CN112321707B

CN112321707B - Specificity recognition antibody and detection kit of machupo virus

Info

Publication number: CN112321707B
Application number: CN202011092262.7A
Authority: CN
Inventors: 徐国东; 幸晓莹; 袁冰
Original assignee: Canvest Wuhan Biotechnology Co ltd
Current assignee: Canvest Wuhan Biotechnology Co ltd
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2021-07-23
Anticipated expiration: 2040-10-13
Also published as: CN112321707A

Abstract

The invention discloses a specificity recognition antibody and a detection kit of a machupo virus, which are prepared by the following method: immunizing a mouse by taking a pseudovirus as an immunogen, then performing a first round of specific binding screening on immune mouse serum by taking eukaryotic-expressed recombinant MACV GP1 protein as an antigen, taking a mouse spleen with high serum specific antibody titer, fusing with mouse myeloma cells, and continuing performing a second round of specific binding screening on the fused cells by taking eukaryotic-expressed recombinant MACV GP1 protein as an antigen to obtain a hybridoma cell strain capable of secreting an antibody specifically recognizing the Machupo virus (MACV); the pseudovirus is a VSV framework pseudovirus with MACV envelope protein. The antibody can be specifically bound to MACV, is different from different types of NW arenaviruses, reduces false positive and false negative, and improves detection accuracy.

Description

Specificity recognition antibody and detection kit of machupo virus

Technical Field

The invention relates to the technical field of virus diagnosis, in particular to an antibody and a detection kit capable of specifically identifying equine autumn wave virus in NW arenavirus.

Background

Pathogenic New World (NW) arenaviruses such as Machupo virus (MACV), junin virus (jun virus, JUNV), guanaretto virus (GTOV), Sabiavirus (SBAV) and charpy virus (Chapare virus, CHAPV) have caused severe hemorrhagic fever epidemics in brazil, borliia, argentina, venezuela, etc., and have caused a great fear in south american society. The lethality rate (35 percent) of the viruses is only second to virulent viruses such as Ebola, Marburg and the like, most of the viruses have no effective control means and are considered as biosafety fourth-level pathogens. In recent years, with the rapid movement of personnel and materials brought by the international economic development and cultural exchange, the viruses are rapidly spread from the americas to europe and asia, and pose a great threat to the global biosafety situation. At present, China is still in the primary stage on the diagnosis and prevention research of the viruses, and the society of China is very easy to cause panic due to the invasion of the viruses. Therefore, the establishment of methods for identifying and diagnosing pathogenic NW arenaviruses in the research of the layout of the pathogenic NW arenaviruses in China is particularly urgent and important for improving the capability of coping with new sudden virulent viruses in China.

Machupo virus (MACV) is an important member of pathogenic NW arenaviruses, causing borlivian hemorrhagic fever with a mortality rate of 10% to 35%. Because of no effective prevention and cure means, MACV is classified as the interpersonal infectious virus with the first kind of harm degree in China. The detection, research and related activities of the infectious materials need to be carried out in a four-level biosafety laboratory. Arenaviruses are double-segmented, negative-strand, ambisense RNA viruses whose genome comprises a large segment l (large) of about 7.2k and a small segment s (small) of about 3.5 k. The L fragment encodes the matrix protein Z and RNA polymerase L, while the S fragment encodes the nucleoprotein NP (nucleoprotein) and the envelope glycoprotein GPC (glycoprotinprecusor). The pathogenic NW arenavirus native host is a wild mouse, but with minor or no symptoms in its body. Humans become infected during work and life by contact with virus-carrying wild mice, or their secretions and excretions, contaminated food, virus-carrying aerosols, and the like. Some people (about 35%) gradually become more severe within 1-2 weeks after infection, showing symptoms of persistent fever, dehydration, renal insufficiency, hypotension, severe bleeding at multiple mucosal sites, neurological disorders, shock, and ultimately death from massive hemorrhage and coma.

