CN114958774A - Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application - Google Patents

Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application Download PDF

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CN114958774A
CN114958774A CN202210493917.4A CN202210493917A CN114958774A CN 114958774 A CN114958774 A CN 114958774A CN 202210493917 A CN202210493917 A CN 202210493917A CN 114958774 A CN114958774 A CN 114958774A
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monoclonal antibody
rabies virus
rabies
antibody
phosphoprotein
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宋杰
李慧
张雪梅
吴忠香
杨燕
朱文兵
张志晓
姬桐
湛小草
赵桂芳
李俊
余越
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Institute of Medical Biology of CAMS and PUMC
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Abstract

The invention relates to an anti-rabies virus monoclonal antibody, a hybridoma cell strain secreting the same and application, and belongs to the technical field of cell engineering and immunology. The invention uses rabies virus as immunogen to prepare monoclonal antibody, and obtains anti-rabies virus phosphoprotein monoclonal antibody and anti-rabies virus glycoprotein monoclonal antibody. The monoclonal antibody obtained by the invention has no cross reaction with other proteins of rabies virus, other antigens and pathogens, has good specificity and sensitivity, has good application prospect in biological diagnosis including ELISA, Western-blot and immunofluorescence aiming at rabies virus, and further expands the application value of the antibody for resisting different epitopes of rabies virus.

Description

Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application
Technical Field
The invention belongs to the technical field of cell engineering and immunology, and particularly relates to a rabies virus resistant monoclonal antibody, a hybridoma cell strain secreting the same and application thereof.
Background
Rabies is an acute highly lethal infectious disease caused by Rabies Virus (RV) infection, which is commonly suffered by both human and animals, and human and almost all warm-blooded animals are susceptible to Rabies virus, and once the disease is dead by almost 100%, no specific therapeutic medicine exists until now. Around 5.9 million deaths worldwide per year are due to rabies, with the majority of cases occurring in developing countries. In recent years, rabies epidemic situation rises year by year, seriously harms the health of the whole people and is an important public health problem which is commonly concerned in the world.
Lyssavirus genus Rhabdoviridae (Rhabboviridae) lyssavirus genus (Lyssa virus) is a single-stranded negative-strand RNA virus. The rabies virus genome contains 5 Open Reading Frames (ORFs), which respectively encode 5 structural proteins, namely nucleoprotein, phosphoprotein, membrane matrix protein, glycoprotein and transcriptase large protein. Glycoprotein is currently considered to be the only rabies virus antigen capable of persistently inducing virus production of neutralizing antibodies, and is also the most extensively and deeply studied antigenic component. Phosphoprotein is a multifunctional protein that plays a role in viral escape, viral transcription replication, and intracellular movement. Researches show that abundant and effective antigen epitopes exist on the phosphoprotein, and more researchers continuously prove the possibility of the phosphoprotein serving as clinical serum detection antigen or superantigen of virus infection. Recently, it has been reported that nucleoprotein and phosphoprotein antigens of rabies virus also induce specific immune response. Therefore, the antigenic components of rabies virus play an indispensable role in participating in the immune response of the organism to rabies virus.
The anti-rabies virus monoclonal antibody is a favorable tool for researching rabies virus diagnosis methods and treating and preventing rabies. Since the monoclonal antibody of rabies virus was prepared by Wiktor and Koprowski for the first time in 1978, more and more laboratories have established monoclonal antibody hybrid strains of rabies virus, and have discussed the application values of monoclonal antibodies in the aspects of immune protection mechanism of rabies virus, development of novel vaccines, epidemiology, protein function, rabies virus infection diagnosis and the like. The research of rabies antibodies directed against different epitopes is also one of the main directions for the research of rabies antibodies. The monoclonal antibody has the incomparable advantages of high homogeneity, specificity and the like of conventional immune serum, becomes the optimal biological probe for detecting pathogeny and the antibody, and is widely applied to virus serological identification, diagnosis and treatment.
Currently, vaccination is the only method to prevent, control and eliminate rabies. The safety, effectiveness and quality stability of the rabies vaccine are key factors of the protective immune effect of the product. The antigen content is used as one of main indexes for evaluating the quality of rabies vaccines, so that a set of simple, convenient, rapid, specific and sensitive rabies virus antigen detection method is important in the research and production processes of rabies vaccines, can replace time-consuming and labor-consuming animal experiments, greatly improves the working efficiency, can rapidly and quantitatively detect the antigen content of each step in the vaccine preparation process in time, and is suitable for monitoring and controlling each stage of virus harvesting, concentration, inactivation, purification processes and the like, so that an internal control standard is established to guide production and quality control, and the consistency and stability among batches are ensured. Therefore, quantitative monitoring of the antigen content in the vaccine by a simple monitoring method has important significance for preventing the epidemic of the disease and establishing a scientific immune program.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a monoclonal antibody capable of being specifically combined with rabies virus phosphoprotein/glycoprotein and a preparation method of a hybridoma cell for producing the antibody aiming at the current situations that rabies still seriously harms the health of the whole population and various difficulties still exist in the prevention and treatment process. The monoclonal antibody has no cross reaction with other proteins of rabies virus and other antigens and pathogens, has the characteristics of high specificity and high sensitivity, can establish a novel, convenient and practical detection method, quantitatively detects the content of antigen components in rabies virus vaccines, is used for timely monitoring the virus content in the vaccine research and development and production processes, and has good application prospect.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a preparation method of a rabies virus resistant phosphoprotein/glycoprotein monoclonal antibody hybridoma cell strain, which comprises the following steps:
step (1), immunogen preparation: taking purified rabies virus as immunogen;
step (2), animal immunization: four immunizations were performed on 6-8 week old Balb/c mice at 2 week intervals, using the following protocol: the first immunization, after fully mixing and emulsifying Freund's complete adjuvant and equivalent immunogen, injecting subcutaneous multiple points on the back; the second immunization and the third immunization, the adjuvant is changed into Freund incomplete adjuvant, and the method and the dosage are the same as those of the first immunization; fourthly, injecting immunogen without adjuvant into the abdominal cavity, wherein the injection amount is the same as that of the first immunization; 3 days later, taking spleen cells of the immunized mice for preparing hybridoma cells;
and (3) preparing and screening hybridoma cell strains: and (3) performing cell fusion on SP2/0 cells in a logarithmic growth phase and spleen cells of the immunized mice in the step (2) under the action of PEG4000, culturing the fused hybridoma cells by using a 1 XHAT selective medium, and detecting cell supernatants by an enzyme-linked immunosorbent assay 10-14 days after cell fusion to screen a hybridoma cell strain capable of specifically secreting the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody.
