CN112159797B - Hybridoma cell strain 3G 71B 10, anti-GII.4 type norovirus P protein monoclonal antibody and application - Google Patents
Hybridoma cell strain 3G 71B 10, anti-GII.4 type norovirus P protein monoclonal antibody and application Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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Abstract
The invention discloses a hybridoma cell strain 3G 71B 10, a GII.4-type norovirus P protein monoclonal antibody and application. The preservation number of the hybridoma cell strain 3G 71B 10 is as follows: GDMCC No: 61139. the hybridoma cell strain 3G 71B 10 can secrete anti-GII.4 norovirus P protein monoclonal antibody. The monoclonal antibody which is marked by horseradish peroxidase and secreted by hybridoma cell strain 3G 71B 10 is used as a detection antibody, and the detection antibody is coded by a preservation number as follows: GDMCC No: 61138 hybridoma cell strain 3G 41D 6 secretes monoclonal antibody as capture antibody, and establishes double antibody sandwich ELISA detection method. The method has the advantages of simple and quick operation, clear result and the like, provides scientific basis for the research of the norovirus in-vitro diagnosis method, and has application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain 3G 71B 10, a GII.4 norovirus-resistant P protein monoclonal antibody and application thereof.
Background
Norovirus (Norovirus, NoV) has become a leading cause of acute nonbacterial gastroenteritis worldwide, infecting people of all ages, especially children under 5 years of age. NoV can be infected at low doses of about 100 viral particles per ml and are readily transmitted through food, water, and vomit and feces of infected persons. To meet public health needs, rapid, sensitive biosensing systems are needed to detect and prevent the spread of viruses.
NoV is a non-enveloped virus having a single positive-stranded RNA genome of about 7.5-7.7kb and belonging to the family Caliciviridae, genus Nore, with a high degree of variability in its genome. NoV consists of three open reading frames (ORF1, ORF2, ORF3), of which ORF2 encodes the major structural protein VP 1. The nucleotide sequence according to VP1 can be divided into GI-GVIII 7 gene groups (Genogroup), wherein the main distribution causing human infection is in the gene groups GI, GII and GIV, wherein GII.4 type has always been the global dominant epidemic. The major structural protein VP1 includes an N-terminal Shell domain S region (Shell domain) and a C-terminal outer protrusion domain P region (Protruding domain) connected by a hinge region, which contains the receptor binding element and the major antigenic site, since the P domain is located outside the viral particle. The P region without the hinge region can be independently expressed in vitro by an escherichia coli expression system to form P particles (P particles), the morphological structure, antigenicity, immunogenicity and receptor binding function of the P particles are very similar to those of natural virus particles, and the prokaryotic expression P particles have the characteristics of short expression period, high stability, simple operation and the like, are used as a substitute tool for researching NoV, and can meet the requirements of polyclonal antibody and monoclonal antibody preparation.
NoV the detection method comprises electron microscopy, molecular biology detection method and immunodetection method, wherein the immunodetection method is widely applied in clinical diagnosis, especially POCT field, such as colloidal gold immunochromatography test strip, enzyme-linked immunosorbent assay (ELISA), etc. The immunoassay method has the advantages of simple operation, short detection time and the like, is particularly suitable for clinical mass detection, but is necessary to detect various genotypes because the humanized norovirus has abundant genetic diversity, comprises three gene groups and more than thirty genotypes, and has new variant strains and causes pandemics every few years. The norovirus immunodetection method, the molecular biology detection method such as RT-PCR and the like lack cross reactivity for different genotypes, detection omission often occurs, and the requirement of clinical detection of multiple genotypes of the human norovirus cannot be met. Therefore, the screening and preparation of the monoclonal antibody with wide cross reactivity for various NoV genotypes is particularly important for establishing an immunodetection method and has important significance for early warning, prevention and control research of different genotypes.