Currently, methods for identifying and detecting pathogenic NW arenaviruses are mainly virus isolation, RT-PCR based on conserved regions of S fragments of the viral genome, Enzyme Linked immunosorbent assay (ELISA) based on serum NP-specific IgM and IgG, or ELISA based on viral NP antigen. However, most of these methods target highly conserved NPs in pathogenic NW arenaviruses, and in practical applications, the false positive rate due to poor specificity is likely to occur, and it is difficult to distinguish five pathogenic NW arenaviruses by a single method. Unlike NP, the GPC subunit GP1 is highly variable in pathogenic NW arenaviruses and relatively conserved between different plant types of the same virus, and is suitable as a target for immunodiagnostics.

Disclosure of Invention

The study aims to establish an immunological diagnosis method of MACV by taking MACV GP1 as a target, and provides reference and supplement for the identification and diagnosis method of MACV. Specifically, provided are an antibody capable of specifically recognizing Machupo virus (MACV) in NW (New world) arenavirus, a hybridoma cell line M88 capable of secreting a specific binding antibody against MACV, and a Machupo virus detection kit.

The technical scheme of the invention is detailed as follows:

in a first aspect, the present invention provides an antibody specifically recognizing machupo virus (MACV), which is prepared by the following method: immunizing a mouse by taking a pseudovirus as an immunogen, then performing a first round of specific binding screening on immune mouse serum by taking eukaryotic-expressed recombinant MACV GP1 protein as an antigen, taking a mouse spleen with high serum specific antibody titer, fusing with mouse myeloma cells, and continuing performing a second round of specific binding screening on the fused cells by taking eukaryotic-expressed recombinant MACV GP1 protein as an antigen to obtain a hybridoma cell strain capable of secreting an antibody specifically recognizing the Machupo virus (MACV); the pseudovirus is a VSV framework pseudovirus with MACV envelope protein.

The antibody is obtained by taking VSV framework pseudovirus with MACV membrane protein as an immunogen and using recombinant MACV GP1 protein as an antigen for screening.

Preferably, the antibody, the pseudovirus are prepared by the following method: constructing the MACV envelope protein gene GPC to a eukaryotic expression vector pCAGGS in an enzyme digestion linkage manner to obtain a recombinant vector pCAGGS-MACV GPC; and (3) transfecting the pCAGGS-MACV GPC to HEK293T cells by adopting a PEI transfection method, culturing for a period of time to complete transfection, infecting the transfected cells by using virus VSV delta G-GFP/VSV G, and collecting cell culture solution supernatant after infection is finished, namely the pseudovirus.

The MACV envelope protein gene GPC nucleotide sequence is preferably shown as SEQ ID NO.1, and is more favorable for expression of eukaryotic expression vectors through codon optimization. The pCAGGS (addgene) eukaryotic expression vector is a commercial expression vector, and MACV GP1 is recombinantly expressed by a 293F eukaryotic expression system. Eukaryotic expression is different from prokaryotic expression, the obtained protein glycosyl is completely modified, the protein structure is closer to membrane protein in a real state, and the subsequent acquisition of antibodies with special properties is facilitated.

The virus VSV Δ G-GFP/VSV G is a commonly used tool virus, and can be self-packaged according to the conventional technical knowledge and raw materials in the field, or can be directly purchased as a commercial product. HEK293T (ATCC) cells are commonly used tool cells for viral packaging.

Preferably, the preparation method of the above antibody and the eukaryotic expression recombinant MACV GP1 protein comprises the following steps:

(1) amplifying GP1 gene, inserting GP1 gene into a secretory eukaryotic expression vector in a gene homologous recombination mode to obtain recombinant plasmid; the nucleotide sequence of the GP1 gene is preferably shown as SEQ ID NO. 2;

(2) amplifying the recombinant plasmid by using escherichia coli DH5 alpha, extracting the plasmid by using an endotoxin-free plasmid large-extraction kit, and transfecting the recombinant plasmid to HEK293F cells by using PEI;

(3) and 7 days after transfection, collecting cell culture supernatant, carrying out affinity chromatography purification on the supernatant by adopting a Ni column, collecting elution components, and dialyzing to PBS to obtain the eukaryotic recombinant expression recombinant MACV GP1 protein.