Further, it is preferable that, in the step (2), the first immunization injection amount is 200ul 10 6 FFU/ml；
In the step (3), SP2/0 cells in logarithmic growth phase and spleen cells of the immunized mice in the step (2) are subjected to cell fusion under the action of PEG4000, and fused hybridoma cells are cultured by using a 1 XHAT selective medium, wherein the specific method comprises the following steps:
selecting 6-8-week-old Balb/c mice, and preparing feeder cells from abdominal macrophages of the mice; mixing SP2/0 cells in logarithmic growth phase with spleen cells of the immunized mouse in the step (2) according to the cell number ratio of 1:10, performing cell fusion by adopting polyethylene glycol 4000, adding hybridoma cells obtained after cell fusion into a culture plate containing feeder layer cells, and culturing by using a selective medium containing 1 XHAT at 37 ℃ under the condition of 5% CO 2;
also included are the cloning of hybridoma cells, specifically: performing subclone culture on the hybridoma cell strain by a limiting dilution method, continuously culturing for multiple times until the antibody in the cloning hole is detected to be 100% positive, performing amplification culture, and freezing and storing.
In a second aspect, the present invention provides an anti-rabies vims phosphoprotein/glycoprotein monoclonal antibody secreted by the hybridoma cell.
The third aspect of the invention provides an anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region of the anti-phosphoprotein monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region of the anti-glycoprotein monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6.
The fourth aspect of the invention provides a coding DNA of a rabies virus resistant phosphoprotein/glycoprotein monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region; the encoding DNA sequence of the light chain variable region of the anti-phosphoprotein monoclonal antibody is shown as SEQ ID NO.3, and the encoding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 4; the encoding DNA sequence of the variable region of the light chain of the anti-glycoprotein monoclonal antibody is shown in SEQ ID NO.7, and the encoding DNA sequence of the variable region of the heavy chain is shown in SEQ ID NO. 8.
The fifth aspect of the invention provides a double-antibody sandwich antigen detection kit for rabies viruses, which adopts the anti-rabies-virus phosphoprotein/glycoprotein monoclonal antibody, takes the anti-rabies-virus phosphoprotein monoclonal antibody as an envelope antibody, and takes an enzyme-labeled anti-rabies-virus glycoprotein monoclonal antibody as a detection antibody, or takes the anti-rabies-virus glycoprotein monoclonal antibody as an envelope antibody, and takes the enzyme-labeled monoclonal antibody phosphoprotein monoclonal antibody as a detection antibody.
Further, preferably, the enzyme is horseradish peroxidase or alkaline phosphatase.
The sixth aspect of the invention provides the use of the above-mentioned anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody in the preparation of a rabies virus vaccine antigen content detection reagent or kit, a rabies virus detection reagent or kit, and a rabies virus antibody detection reagent or kit.
The seventh aspect of the invention provides an application of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody in preparation of a rabies virus vaccine production product quality detection reagent or kit.
The eighth aspect of the invention provides an application of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody in preparation of a medicament for preventing or treating rabies.
In the kit of the present invention, the monoclonal antibody may be a labeled complex obtained by labeling with a biomarker or a chemical marker. The biomarker is enzyme label, and the enzyme is horseradish peroxidase or alkaline phosphatase.
The monoclonal antibody capable of identifying the rabies virus phosphoprotein and the monoclonal antibody capable of identifying the rabies virus glycoprotein have good specificity. The recognition proteins and the recognition sites among all clones have no cross reaction, and indirect ELISA shows that the two antibodies have higher sensitivity, so the antibody can be used for detecting antigens in rabies viruses and vaccines. In addition, the two monoclonal antibodies respectively recognize different antigenic determinants on different proteins of the rabies virus. Therefore, the double-antibody sandwich method can be adopted to respectively utilize two monoclonal antibodies which are specifically identified to carry out timely quantitative detection on the rabies virus antigen.
The double-antibody sandwich ELISA method constructed based on the method shows good sensitivity and specificity, can be used for timely monitoring rabies virus antigens in each stage in the vaccine production process, and can be used as an internal control standard for guiding the screening, determination and optimization of a preparation method and a process of a rabies virus vaccine.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
In the invention, the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody hybridoma cell strain is two strains of the anti-rabies virus phosphoprotein monoclonal antibody and the anti-rabies virus glycoprotein monoclonal antibody.
Compared with the prior art, the invention has the beneficial effects that:
based on the anti-rabies virus monoclonal antibody provided by the invention, different antigenic determinants on different proteins of rabies virus, including rabies virus phosphoprotein and rabies virus glycoprotein, are respectively identified, so that the possibility of applying the rabies monoclonal antibody aiming at different antigenic epitopes in the aspects of diagnosis, prevention and treatment of rabies is expanded, and a solid foundation is laid for the establishment of a detection quantitative method of rabies virus and the research of functions of different proteins.