Disclosure of Invention
The first purpose of the invention is to provide two hybridoma cell strains for producing monoclonal antibodies against GII.4 type norovirus P protein, which are respectively: hybridoma cell line 3G 41D 6 and hybridoma cell line 3G 71B 10, wherein the preservation number of the hybridoma cell line 3G 41D 6 is as follows: GDMCC No: 61138; the accession number of the hybridoma cell line 3G 71B 10 is: GDMCC No: 61139.
the second purpose of the invention is to provide two monoclonal antibodies against GII.4 type norovirus P protein, which are secreted and generated by the hybridoma cell line 3G 41D 6 and the hybridoma cell line 3G 71B 10 respectively, and the indirect ELISA titer of the two monoclonal antibodies is 10 -6 The aboveThe subtype of the monoclonal antibody secreted by the hybridoma cell line 3G 41D 6 is IgG2a, the subtype of the monoclonal antibody secreted by the hybridoma cell line 3G 71B 10 is IgG1, and both monoclonal antibodies can be specifically combined with GII.4 type norovirus P protein in ELISA and Western-blot detection.
The third purpose of the invention is to provide the application of the hybridoma cell strain 3G 41D 6 and the hybridoma cell strain 3G 71B 10 in the production of the anti-GII.4 norovirus P protein monoclonal antibody.
The fourth purpose of the invention is to provide the application of the anti-GII.4 type norovirus P protein monoclonal antibody secreted by the hybridoma cell strain 3G 41D 6 and the hybridoma cell strain 3G 71B 10 in preparing a detection reagent for different genotype P proteins of norovirus.
The monoclonal antibody for resisting the GII.4 type norovirus P protein can have immunoreaction with samples of common genotypes of norovirus GII.2, GII.3, GII.4, GII.6 and GII.17, and has no cross reaction with rotavirus, astrovirus, enterovirus and Sapovirus.
The fifth purpose of the invention is to provide a kit, which comprises an anti-GII.4 type norovirus P protein monoclonal antibody secreted by a hybridoma cell line 3G 41D 6 and a monoclonal antibody secreted by a hybridoma cell line 3G 71B 10 and marked by horse radish peroxidase.
The sixth purpose of the invention is to provide a double-antibody sandwich ELISA detection method for different genotype P proteins of norovirus, which uses an anti-GII.4 type norovirus P protein monoclonal antibody secreted by a hybridoma cell strain 3G 41D 6 as a capture antibody, and uses a monoclonal antibody labeled by horseradish peroxidase and secreted by a hybridoma cell strain 3G 71B 10 as a detection antibody.
Preferably, in the above-mentioned double-antibody sandwich ELISA detection method, the capture antibody is coated on the ELISA plate, the optimal coating concentration of the capture antibody is 2 μ g/mL, and the working concentration of the detection antibody is 1: 5000.
Compared with the prior art, the invention has the following beneficial effects:
1. the two hybridoma cell strains can stably secrete antibodies, the two secreted monoclonal antibodies have good pairing effect, and the method is suitable for various double-antibody sandwich detection methods.
2. Aiming at the clinical excrement sample detection of GII type norovirus, the kit is characterized by aiming at all current norovirus epidemic strains, and having good cross reactivity, strong specificity, high sensitivity and good stability. The method overcomes the defect of narrow coverage of the detection method generally caused by complicated norovirus typing.
3. Simple operation and clear result, and is suitable for clinical on-site diagnosis and large-scale epidemiological investigation. And the method can also be used in institutions with norovirus detection requirements such as medical health, inspection and quarantine, and the like, and the corresponding scientific research field.
The Mus musculus hybridoma 3G 41D 6 of the present invention was deposited in Guangdong province culture Collection (GDMCC) at 8/12/2020 with addresses: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC NO: 61138.
the Mus musculus hybridoma 3G 71B 10 of the present invention was deposited in Guangdong province microorganism culture Collection (GDMCC) at 8/12/2020 with addresses: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC NO: 61139.
drawings
FIG. 1 shows PCR amplification of GII.4 type P fragment (A) and PCR identification of recombinant plasmid pGEX-4T-1-GII.4-L307-P colony (B).
FIG. 2 shows SDS-PAGE (A) of purified GST-P binding protein and SDS-PAGE (B) of GST-tagged P particles.
FIG. 3 is SDS-PAGE of the purification of two monoclonal antibodies (3G 71B 10 and 3G 41D 6).
