PEG at a concentration of 40% was added. 8000 / NaCl solution, mix well and place at 4℃ for precipitation overnight, centrifuge at 4℃ and 7000rpm for 30min, discard the supernatant, resuspend the precipitate with 3-4mL PBS respectively to obtain concentrated and purified rabies virus CVS strain and Flury strain virus particles, determine the concentration and then divide into 1.5mL centrifuge tubes, and store at -80℃ for later use.

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

[0075] After the cells of the single-chain antibody gene library obtained in Example 2 were revived and activated, the culture medium was added to 400 mL of ampicillin-resistant 2×YT medium containing 1% glucose, and cultured at 37°C and 200 rpm until the logarithmic growth phase. 200 μL of helper phage M13K07 (titer of 3.9×10 12 pfu / mL), mixed well, incubated at 37°C without shaking for 30 min, then cultured at 37°C and 200 rpm for 30 min, then centrifuged at 3300 g for 10 min at room temperature, discarded the supernatant, and resuspended the cell pellet in 400 mL of 2×YT medium containing ampicillin resistance and kanamycin resistance; cultured at 37°C and 200 rpm overnight. The next day, centrifuge at 4°C, collect the supernatant, add 1 / 4 volume of sterile PEG8000 / NaCl solution, precipitate phage on ice, centrifuge at 4°C and 10800g for 30 min, discard the supernatant, resuspend the precipitate with sterile water, add 1 / 5 volume of PEG8000 / NaCl solution, precipitate phage on ice for at least 30 min, centrifuge at 4°C and 10800g for 10 min, discard the supernatant, resuspend the phage precipitate with PBS, centrifuge at 11600g for 10 min at room temperature to remove residual cell debris, collect the supernatant as the ScFv phage supernatant, and store at 4°C for subsequent bio-screening.

[0076] Take 10-20 μL of supernatant and use gradient dilution method and Amp resistance plate to screen phage monoclonal colonies for phage titer detection. The results (3×10 10 pfu) is the phage input amount (Input) of the first round of bio-panning.

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

[0078] The purified CVS virus particles in Example 3 were diluted to 20 μg / mL with the coating solution, added to the ELISA plate, and coated overnight at 4°C. The next day, the coating solution was discarded, 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 discarding the blocking solution and washing the plate, the excess liquid was patted dry on sterile paper, and 100 μL of the ScFv phage library enriched in Example 4 (i.e., the input of the first round was 3×10 10 pfu), incubate at room temperature with gentle shaking for 1-2h, discard the antibody library solution, wash 10 times with PBST, pat dry the excess liquid, add 50μL of 2M Gly-HCl (pH=2.2, containing 1mg / mL BSA) to each well, shake and elute at room temperature for 10min, then add 150μL of neutralizing solution to each well for rapid neutralization of the eluate, collect the neutralized solution, which is the first round of screening product; take out 10-15μL of the first round of screening product for titer detection, so as to calculate the phage output (Output) of this round of panning, that is, 4×10 6 pfu, the remaining screening products infect the TG1 bacterial solution in the logarithmic growth phase for 30 minutes, centrifuge at 3300g for 5 minutes, discard the supernatant, resuspend the cell pellet with 4-5mL of non-resistant 2×YT medium, spread all on the YTAG plate, and invert and culture overnight (12-16h) in a 37°C incubator. The next day, add 2-3mL of non-resistant 2×YT medium to the surface of the plate, gently scrape the single colony on the surface with a coating rod, collect all in a 50mL centrifuge tube, and centrifuge at 3300g for 5 minutes; discard the supernatant, resuspend the pellet with 5mL of non-resistant 2×YT medium, take 2mL of bacterial solution for expansion culture, and enrich with reference to the rescue and titer detection steps of the phage library in Example 4, and the enriched phage is used for the next round of biological screening. The biopanning steps of “adsorption-washing-elution-enrichment” were repeated for a total of 3 rounds. The antigen coating concentration was reduced to 15 μg / mL and 10 μg / mL in each subsequent round, and the number of plate washes was increased to 15 and 20 times. The preliminary screening of the anti-RABV ScFv phage library was completed. The screening results are shown in Table 10. The recovery rate of eluted phages after each round of panning increased from 1.33×10 -4 Increased to 5.11×10 -1 The enrichment factor 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 phages after three rounds of bio-panning;

[0079] Dot blot experimental operation steps: cut 2 NC membranes of appropriate size according to experimental requirements, press three circular holes of appropriate size on the membrane, and then cut the upper left corner of the membrane for marking. Take the purified CVS and Flury virus particles frozen at -80℃ and the supernatant of N2a cells that are not infected with the virus, take 20μL of each sample and spot it in the center of the circular hole of one NC membrane after thawing, and take 20μL of the above-enriched library phage and 15μL of auxiliary phage to spot it on another NC membrane, spot it several times, set up PBS control wells, and wait for the membrane to be completely air-dried. Place the membrane in an incubation box, add 5% skim milk, and block it at 37℃ overnight. Discard the blocking solution and wash the membrane 5 times with PBST, each time for 5 minutes. Pipette 2-3mL of the above-precipitated enriched library phage into the NC membrane and incubate at 37℃ for 2h. Discard the phage solution and wash the membrane 5 times with PBST, each time for 5 minutes. After diluting HRP-labeled Anti-M13 monoclonal antibody (1:5000, volume ratio) with 5% skim milk, add it to the NC membrane and incubate at 37°C for 1 hour. Discard the antibody diluent, wash the membrane 5 times with PBST, each time for 5 minutes, prepare ECL colorimetric solution, and perform chemiluminescence imaging. The results are shown in Figure 7 As shown in the figure, the results show that the phages in the screened library can specifically recognize rabies virus particles, and there are detectable phages in the enriched library.