At present, the rabies virus detection kit based on double antibodies is limited to rabies virus nucleoprotein and glycoprotein, and the rabies virus antibody detection kit based on the phosphoprotein monoclonal antibody is rarely reported. A hybridoma cell strain and a rabies virus phosphoprotein monoclonal antibody (201611157550.X) generated by the hybridoma cell strain are disclosed, the rabies virus phosphoprotein monoclonal antibody generated by the hybridoma cell strain is screened and applied to preparation of a rabies virus detection reagent, and the detection sensitivity can reach 0.1 IU/ml. The monoclonal antibody capable of identifying the rabies virus phosphoprotein and the monoclonal antibody capable of identifying the rabies virus glycoprotein both have good specificity, have no cross reaction with other rabies virus proteins, other antigens and pathogens, and have the advantages of high specificity and sensitivity when used for detection, so that a double-antibody sandwich method for quickly and quantitatively detecting the content of rabies virus antigens is established, the sensitivity (0.02IU/ml) of the rabies virus double-antibody sandwich ELISA antigen detection method is greatly improved, the method is particularly important for quality control and timely monitoring of the virus content in the development and production processes of rabies virus vaccines, and the method can be widely applied to vaccines and clinical detection and has important guiding significance for detection and prevention and control of rabies.
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FIG. 1 is an SDS-PAGE electrophoresis of the anti-rabies monoclonal antibody, wherein M is a protein molecular weight standard (KDa), lane 1 is the anti-rabies phosphoprotein monoclonal antibody 112E3, lane 2 is the anti-rabies glycoprotein monoclonal antibody 119A 4;
FIG. 2 is a Western blot result chart of the anti-rabies vims phosphoprotein monoclonal antibody 112E3, wherein M is a protein molecular weight standard (KDa), lane 1 is RV G protein, and lane 2 is rabies vims;
FIG. 3 is a graph showing the results of Western blot analysis of the anti-rabies glycoprotein monoclonal antibody 119A4, wherein M is a protein molecular weight standard (KDa); a is a multi-anti-pAb control, and b is an anti-rabies virus glycoprotein monoclonal antibody 119A 4;
FIG. 4 is a graph showing the protein recognition and localization results of the anti-rabies virus monoclonal antibody; a is rabies virus glycoprotein polypeptide scanning, and b is rabies virus phosphoprotein polypeptide scanning;
FIG. 5 is a PCR gel electrophoresis diagram of the light chain-heavy chain variable region of the anti-rabies vims phosphoprotein monoclonal antibody 112E3, wherein a is the light chain variable region, and b is the heavy chain variable region; m is a protein molecular weight standard (KDa), light chain PCR product 1 at 112E3 in lane 1 and light chain PCR product 2 at 112E3 in lane 2;
FIG. 6 shows PCR gel electrophoresis of the light chain-heavy chain variable region of anti-rabies glycoprotein monoclonal antibody 119A4, wherein a is the light chain variable region and b is the heavy chain variable region; m is a protein molecular weight standard (KDa), light chain PCR product 1 at 119a4 in lane 1 and light chain PCR product 2 at 119a4 in lane 2;
FIG. 7 shows the antigen detection specificity of the double antibody sandwich ELISA method established in the present invention;
FIG. 8 shows the results of the linear test and sensitivity of the double antibody sandwich ELISA method established in the present invention, wherein a is the linear test of the double antibody sandwich ELISA method, the abscissa is the titer (IU/ml) of the vaccine, and the ordinate is the OD450 values corresponding to vaccines with different titers; b is a sensitivity result, Human Serum Albumin (HSA) is used as a detection baseline, samples RV-1, RV-2 and RV-3 are virus harvesting solutions of different batches, and the final dilution reaches 2 by gradient dilution -7 The corresponding diluted reference vaccine is used as a quantitative standard to quantify the antigen content; the dotted line is the detection threshold (cutoff P/N)>2.1)。
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
The percentage numbers represent volume percentages and the ratios represent volume ratios, unless otherwise indicated.
1 × HAT selective medium: 50 XHAT (purchased from sigma), 20% (V/V) fetal bovine serum (purchased from Biological Industries), 1640 medium (purchased from Corning), 1% (V/V) diabody (penicillin-streptomycin solution);
1 × HT complete medium: (50 XHT (from sigma), 20% (V/V) fetal bovine serum (from Biological Industries), 1640 medium (from Corning), 1% (V/V) diabody (penicillin-streptomycin solution);
common complete medium: 20% (V/V) fetal bovine serum (purchased from Biological Industries), 1640 medium (purchased from Corning), 1% (V/V) diabody (penicillin-streptomycin solution).
Example 1 establishment of anti-rabies-Virus monoclonal antibody hybridoma cell line
1. Preparing immunogen: taking purified rabies virus (rabies virus CTN cell strain) as immunogen;
2. animal immunization: taking Balb/c mice with the age of 6-8 weeks, and carrying out four times of immunization at intervals of 2 weeks, wherein the procedures are as follows: the first immunization injection amount is 200ul 10 6 FFU/ml, Freund's complete adjuvant and equivalent immunogen fully mix and emulsify, subcutaneous multiple spot injection of back; the second immunization and the third immunization, the adjuvant is changed into Freund incomplete adjuvant, and the method and the dosage are the same as those of the first immunization; fourthly, injecting immunogen without adjuvant into the abdominal cavity, wherein the injection amount is the same as that of the first immunization; 3 days later, taking spleen cells of the immunized mice for preparing hybridoma cells;
3. preparation of hybridoma cell strain: the method comprises the following steps: 2 days before cell fusion, spleen cells of immunized mice are taken to prepare hybridoma cells. Selecting 6-8 weeks old Balb/c mice, taking abdominal cavity macrophages of the mice to prepare feeder cells, and enabling the cell concentration to be 10 5 Adding into 96-well plate (100 μ l per well), standing at 37 deg.C and 5% CO 2 Culturing under the condition for later use. SP2/0 cells (purchased from ATCC) in the logarithmic growth phase were mixed with spleen cells of the immunized mouse of step 2 at a cell number ratio of 1:10, and hybridoma cells obtained after fusion of 45% polyethylene glycol 4000(PEG) (purchased from sigma) cells were added to a 96-well plate already containing feeder cells, and cultured at 37 ℃ under 5% CO2 using a medium containing 1 XHAT selection.