FIG. 4 shows the specificity of two monoclonal antibodies (3G 71B 10 and 3G 41D 6) detected by Western-Blot.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The following examples are given without specifying the particular experimental conditions and methods, and the technical means employed are generally conventional means well known to those skilled in the art.
Example 1 preparation of hybridoma cell lines and monoclonal antibody obtaining
Preparation of GII.4 norovirus P protein immunogen
The amplification primers are designed according to the P region of the GII.4-2012 type GZ2014-L307 strain (GenBank KT202798), the BamHI and EcoRI cutting sites (underlined parts) are respectively introduced into the upstream primer P1 and the downstream primer P2, and CDCRGDCFC amino acid sequences (italic parts) are added to the 5' end of the downstream primer, so that the formation of norovirus P particles can be promoted, as shown in the table 1. PCR amplification is carried out by taking GII.4-2012 GZ2014-L307 strain cDNA as a template, and BamHI and EcoRI double enzyme digestion is respectively carried out on an amplification product and a pGEX-4T-1 vector (GE general medicine, USA). Connecting the amplified product to pGEX-4T-1 vector, transforming to escherichia coli BL21 competent cells, plating, and then carrying out colony PCR and sequencing verification. The recombinant plasmid (pGEX-4T-1-GII.4-L307-P) is subjected to prokaryotic expression, and the expression condition of the recombinant protein is identified by SDS-PAGE electrophoresis. The PCR amplification of GII.4 type P fragment and the PCR identification of recombinant plasmid pGEX-4T-1-GII.4-L307-P colony are shown in FIG. 1. The recombinant fusion protein was purified using a GST affinity purification column, and after purification, the GST tag was cleaved with Thrombin (Thrombin). The buffer was changed to 0.01M PBS by centrifugation for 3 salt changes, purified again using GST affinity column and the flow-through was collected. The recombinant protein was 62kDa in size as analyzed by SDS-PAGE electrophoresis. After cleaving off the GST tag protein by Thrombin, the desired protein (P protein antigen) of about 36kDa was obtained, consistent with the expected size (FIG. 2).
TABLE 1 primer sequences for amplification of GII.4-2012 type norovirus P particles
2. Immunization of mice
Selecting female Balb/c mice about 8 weeks, mixing and emulsifying a solution containing 50 mu g of protein P antigen and an equal volume of Freund complete adjuvant into a water-in-oil state for first immunization, fully mixing and emulsifying the antigen and the Freund incomplete adjuvant with equal doses for boosting immunization for the first immunization, wherein the immunization mode is subcutaneous multi-point injection. Boosting immunization for three times, wherein each immunization is separated by 2 weeks, then blood collection is carried out, the serum titer is measured by an indirect ELISA method, and mice with the titer higher than 1:10000 are selected to carry out intraperitoneal impact at the dose of 50 mu g P protein antigen/mouse within 1 week, and no adjuvant is used at this time.
3. Cell fusion and selection
After 3 days of abdominal shock in mice, the eyes were removed and sacrificed, and positive blood was collected, spleens were removed and prepared into single cell suspensions, and splenocytes were fused with SP2/0 cells (mouse myeloma cells) at log phase with 50% polyethylene glycol (PEG 1450). And cloning and screening the fused hybridoma cells by a conventional method to finally obtain hybridoma cell strains which grow well and can stably secrete antibodies, wherein the hybridoma cell strains are hybridoma cell strains 3G 41D 6 and 3G 71B 10 respectively. Hybridoma cell lines 3G 41D 6, 3G 71B 10 were deposited at the guangdong province collection of microorganisms (GDMCC) at 12 months 8 in 2020, addresses: the Guangzhou city Pielizhou No. 100 college No. 59 floor 5, wherein the preservation number of the hybridoma cell strain 3G 41D 6 is as follows: GDMCC NO: 61138; the preservation number of the hybridoma cell strain 3G 71B 10 is as follows: GDMCC NO: 61139.