In a second aspect, the invention provides a hybridoma cell strain M88, which is used for secreting monoclonal antibodies capable of identifying different NW arenaviruses and monoclonal antibodies capable of identifying McFabo viruses, and is deposited in China center for type culture Collection with the preservation number of CCTCC No. C2020189.

In a third aspect, the present invention also provides a monoclonal antibody (i.e., mAb) secreted by the hybridoma cell line M88.

In a fourth aspect, the present invention also provides a kit for detecting equine autumn wave virus (MACV), which contains the monoclonal antibody.

Preferably, the kit is used in combination with a TMB color development kit, and comprises the following components: a pore plate coated with the monoclonal antibody of the equine autumn wave virus, FBS confining liquid, washing liquid and HRP coat anti-mouse secondary antibody; the FBS sealing liquid is an aqueous solution with the FBS content of 5% by volume; the washing solution was a PBS solution containing 0.05% Tween20 by volume.

Compared with the prior art, the invention has the following beneficial effects:

the eukaryotic expression recombinant GP1 antigen used by the invention is highly variable in pathogenic NW arenaviruses, but is relatively conserved among different plant types of the same virus, so the eukaryotic expression recombinant GP1 antigen is very suitable to be used as a target antigen for immunodiagnosis, and can obviously distinguish MACV from other different types of NW arenaviruses. Eukaryotic expression ensures proper folding and conformation naturalness of GP1 protein after expression.

The monoclonal antibody secreted by the hybridoma cell strain M88 can effectively distinguish MACV from other pathogenic NW arenaviruses, and different strains of the same MACV are highly conserved, so that the occurrence of false positive results and false negative results is greatly reduced, and the detection accuracy is improved.

Preservation information:

mouse hybridoma cell line M88, deposited in China center for type culture Collection at 10/9/2020 with the deposition address: wuhan university preservation center 430072 with preservation number CCTCC No. C2020189.

Drawings

FIG. 1 shows the binding results of M88 monoclonal antibody to different antigens in example 3;

FIG. 2 shows the specific binding assay results of monoclonal antibody M88 for five NW arenaviruses GP 1;

FIG. 3 shows the results of specific binding assays of mAb M88 to five NW arenavirus GPCs.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

The techniques used in the following examples are, unless otherwise specified, conventional techniques known to those skilled in the art; the instruments, reagents, etc. used, unless otherwise specified in this specification, are publicly available to those of skill and research in the art.

Example 1 preparation of recombinant GP1 antigen

a) Firstly, MACV envelope protein gene GPC is obtained through whole gene synthesis, the gene sequence is shown in SEQ ID NO.1, and the MACV envelope protein gene GPC is constructed to a eukaryotic expression vector pCAGGS through enzyme digestion linkage, so that a recombinant expression vector pCAGG S-MACV GPC is obtained.

b) PCR was performed on GP1 gene by designing specific primers, the nucleotide sequence of GP1 gene is shown in SEQ ID NO.2, and GP1 was inserted into a secretory eukaryotic expression vector pCDNA3.1(Invitrogen) using a gene homologous recombination method to obtain a recombinant plasmid pCDNA3.1-CD5-GP1-His with secretory peptide CD5 and purification tag His.

c) The recombinant plasmid of step b) was amplified by E.coli DH5 α and plasmid extraction was performed according to the procedures described in the specification using endotoxin-free plasmid Mass extraction kit (product of TIANGEN Co., Ltd., cat # DP117), followed by transfection of the recombinant plasmid into HEK293F cells using PEI transfection method.

PEI: a polyethyleneimine. The PEI transfection method adopts the following reagent formula: PEI stock (100 μ M): 125mg of PEI powder was weighed out and dissolved in 50ml of 1 XHBS (pH7.4), filtered through a 0.2 μm filter and stored at 4 ℃ until use. 1 × HBS (pH7.4): 8.76g NaCl dissolved in 900ml ultrapure water, added with 20ml 1M HEPES, adjusted pH to 7.4, constant volume to 1L, filter (0.2 μ M filter membrane) and stored at 4 ℃ for use.

d) 7 days after transfection, cell culture supernatants were harvested. And (3) carrying out affinity chromatography purification on the supernatant by adopting a Ni column, collecting an eluted component, and dialyzing to PBS to obtain the eukaryotic recombinant expression GP1 antigen named MACV GP1 antigen.