4. Screening of hybridoma cell strains specifically comprises the following steps: the hybridoma cells obtained in step 3 were cultured in the presence of 1 XHAT selective medium for 10 to 14 days, and half of the 1 XHAT selective medium was replaced every 4 days. After 12 days of culture, the supernatant of the hybridoma cells was subjected to indirect ELISA to primarily screen for hybridoma cells that specifically secrete anti-rabies phosphoprotein/glycoprotein monoclonal antibody. The method comprises the following specific steps: respectively coating an enzyme label plate with purified rabies virus and RV G protein, setting a blank control, taking culture supernatant as a primary antibody, taking SP2/0 cell culture supernatant as a negative control, taking a secondary antibody which is HRP-labeled goat anti-mouse IgG (1:6000) (Thermo Scientific), TMB (Solarbio) for color development termination, detecting an absorbance value (A450 value) of 450nm by using an enzyme label instrument, taking a hole which is more than 2.1 times higher than the A450 value of the negative control as a positive cell hole, and carrying out subcloning in the next step.
Among them, RV G protein is an existing protein, and can be found in the prokaryotic expression and basic function evaluation of G protein of the CTN-1 strain of rabies virus [ J ]. J, J.Biometrics, 2016,29(10):1032-1036, DOI:10.13200/j.cnki.cjb.001471, Lei-Zhang-Xuanxi, Wu-faixiang, Lu-kongjie, Suming, and Dong Shao-fai.
5. Cloning of hybridoma cells, specifically: selecting the positive cell wells obtained by screening in the step 4, cloning the cells of the positive wells by adopting a limiting dilution method, sucking 200 mu l of cells in each well to a sample adding groove, diluting the cells to 20ml by using a 1 XHT complete culture medium, and uniformly adding the diluted cells to a new 96-well plate, wherein each well is 200 mu l, the temperature is 37 ℃, and the CO content is 5 percent 2 Culturing for 5 days under the condition, performing indirect ELISA detection, and selecting the cells with good growth state which are more than 2.1 times higher than the A450 value of the negative controlCell wells were cloned into another 96-well plate and cultured in 1 × HAT selective medium, and this was repeated three to four rounds until the antibody detection in the wells was 100% positive. Selecting cells with good growth state, gradually expanding and culturing by using a common complete culture medium, and freezing and storing by using liquid nitrogen.
In the process of subclone culture and screening, purified rabies virus and RV G protein are respectively used for coating an ELISA plate, hybridoma fine culture supernatant is taken for indirect ELISA after being closed, blank control is set, SP2/0 cell culture supernatant is negative control, goat anti-mouse IgG (1:6000) marked by HRP is used as a secondary antibody, monoclonal cells capable of identifying rabies virus phosphoprotein and glycoprotein are screened according to the reading of the value of A450 of the ELISA plate, and the hybridoma cell strains are further subjected to subclone and amplification culture.
As a result, the positive hybridoma cells are subcloned for 4 times continuously to obtain 2 cell strains which stably secrete the antibody, wherein the cell strains are numbered as RV 112E3 and RV 119A4, and the obtained hybridoma cell lines can be stably passaged and can continuously and stably secrete the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody.
EXAMPLE 2 Mass production and characterization of monoclonal antibodies against rabies phosphoprotein/glycoprotein
Mass preparation of rabies virus phosphoprotein/glycoprotein resisting monoclonal antibody
Taking 8-10 weeks old healthy Balb/c mice, injecting 500ul of liquid paraffin oil into the abdominal cavity, injecting the positive clone cells obtained by the expanded culture in the steps into the abdominal cavity of the mice after two weeks, wherein the injection amount of each mouse cell is 10 6 -10 7 And taking ascites samples after 10-14 days, and subpackaging for freezing or purifying. Centrifuging the mouse ascites at 8000rpm/min for 30 min, removing cell components and other precipitates, purifying the collected supernatant with Protein A column (purchased from whole formula gold), analyzing antibody purity and integrity with 12% SDS-PAGE, and as shown in figure 1, the heavy chain and light chain of 2 monoclonal antibodies are respectively about 55KD and 28KD, and the finally obtained mouse anti-rabies virus phosphoprotein/glycoprotein monoclonal antibodies are ultrafiltered and concentrated to 1mg/ml with a centrifuge tube, and are respectively numbered as 112E3 and 119A4, and are frozen for later use.
Second, biological characteristic analysis and identification of monoclonal antibody
1. Identification of monoclonal antibodies
The Mouse Monoclonal Antibody Isotyping Kit (Roche) was used to identify the type of Antibody, as described in the specification. The result proves that the Mab 112E3 is of IgG2b subtype, the Mab 119A4 is of IgG1 subtype, and the light chains are all of Kappa type.