4. preparation and purification of monoclonal antibodies
Injecting liquid paraffin into abdominal cavity one week before Balb/c mouse immunization, selecting 10% fetal calf serum culture medium for expanding culture after cell strain determination, and culturing when cell density reaches 1 × 10 6 -2×10 6 at/mL, the cells were centrifuged at 1000rpm, and the cell pellet was collected and resuspended in PBS and injected into the abdominal cavity of mice. Ascites is collected after 10 days, Protein G affinity chromatography column is selected for purification, two anti-GII.4 type norovirus P Protein monoclonal antibodies secreted by hybridoma cell 3G 41D 6 and hybridoma cell 3G 71B 10 are respectively obtained and named as 3G 41D 6 monoclonal antibody and 3G 71B 10 monoclonal antibody, the subtype of the monoclonal antibody (3G 41D 6 monoclonal antibody) secreted by hybridoma cell strain 3G 41D 6 is identified as IgG2a type, and the subtype of the monoclonal antibody (3G 71B 10 monoclonal antibody) secreted by hybridoma cell strain 3G 71B 10 is identified as IgG1 type. SDS-PAGE analysis shows that the monoclonal antibody of 2 strains has purityThe total content of the monoclonal antibody is more than 90%, and two bands are obvious at about 25 kD and 50kD, namely a monoclonal antibody light chain and a monoclonal antibody heavy chain, and WB experiments show that the monoclonal antibody light chain and the monoclonal antibody heavy chain both have obvious single target bands. SDS-PAGE of the two monoclonal antibodies (3G 41D 6 monoclonal antibody, 3G 71B 10 monoclonal antibody) is shown in FIG. 3. The reactivity of the two antibodies with the P protein antigen is verified by Western-Blot, and the result shows that both antibodies have obvious single target bands, as shown in FIG. 4.
5. Evaluation of monoclonal antibodies
The antibody concentration is quantitatively determined by NanoDrop 2000 protein, then an indirect ELISA method is applied, 1 mu G/mL of GII.4P particles are used as coating antigen, 5% skimmed milk powder is closed, 2 monoclonal antibodies (3G 41D 6 monoclonal antibody and 3G 71B 10 monoclonal antibody) which are diluted by fold by PBS solution are used as primary antibodies, horseradish peroxidase (HRP) marked goat anti-mouse IgG is diluted by 1:3000 times and used as secondary antibodies, the OD450 value is determined after TMB color development, the P/N value (P is the value of the OD450 of a detection sample, N is the value of a negative control OD 450) is calculated, and the minimum dilution degree that the P/N is more than 2.1 is the antibody titer. And determining the reaction condition of the 2 monoclonal antibodies and NoV samples with different genotypes by using the same indirect ELISA method and using GI.6, GII.2, GII.3, GII.4-2012, GII.6 and GII.17 positive samples as coating antigens. The concentrations of 3G 41D 6 and 3G 71B 10 antibodies were 2.2mg/mL and 5.5mg/mL, respectively, as determined by NanoDrop 2000. The titers of the purified 3G 41D 6 and 3G 71B 10 antibodies are respectively 10 by indirect ELISA assay -6 The above. The 2 antibodies all show broad-spectrum binding activity, and the reaction results with GII.2, GII.3, GII.4, GII.6 and GII.17 samples are positive, and the results are shown in Table 2.
TABLE 2 evaluation of monoclonal antibody reaction with norovirus samples
Example 2 establishment of double antibody Sandwich ELISA method
1. Paired antibody screening and optimal working concentration determination
Firstly, 2 monoclonal antibodies are labeled according to the specification of a horseradish peroxidase (HRP) coupling labeling kit, the sensitivity of the labeled antibodies is measured by adopting direct ELISA, and the detection antibodies are determined. The optimal pairing antibody and the optimal working concentration are determined by using a double-antibody sandwich ELISA method, using 1 mu G/mL of GII.4P particles as detection antigens, using determined HRP-labeled monoclonal antibodies (HRP-3G 71B 10 and HRP-3G 41D 6) as detection antibodies, and using the other two monoclonal antibodies (3G 71B 10 and 3G 41D 6) as capture antibodies to pair with the detection antibodies. The results showed that the sensitivity of the HRP-labeled 3G 71B 10 antibody (HRP-3G 71B 10) was optimal, and therefore HRP-3G 71B 10 was selected as the detection antibody. The matched antibody screening result shows that the HRP-3G 71B 10 and 3G 41D 6 are matched better. The antibody is diluted in a checkerboard gradient way, the working concentration of the detection antibody HRP-3G 71B 10 is determined to be 1:5000, and the optimal coating concentration of the capture antibody 3G 41D 6 is determined to be 2 mug/mL.