Example 2 preparation of monoclonal antibodies targeting recombinant GP1

a) Pseudovirus packaging: transfecting the recombinant expression vector pCAGG S-MACV GPC prepared in the step a) of the example 1 into HEK293T cells by using a PEI transfection method; after 24 hours, the transfected cells were infected with the tool virus VSV Δ G-GFP/VSV G; after 36 hours, the cell culture supernatant was collected, which was a VSV framework pseudovirus with MACV membrane protein, designated VSV Δ G-GFP/MACV GPC, and used as an immunogen.

b) Will 10⁶FFU VSV Δ G-GFP/MACV GPC diluted in 100 μ l PBS, mixed with 100 μ l Freund's adjuvant, and mice were immunized by multiple injections. Immunizations were performed 2 weeks apart and 3 times total.

c) Serum specific antibody titer ELISA assay: taking orbital blood of a mouse after three times of immunization, standing for 30 minutes at room temperature, centrifuging and collecting supernatant to obtain serum; MACV GP1 antigen was coated onto ELISA plates and the serum after gradient dilution was used as primary antibody followed by routine ELISA assays for the first round of specific binding screening.

d) Fusing: taking mouse spleen with high serum GP1 specific antibody titer, fusing the mouse spleen with mouse myeloma cells, and performing HAT selective culture on the fused cells.

e) Screening: screening the fused cells by ELISA by taking MACV GP1 as an antigen; subsequently, cloning culture is carried out on the strong positive hole cells, and a second round of GP1 specific screening is carried out on the monoclonal cell supernatant, so as to obtain a candidate hybridoma cell strain M88.

f) Preparing an antibody: monoclonal cells M88 secreting GP1 specific antibodies were expanded in vitro and injected into the abdominal cavity of mice pre-stimulated with paraffin oil, and ascites were harvested 10 days later. And (3) carrying out affinity chromatography purification on the ascites by adopting a proteinA column, wherein an elution component is the monoclonal antibody M88 of GP1, which is called M88 monoclonal antibody for short.

Example 3 detection of different viruses by monoclonal antibody M88

The binding capacity of M88 monoclonal antibody was determined by ELISA using recombinant GP1 antigen, pseudoviral VSV Δ G-GFP/MACV GPC, and HeLa cells overexpressing MACV GPC, respectively, as antigens. The specific operation of ELISA was as follows:

A. antigen coating: recombinant GP1 antigen was coated in 96 well plates at 10. mu.g/ml, 100. mu.l/well; 10⁶FFU/ml VSV Δ G-GFP/MACV GPC coated in 100. mu.l/well 96-well plates; HeLa cells seeded in 96-well plates, transfected with 0.1. mu.g of pCAGGS-MACV GPC per well, 24hrs after which the supernatant was removed, fixed with 4% paraformaldehyde, and washed free of paraformaldehyde.

B. And (3) sealing: the wells were discarded and patted dry on filter paper, 200. mu.l of 5% FBS blocking solution was added to each well for 1-2hrs at room temperature.

C. Washing: the well was discarded and patted dry on filter paper. Add 200. mu.l of washing solution (PBS + 0.05% Tween20), let stand at room temperature for 5mins, discard the liquid, and pat dry on filter paper. This step was repeated 3 times.

D. Primary antibody incubation: serum was diluted in 10-fold gradients and 100. mu.l was added to each well. Incubate at room temperature for 2 hrs.

E. Washing: the well was discarded and patted dry on filter paper. Adding 200 μ l of washing solution, standing at room temperature for 5mins, discarding the solution, and patting dry on filter paper. This step was repeated 5 times.

F. And (3) secondary antibody incubation: HRP coat anti-mouse secondary antibody was diluted in PBS and 100. mu.l was added to each well. Incubate at room temperature for 1 h.

G. Washing: the well was discarded and patted dry on filter paper. Adding 200 μ l of washing solution, standing at room temperature for 5mins, discarding the solution, and patting dry on filter paper. This step was repeated 5 times.