2. Identification of monoclonal antibody specificity
Respectively taking purified RV G protein and rabies virus samples, adding a 5 xSDS-PAGE protein loading buffer solution [250Mm Tris-HCl (pH6.8), 10% (W/V) SDS, 0.5% (W/V) BPB, 50% (V/V) glycerol and 5% (W/V) beta-mercaptoethanol ] according to a ratio of 4:1, adding the sample, and heating the sample at 95 ℃ for 10 minutes to fully denature the protein, wherein the final concentration of the buffer solution is 1 x. And cooling to room temperature, and directly loading samples treated by the RV G protein and the rabies viruses into SDS-PAGE gel sample loading holes respectively. 80V for 30 min and 150V for 50 min. Using a semidry membrane transfer apparatus (BIO-RAD), a membrane transfer current of 100mA and a membrane transfer time of 45min were set, and the electrophoresed proteins were transferred onto a PVDF membrane (purchased from Millipore) according to the instructions of a membrane transfer apparatus. Immediately after the completion of the membrane transfer, the PVDF membrane subjected to the membrane transfer was placed in 5% (W/V) skimmed milk powder TBS [20mM Tris-HCl, 500mM NaCl (pH7.5) ] prepared in advance, and the mixture was gently shaken (40rpm/min) on a shaker and blocked at room temperature for 60 minutes. The purified monoclonal antibody was used as a primary antibody, which was diluted to 1:1000 with TBST membrane-washing solution [20mM Tris-HCl, 500mM NaCl (pH7.5) 0.01% Tween-20(V/V) ], and added to the PVDF membrane after completion of blocking for overnight incubation. Discard the primary anti-dilution solution. 5ml of TBST membrane washing liquid is added. Shaking on a side shaking table (speed 80rpm/min) was repeated 5 times. Followed by incubation at room temperature for 1h with HRP-labeled goat anti-mouse IgG as secondary antibody (diluted 1:10000 with TBST wash solution). The secondary antibody diluent was discarded, the membrane washing with TBST was repeated 5 times, ECL (purchased from Millipore) reagent was added, the plate was pressed in a dark room, and the plate was washed with developer fixer or machine-exposed to detect the monoclonal antibody recognizing effect. As a result, the 112E3 monoclonal antibody screened by ELISA for multiple times can specifically recognize the hydrophobin of rabies virus as shown in FIG. 2. WB identification was performed on the 119A4 monoclonal antibody by using Native-Page without denaturation, and the results are shown in FIG. 3, and the 119A4 and the polyclonal antibody can specifically recognize the natural rabies virus glycoprotein.
3. Monoclonal antibody protein recognition and localization
RV P protein and G protein segmented polypeptides (15-16 aa/peptide) were synthesized by Kunming Zanna Biotechnology, Inc., see Table 1. After all the polypeptides are dissolved, the polypeptides are respectively diluted to 10 mu g/ml by PBS buffer solution, each hole is coated with a 96-hole enzyme label plate in an amount of 100 mu l, and the polypeptide is incubated overnight at 4 ℃; discarding the solution in the wells, washing the plate for 3 times by 0.05 percent PBST, adding PBST of 5 percent skim milk powder into the enzyme-labeled wells in an amount of 200 mu l per well, incubating for 2h at 37 ℃, and sealing; discarding the solution in the wells, washing the plate for 3 times by using 0.05 percent PBST, diluting the obtained purified ascites monoclonal antibody to 10 mu g/ml by using PBS buffer solution, adding 100 mu l of the purified ascites monoclonal antibody into the coated reaction wells respectively, incubating for 1h at 37 ℃, and simultaneously making blank wells and negative control wells, wherein the blank wells are non-coated wells, and the negative control wells use the PBS solution to replace the ascites monoclonal antibody as a primary antibody. Discarding the well solution, washing the plate 3 times with 0.05% PBST, adding fresh diluted HPR-goat anti-mouse IgG (1:6000) to each reaction well, 100. mu.l per well, incubating at 37 ℃ for 1 h; the well solution was discarded, the plate was washed 3 times with 0.05% PBST, 100. mu.l of TMB substrate coloring solution was added to each well, and after 10min of color development in the dark at 37 ℃ 100. mu.l of 2M sulfuric acid was added to terminate the reaction, and the absorbance (A450 value) at 450nm was measured. The results are shown in FIG. 4, 112E3 can specifically recognize P3(NIEDNQAHLQGEPIE) of rabies virus phosphoprotein, 119A4 can specifically recognize rabies virus glycoprotein G15(RGLYKSLKGACKLKL), 112E3 and 119A4 respectively recognize different antigenic determinants on different proteins of rabies virus, and the recognition protein and the recognition site between clones have no cross reaction and are used for detection, so that the detection has the advantages of high specificity and sensitivity.
TABLE 1 RV P protein and G protein segmented polypeptide (15-16 aa/peptide)
Figure RE-GDA0003752531170000051
Figure RE-GDA0003752531170000061
4. Monoclonal antibodies 112E3 and 119A4 variable region sequencing
1) RNA extraction: extracting RNA of the screened hybridoma cell for identifying the rabies virus phosphoprotein/glycoprotein by TRNzol-A + (TIANGEN), wherein the cell number is 10 5 。
2) Reverse transcription to obtain cDNA: the obtained RNA was reverse-transcribed into cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Dalian).
3) Amplifying the heavy chain variable region of the antibody by nested PCR: the nested PCR amplification system is 56 ul:
PrimeSTAR Max premixM(Takara,Dalian))28ul；
5ul of cDNA obtained in the step 2);
VH Forward (2 pM per primer concentration): 1ul each, 4 in total, for a total of 4 ul;
VH Reverse (2 pM concentration per primer): 1ul each, 19 in total, for a total of 19 ul.
The amplification procedure was: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 deg.C for 10 seconds, renaturation at 65 deg.C for 10 seconds, extension at 72 deg.C for 10 seconds, and circulation for 35 times; extension at 72 ℃ for 5 minutes.
4) Nested PCR amplification of antibody light chain variable region: nested PCR amplification system 52 ul:
PrimeSTAR Max premixM(Takara,Dalian)26ul；
5ul of cDNA obtained in the step 2);
VL (kappa) Forward (2 pM per primer concentration): 1ul each, 4 in total, for a total of 4 ul;
VL (κ) Reverse (2 pM per primer concentration): 1ul each, 17 in total, for a total of 17 ul.
The amplification method comprises the following steps: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 deg.C for 10 seconds, renaturation at 60 deg.C for 10 seconds, extension at 72 deg.C for 10 seconds, and circulation for 35 times; extension at 72 ℃ for 5 min; the PCR primers are shown in Table 2.
5) Identifying the nested PCR product by agarose gel electrophoresis: sample loading: marker (DL2000), VH, VL; the results are shown in FIG. 5 and FIG. 6 using 1% agarose gel, 100mA, 20 min.