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

[0081]

[0082] At the same time, 20 phage monoclonal colonies after the third round of panning were randomly selected, and the bacterial solution PCR was used to determine the gene fragment deletion of the ScFv library after panning. The results are as follows: Figure 8 As shown, the results showed that the insert fragment sizes of the 20 monoclonal strains were completely correct, and there was no insertion fragment missing, which indicated that the three rounds of specific panning did not cause the loss of the insert fragment. Instead, the positive strains in the primary library that were successfully connected to the vector and had the correct fragment insertion were screened and enriched.

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

[0084] After infecting TG1 cells with the third round of screening products, spread them on YTAG plates and incubate them inverted in a 37°C constant temperature incubator overnight; or take the third round of panning phage library frozen at -80°C, thaw it at low temperature, inoculate it on ampicillin-resistant 2×YT plates by plate streaking method, and incubate them inverted in a 37°C constant temperature incubator overnight. In the clean bench, randomly pick 100 monoclonal colonies in 1mL of ampicillin-resistant 2×YT medium, incubate at 37°C and 200rpm for 3-4h until the bacterial solution becomes turbid, and then add 10μL of a titer of about 1.5×10 12 pfu / mL of helper phage M13K07, after mixing, let stand at 37°C for 30min, then culture at 37°C and 180rpm for 30min, centrifuge at 3300g for 5min at room temperature, discard the supernatant, resuspend the precipitate in 1mL of 2×YT medium with ampicillin resistance and kanamycin resistance, and culture at 37°C and 180rpm overnight (10-12h); centrifuge at 3300g for 5min, take the supernatant for ELISA detection, the operation steps refer to Example 1, incubate the secondary antibody with anti-M13 (volume ratio 1:5000) monoclonal antibody diluted with 5% skim milk, and measure OD with an enzyme marker 450nm The absorbance value is as follows Fig. 9 As shown; the results showed that most of the screened monoclonal phages had a strong affinity for rabies virus particles, and then 15 monoclonal phages with strong affinity were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. After comparing and analyzing the sequencing results, 6 monoclonal phages with correct and complete single-chain antibody sequences were screened out, and they were named phage No. 1, phage No. 3, phage No. 6, phage No. 7, phage No. 9 and phage No. 1-9, and the affinity and neutralization activity of the 6 monoclonal phages were identified again;

[0085] 1. Use ELISA and Dot blot to identify the affinity of monoclonal phage

[0086] The purified rabies virus particles CVS and Flury were coated on the enzyme-linked immunosorbent assay plate and ELISA was performed. The operation steps were referred to Example 1. The secondary antibody was also incubated with anti-M13 (volume ratio 1:5000) monoclonal antibody diluted with 5% skim milk. The results are shown in Figure 1. Fig.10 As shown, phage No. 3 has the highest binding activity to rabies virus particles; Dotblot experiment verification was also performed, and the results are as follows Fig.11 As shown, monoclonal phage No. 3 can also specifically recognize rabies virus particles, and the results in Figure 11B show that the phage precipitation amount of phage No. 3 is moderate, but the recognition ability with rabies virus particles is higher, which is consistent with the results of ELISA.

[0087] 2. Western blot verification of monoclonal phage neutralization activity

[0088] The six phage monoclonal colonies were expanded and cultured. 8000 After precipitation and enrichment with NaCl, dialysis was performed to remove endotoxins, and the phage content was determined. 50 μg of monoclonal phage was mixed with 10 μL (calculated according to the infection multiplicity MOI = 0.01) of rabies virus, and DMEM culture medium was added to make up to 500 μL. At the same time, rabies virus (RABV) diluted in DMEM and normally cultured mouse brain neuroma cells (N2a) were set as controls. After vortex mixing, the mixture was incubated at 37°C for 2 hours. After the incubation was completed, the mixture was added to the mouse brain neuroma cells (N2a) cultured in a 12-well cell culture plate. After culturing for 4-6 hours, the corresponding monoclonal phage and DMEM culture medium mixture was replaced with 1 mL, and the serum concentration was adjusted to 2%. The culture was continued under the same conditions. After 48 hours of virus infection of the cells, the cell lysate was collected, and the rabies virus (RABV) nucleoprotein in the cells was detected by Western blot (the primary antibody was monoclonal antibody 1N1: Anti-RABV-N). The results are as follows Fig.12 As shown, the results showed that the viral protein content corresponding to phage No. 3 was the lowest and the corresponding quantitative statistical results were the same, which indicated that phage No. 3 had good neutralizing activity against rabies virus.

[0089] 3. IFA identification of monoclonal phage neutralizing activity

[0090] The monoclonal phage No. 3 was incubated with rabies virus and infected with N2a cells in the same manner as above. After 48 hours of cell culture, the cell supernatant was rendered harmless and a 4% paraformaldehyde fixative was added. The cells were fixed at room temperature for 15-30 minutes and the neutralization ability of the monoclonal phage against rabies virus was detected by IFA. The virus-infected N2a cells and the virus-uninfected N2a cells were incubated with monoclonal antibody 1N1 (Anti-RABV-N) as controls.

[0091] The results are as follows Fig.13 As shown, compared with the control, phage No. 3 also has good neutralizing activity, and the neutralizing ability increases with the increase of its concentration.

[0092] 4. The monoclonal phage No. 3 was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The nucleotide sequence obtained was compared and analyzed again. The sequence is shown in SEQ ID NO: 2. The single-chain antibody sequence is complete and correct. 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. Use of the single-chain antibody according to claim 1 in the preparation of anti-rabies virus drugs.

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