2. Drawing of standard curve and determination of lowest detection limit
The OD450 values were read using a double antibody sandwich ELISA method to detect the gradient dilution of P-granule protein (0.125, 0.25, 0.5, 1, 2. mu.g/mL). Taking P-granular protein concentration (mu g/mL) as an abscissa and OD450 as an ordinate, drawing a standard curve, calculating a P/N value, judging that the P/N is positive when the P/N is more than 2.1, and obtaining good linearity in the range of 0.125-2 mu g/mL according to the curve, wherein the standard curve equation is that y is 1.8032x +0.2695, R is 2 0.9938, the lowest detection limit was 125 ng/mL.
Example 3 Performance testing of the method
The performance test of the double-antibody sandwich ELISA method established in example 2 is as follows:
1. specificity test
The method is used for detecting the reactivity of the diarrhea virus, such as rotavirus, astrovirus, enterovirus and Saporo virus, and the result shows that the method has no cross reaction with the diarrhea virus, such as rotavirus, astrovirus, enterovirus and Saporo virus.
2. Repeatability test
Enzyme label coated with 3G 41D 6 antibody in the same batch and different batchesPlate, 3 GII norovirus positive samples were tested in batch and in batch, each 3 replicates, using the formula for coefficient of variation
And calculating the variation coefficient between batches and in batches. The results show that the inter-batch and intra-batch coefficient of variation CV is less than 7% (see Table 3), which indicates that the three batches have better repeatability.
TABLE 3 results of the repeatability tests
3. Clinical sample testing
And simultaneously, the established double-antibody sandwich ELISA method and the fluorescent quantitative RT-PCR method are applied to detect 24 clinical excrement samples, the results are shown in table 4, wherein 13 positive samples are detected by the fluorescent quantitative RT-PCR method, 11 positive samples are detected by the double-antibody sandwich ELISA method in the 13 positive samples, and the rest results are negative. The positive match rate of the two is about 84.6%, and the results are shown in Table 4.
TABLE 4 double antibody sandwich ELISA and fluorescent quantitative RT-PCR detection results
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (9)
1. A hybridoma cell line 3G 71B 10, having a accession number of: GDMCC No: 61139.
2. a monoclonal antibody against GII.4-type norovirus P protein, which is produced by the hybridoma cell line 3G 71B 10 according to claim 1.
3. The use of the hybridoma cell line 3G 71B 10 of claim 1 for producing a monoclonal antibody against GII.4 norovirus P protein.
4. Use of the monoclonal antibody against the P protein of gii.4 norovirus of claim 2 for preparing a reagent for detecting P proteins of different genotypes of norovirus; the different genotypes of the norovirus include GII.2, GII.3, GII.4, GII.6 and GII.17.
5. The use of claim 4, wherein the detection reagent detects the expression of the P protein of different genotypes of norovirus in the sample by ELISA.
6. A kit comprising the anti-gii.4 norovirus P protein monoclonal antibody of claim 2.
7. The kit of claim 6, further comprising a peptide encoded by the accession number: GDMCC No: 61138 hybridoma cell line 3G 41D 6.
8. A double antibody sandwich ELISA detection method of different genotype P proteins of norovirus for non-disease diagnosis and treatment purposes is characterized in that a monoclonal antibody against GII.4 type norovirus P protein, which is labeled with horseradish peroxidase, is used as a detection antibody, and the detection antibody is represented by the following accession number: GDMCC No: 61138 hybridoma cell line 3G 41D 6 secretion of monoclonal antibody as capture antibody; the different genotypes of the norovirus include GII.2, GII.3, GII.4, GII.6 and GII.17.
9. The detection method according to claim 8, wherein the capture antibody is coated on an enzyme label plate, the optimal coating concentration of the capture antibody is 2 μ g/mL, and the working concentration of the detection antibody is 1: 5000.
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