6) Antibody variable region sequencing and sequence analysis: and (3) sending the gel recovery product in the step 5) into a linked T vector to Tsingerke company for sequencing, comparing and analyzing the sequence by IgBLAST according to the sequencing result, and translating the obtained sequence into the amino acid sequence of the protein. The results are shown below, where the amino acid sequence of 112E3 includes the light chain variable region shown in SEQ ID No.1 and the heavy chain variable region shown in SEQ ID No. 2; the coding DNA sequence of the light chain variable region is shown in SEQ ID NO. 3; the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 4; 119A4 has an amino acid sequence comprising a light chain variable region as shown in SEQ ID No.5 and a heavy chain variable region as shown in SEQ ID No. 6; the coding DNA sequence of the light chain variable region is shown as SEQ ID NO. 7; the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the alignment of the sequences did not show the same sequence, indicating that the sequences obtained were specific to each of the 2 clones.
TABLE 2 PCR primers
Figure RE-GDA0003752531170000062
Figure RE-GDA0003752531170000071
Example 3 establishment of rabies virus double antibody sandwich ELISA antigen detection method
1. Establishment of rabies virus double-antibody sandwich ELISA antigen detection method
1) Coating: diluting 100 μ l/well of anti-rabies virus protein monoclonal antibody with 0.05M carbonate buffer solution with pH9.6, and coating overnight at 4 ℃;
2) washing: discard well solution, wash plate 3 times with 0.05% PBST;
3) and (3) sealing: adding sealing solution, i.e. sample diluent, into the mixture at a concentration of 200. mu.l/hole, and sealing the mixture at 37 ℃ for 2 hours;
4) washing: discard well solution, wash plate 3 times with 0.05% PBST;
5) adding an antigen: diluting the antigen with sample diluent, incubating at 37 deg.C for 1h at 100 μ l/well;
6) washing: discard well solution, wash plate 3 times with 0.05% PBST;
7) adding enzyme-labeled monoclonal antibody: adding horseradish peroxidase-labeled antibody into the reaction hole, incubating for 50min at the temperature of 37 ℃ at a concentration of 100 mu l/hole;
8) washing: discard well solution, wash plate 3 times with 0.05% PBST;
9) adding a substrate: developing TMB substrate color solution (from Solarbio)100 μ l in each well at 37 deg.C for 10min in dark;
10) adding a stop solution: the reaction was stopped with 100. mu.l of 2M sulfuric acid, and the absorbance (A450 value) at a wavelength of 450nm was measured.
2. Selection of coated monoclonal antibody and enzyme-labeled monoclonal antibody
The 2 anti-rabies virus monoclonal antibodies 112E3 and 119A4(10 mu g/ml) prepared by the invention are respectively used as coated antibodies to coat a 96-well enzyme label plate, a positive control (rabies virus sample) and a negative control (cell culture supernatant) are added to be detected, the 2 anti-rabies virus monoclonal antibodies are respectively marked and diluted to 0.5 mu g/ml by horseradish peroxidase, the operation is carried out according to the ELISA step, and the matched combination of the coated monoclonal antibodies and the enzyme-labeled monoclonal antibodies is selected according to OD 450.
3. Selection of working concentration of coating monoclonal antibody and enzyme-labeled monoclonal antibody
The combination of the anti-rabies virus phosphoprotein coated monoclonal antibody 112E3 and the HRP-labeled anti-rabies virus glycoprotein enzyme-labeled monoclonal antibody 119A4 was used to investigate the respective use concentrations of the monoclonal antibodies. 112E3, and respectively coating enzyme label plates, adding a positive control (rabies virus sample) and a negative control (PBS) as samples to be detected, diluting an enzyme-labeled antibody to 0.5 mu g/ml, and detecting according to the ELISA step; the detection working concentration of the enzyme-labeled monoclonal antibody 119A4 is determined by comparing the detection results of antibodies with different concentrations including 1, 0.5, 0.1 and 0.01 mu g/ml, the coating concentration of the selected coated monoclonal antibody 112E3 is 2.5 mu g/ml by comprehensive evaluation, and the working concentration of the enzyme-labeled monoclonal antibody 119A4 is 0.5 mu g/ml.
4. Experiment of specificity
The inactivated poliovirus type I (IPV-I), IPV-II, IPV-III, Hepatitis A Virus (HAV), Enterovirus (Enterovirus 71, EV71), rabies virus reference vaccine, rabies virus preparation sample and PBS are respectively detected according to the established double-antibody sandwich ELISA antigen detection method, and the rabies virus reference vaccine is used as a positive control, and the PBS is used as a negative control to evaluate the specificity of the method. The results show that the OD450 values of other 5 virus antigens detected by the double-antibody sandwich ELISA method established by the invention are all lower than the positive and negative critical values, namely the detection results are all negative, which indicates that the detection specificity of the method for rabies virus antigens is good, and the results are shown in figure 7.
5. Sensitivity and linear range testing
The rabies virus vaccine reference substances are respectively diluted to 2.0, 1.75, 1.5, 1.25, 0.625, 0.313 and 0.156IU/ml, experiments are carried out according to the rabies virus double-antibody sandwich ELISA antigen detection method in the example 3, and finally the linear result is shown in FIG. 8 a. Different batches of rabies virus harvest liquid RV-1, RV-2, RV-3 and rabies virus vaccine are diluted to 2 times in a gradient way -7 The initial concentration of the vaccine is 1.25IU/mL, the sensitivity of the method is 0.02IU/mL, the linear range is 0.15-2.0IU/mL, and as shown in FIG. 8b, the experimental result shows that the kit has good sensitivity and linear range.
The results show that the anti-rabies-virus glycoprotein/phosphoprotein monoclonal antibody has strong specificity and good sensitivity, can be used for detecting, preventing and treating rabies, investigating epidemic diseases and the like through further development, and has important practical significance. The double-antibody sandwich ELISA antigen detection method constructed based on the monoclonal antibody has no cross reaction to other epidemic virus antigens, has good specificity and sensitivity, can be used for quickly and quantitatively detecting the antigen content of each stage in the vaccine preparation process in time by establishing an internal control standard, is suitable for monitoring and controlling virus harvesting, concentrating, inactivating and purifying process steps, and provides a basis for screening, determining and optimizing a rabies virus vaccine preparation method and process.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (10)

1. The preparation method of the rabies virus phosphoprotein/glycoprotein monoclonal antibody hybridoma cell strain is characterized by comprising the following steps:
step (1), immunogen preparation: taking purified rabies virus as immunogen;
step (2), animal immunization: four immunizations were performed on 6-8 week old Balb/c mice at 2 week intervals, using the following protocol: for the first immunization, Freund's complete adjuvant and an equivalent immunogen are fully mixed and emulsified, and then are injected subcutaneously at multiple points on the back; the second immunization and the third immunization, the adjuvant is changed into Freund incomplete adjuvant, and the method and the dosage are the same as those of the first immunization; fourthly, injecting immunogen without adjuvant into the abdominal cavity, wherein the injection amount is the same as that of the first immunization; 3 days later, taking spleen cells of the immunized mice for preparing hybridoma cells;
and (3) preparing and screening hybridoma cell strains: and (3) performing cell fusion on SP2/0 cells in a logarithmic growth phase and spleen cells of the immunized mice in the step (2) under the action of PEG4000, culturing the fused hybridoma cells by using a 1 XHAT selective culture medium, and detecting cell supernatants by an enzyme-linked immunosorbent assay 10-14 days after cell fusion so as to screen hybridoma cell strains capable of specifically secreting the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody.
2. The method for preparing the rabies virus resistant phosphoprotein/glycoprotein monoclonal antibody hybridoma cell strain according to claim 1, wherein the first immunization injection amount in the step (2) is 200ul 10 6 FFU/ml；
In the step (3), SP2/0 cells in logarithmic growth phase and spleen cells of the immunized mice in the step (2) are subjected to cell fusion under the action of PEG4000, and fused hybridoma cells are cultured by using a 1 XHAT selective medium, wherein the specific method comprises the following steps:
selecting 6-8-week-old Balb/c mice, and preparing feeder cells from abdominal macrophages of the mice; mixing SP2/0 cells in logarithmic growth phase with spleen cells of the immunized mouse in the step (2) according to the cell number ratio of 1:10, performing cell fusion by adopting polyethylene glycol 4000, adding hybridoma cells obtained after cell fusion into a culture plate containing feeder layer cells, and culturing by using a selective medium containing 1 XHAT at 37 ℃ under the condition of 5% CO 2;
also included are the cloning of hybridoma cells, specifically: performing subclone culture on the hybridoma cell strain by a limiting dilution method, continuously culturing for multiple times until the antibody in the cloning hole is detected to be 100% positive, performing amplification culture, and freezing and storing.
3. An anti-rabies vims phosphoprotein/glycoprotein monoclonal antibody secreted by the hybridoma cell of claim 1 or 2.
4. The anti-rabies-virus phosphoprotein/glycoprotein monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region of the anti-rabies-virus phosphoprotein monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region of the anti-glycoprotein monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6.
5. A DNA encoding a rabies virus phosphoprotein/glycoprotein monoclonal antibody, comprising a heavy chain variable region and a light chain variable region; the encoding DNA sequence of the light chain variable region of the anti-phosphoprotein monoclonal antibody is shown as SEQ ID NO.3, and the encoding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 4; the encoding DNA sequence of the variable region of the light chain of the anti-glycoprotein monoclonal antibody is shown in SEQ ID NO.7, and the encoding DNA sequence of the variable region of the heavy chain is shown in SEQ ID NO. 8.
6. A double-antibody sandwich antigen detection kit for rabies viruses, which adopts the anti-rabies-virus phosphoprotein/glycoprotein monoclonal antibody of claim 4, is characterized in that the anti-rabies-virus phosphoprotein monoclonal antibody is used as a coating antibody, an enzyme-labeled anti-rabies-virus glycoprotein monoclonal antibody is used as a detection antibody, or the anti-rabies-virus glycoprotein monoclonal antibody is used as a coating antibody, and the enzyme-labeled monoclonal antibody phosphoprotein monoclonal antibody is used as a detection antibody.
7. The double-antibody sandwich antigen detection kit for rabies virus according to claim 6, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.
8. The use of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody of claim 4 in the preparation of a rabies virus vaccine antigen content detection reagent or kit, a rabies virus antibody detection reagent or kit.
9. The use of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody of claim 4 in the preparation of a rabies virus vaccine production product quality detection reagent or kit.
10. The use of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody according to claim 4 in the preparation of a medicament for preventing or treating rabies.
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Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030165507A1 (en) * 2000-05-16 2003-09-04 Hooper Douglas C. Rabies virus-specfic neutralizing human monoclonal antibodies and nucleic acids and related methods
CN103232975A (en) * 2013-04-03 2013-08-07 中国农业科学院油料作物研究所 Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody
CN103954777A (en) * 2014-05-20 2014-07-30 北京凯思百奥科技发展有限公司 Rabies virus monoclonal antibody and application thereof
CN105504051A (en) * 2015-12-17 2016-04-20 北京天成新脉生物技术有限公司 Rabies virus nucleoprotein monoclonal antibody and application thereof
KR20160074756A (en) * 2014-12-18 2016-06-29 대한민국(관리부서 질병관리본부장) Monoclonal antibody targeting rabies virus P protein or use for detection test of antigen and neutralized antibody
CN106632674A (en) * 2015-10-30 2017-05-10 中山康方生物医药有限公司 Anti-PD-1 (programmed cell death 1) monoclonal antibody as well as pharmaceutical composition and application thereof
CN106701687A (en) * 2016-12-15 2017-05-24 昆明理工大学 Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same
US20180105579A1 (en) * 2014-11-18 2018-04-19 Humabs Biomed Sa Antibodies that potently neutralize rabies virus and other lyssaviruses and uses thereof
CN108203716A (en) * 2016-12-20 2018-06-26 深圳市雅臣爱己生物工程有限公司 Anti- Pseudorabies virus pig source monoclonal antibody preparation method and its application
CN109970852A (en) * 2019-04-01 2019-07-05 浙江大学 Secrete hybridoma cell strain and the application of rabies poison M protein monoclonal antibody
CN110218704A (en) * 2019-05-21 2019-09-10 华中农业大学 A kind of monoclonal antibody and its application of rabies poison G-protein
CN110551212A (en) * 2019-09-29 2019-12-10 中国医学科学院医学生物学研究所 Preparation method and application of anti-GII.4 norovirus capsid protein VP1 and VLP (virus-like particle) monoclonal antibody
CN111139233A (en) * 2020-01-19 2020-05-12 中国医学科学院医学生物学研究所 Broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM monoclonal antibody and application
CN111999497A (en) * 2020-08-24 2020-11-27 中牧实业股份有限公司 Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application thereof
CN115260308A (en) * 2021-05-08 2022-11-01 南昌大学 High-affinity rabies virus-resistant fully-humanized monoclonal antibody and application thereof

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030165507A1 (en) * 2000-05-16 2003-09-04 Hooper Douglas C. Rabies virus-specfic neutralizing human monoclonal antibodies and nucleic acids and related methods
CN103232975A (en) * 2013-04-03 2013-08-07 中国农业科学院油料作物研究所 Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody
CN103954777A (en) * 2014-05-20 2014-07-30 北京凯思百奥科技发展有限公司 Rabies virus monoclonal antibody and application thereof
US20180105579A1 (en) * 2014-11-18 2018-04-19 Humabs Biomed Sa Antibodies that potently neutralize rabies virus and other lyssaviruses and uses thereof
KR20160074756A (en) * 2014-12-18 2016-06-29 대한민국(관리부서 질병관리본부장) Monoclonal antibody targeting rabies virus P protein or use for detection test of antigen and neutralized antibody
CN106632674A (en) * 2015-10-30 2017-05-10 中山康方生物医药有限公司 Anti-PD-1 (programmed cell death 1) monoclonal antibody as well as pharmaceutical composition and application thereof
CN105504051A (en) * 2015-12-17 2016-04-20 北京天成新脉生物技术有限公司 Rabies virus nucleoprotein monoclonal antibody and application thereof
CN106701687A (en) * 2016-12-15 2017-05-24 昆明理工大学 Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same
CN108203716A (en) * 2016-12-20 2018-06-26 深圳市雅臣爱己生物工程有限公司 Anti- Pseudorabies virus pig source monoclonal antibody preparation method and its application
CN109970852A (en) * 2019-04-01 2019-07-05 浙江大学 Secrete hybridoma cell strain and the application of rabies poison M protein monoclonal antibody
CN110218704A (en) * 2019-05-21 2019-09-10 华中农业大学 A kind of monoclonal antibody and its application of rabies poison G-protein
CN110551212A (en) * 2019-09-29 2019-12-10 中国医学科学院医学生物学研究所 Preparation method and application of anti-GII.4 norovirus capsid protein VP1 and VLP (virus-like particle) monoclonal antibody
CN111139233A (en) * 2020-01-19 2020-05-12 中国医学科学院医学生物学研究所 Broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM monoclonal antibody and application
CN111999497A (en) * 2020-08-24 2020-11-27 中牧实业股份有限公司 Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application thereof
CN115260308A (en) * 2021-05-08 2022-11-01 南昌大学 High-affinity rabies virus-resistant fully-humanized monoclonal antibody and application thereof

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
JIHY UM等: "Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT", 《PLOS NEGL TROP DIS.》, vol. 11, no. 12, pages 0006084 *
LIU, R等: "A novel rabies virus lipopeptide provides a better protection by improving the magnitude of DCs activation and T cell responses", 《VIRUS RESEARCH》, vol. 221, pages 66 - 73, XP029592966, DOI: 10.1016/j.virusres.2016.05.007 *
N DORFMAN等: "Development of human monoclonal antibodies to rabies", 《HYBRIDOMA》, vol. 13, no. 5, pages 397 - 402 *
PAN, Y等: "Expression and purification of rabies virus protein P: production and characterization of anti-P polyclonal antibodies", 《THAI JOURNAL OF VETERINARY MEDICINE》, vol. 48, no. 2, pages 257 - 264 *
严丽蔚;巩蔚;朱文兵;张雪梅;徐婧雯;吴忠香;卢孔杰;孙明;董少忠;: "狂犬病病毒糖蛋白表达及纯化及其记忆性B细胞结合能力的分析", 生物工程学报, no. 11, pages 1840 - 1849 *
严丽蔚等: "狂犬病病毒糖蛋白表达及纯化及其记忆性B细胞结合能力的分析", 《生物工程学报》, vol. 33, no. 11, pages 1840 - 1849 *
何肖芳: "抗狂犬病毒单克隆抗体的制备及胶体金试剂盒的建立", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》, no. 8, pages 006 - 392 *
巩蔚;严丽蔚;朱文兵;张雪梅;徐婧雯;吴忠香;卢孔杰;孙明;董少忠;: "狂犬病病毒CTN-1株G蛋白的原核表达及基本功能评价", 中国生物制品学杂志, no. 10, pages 29 - 33 *
巩蔚等: "狂犬病病毒CTN-1株G蛋白的原核表达及基本功能评价", 《中国生物制品学杂志》, vol. 29, no. 10, pages 1032 - 1036 *
米立娟等: "狂犬病病毒磷蛋白单抗的制备与应用", 《中国动物传染病学报》, vol. 22, no. 1, pages 1 - 2 *
隋欣: "狂犬病病毒G蛋白单克隆抗体的制备", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》, no. 2, pages 006 - 535 *

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