H. Color development: adopting a TMB color development kit, and mixing the components in solution A: solution B was prepared at a ratio of 1:100, and 100 μ l was added to each well. After standing at room temperature for 5-15mins, the blue color slowly appeared in the wells.

I. Stopping the reaction: add 100. mu.l stop solution (1M H) to each well₂SO₄) The color in the wells turned from blue to yellow.

J. Reading: OD450 was measured on a microplate reader.

The results of the binding of the M88 monoclonal antibody to different antigens are shown in FIG. 1, and the results suggest that the monoclonal antibody M88 prepared by the method described in the patent has strong binding force to MACV GP1 subunit, and has good binding effect to MACV complete envelope protein over-expressed on the cell surface and VSV framework pseudovirus with MACV complete envelope protein.

Example 4 specific recognition assay of monoclonal antibody M88 against different NW arenaviruses

Monoclonal antibody M88 was tested for binding ability to five NW arenaviruses (MACV, JUNV, GTOV, SBAV and CHAPV) GP 1: (same procedure as in example 3).

The detection results are shown in FIG. 2, and the results show that: m88 has significant binding to MACV GP1, and little binding to JUNV, GTOV, SBAV and CHAPV GP 1. The M88 monoclonal antibody was suggested to be selective for binding to the GP1 subunit of five NW arenaviruses (MACV, JUNV, GTOV, SBAV and CHAPV), and specifically recognized for MACV.

Example 5

5. Detection of binding capacity of monoclonal antibody M88 to five NW arenavirus VSV framework pseudoviruses

The binding capacity of five NW arenaviruses (MACV, JUNV, GTOV, SBAV, and CHAPV) VSV framework pseudoviruses was tested as in example 3.

As MACV, JUNV, GTOV, SBAV and CHAPV are four-class pathogens and no live virus exists in China, the pseudo virus with the VSV framework is adopted as a substitute antigen to carry out specificity verification on the antibody.

The results are shown in fig. 3, which suggests: monoclonal antibody M88 has significant binding effect on MACV pseudoviruses, has almost no binding effect on JUNV, GTOV, SBAV and CHAPV pseudoviruses, and can distinguish MACV viruses from five NW arenaviruses.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

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<120> specific recognition antibody of machupo virus and detection kit

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Claims

1. A specific recognition antibody of Machupo virus (MACV), which is prepared by the following method: immunizing a mouse by using a pseudovirus as an immunogen, then performing a first round of specific binding screening on immune mouse serum by using eukaryotic-expressed recombinant MACV GP1 protein as an antigen, taking a mouse spleen with the highest serum specific antibody titer, fusing with mouse myeloma cells, and continuing performing a second round of specific binding screening on fused cells by using eukaryotic-expressed recombinant MACV GP1 protein as an antigen to obtain a hybridoma cell strain, wherein the hybridoma cell strain is preserved in China center for type culture collection with the preservation number of CCTCC No. C2020189, and can secrete an antibody for specifically recognizing Machupo virus (MACV);

the pseudovirus is a VSV framework pseudovirus with MACV envelope protein.

2. The antibody of claim 1, wherein the pseudovirus is prepared by: constructing the MACV envelope protein gene GPC to a eukaryotic expression vector pCAGGS in an enzyme digestion linkage manner to obtain a recombinant vector pCAGGS-MACV GPC; and (3) transfecting the pCAGGS-MACV GPC to HEK293T cells by adopting a PEI transfection method, culturing for a period of time to complete transfection, infecting the transfected cells by using virus VSV delta G-GFP/VSV G, and collecting cell culture solution supernatant after infection is finished, namely the pseudovirus.

3. The antibody according to claim 1, characterized in that the method for the preparation of the eukaryotic recombinant MACV GP1 protein comprises the following steps:

(1) amplifying GP1 gene, inserting GP1 gene into a secretory eukaryotic expression vector in a gene homologous recombination mode to obtain recombinant plasmid;

4. The hybridoma cell strain M88 is characterized in that the hybridoma cell strain M88 is used for secreting monoclonal antibodies capable of identifying equine autumn wave virus, and is preserved in China center for type culture Collection with the preservation number of CCTCC No. C2020189.

5. A monoclonal antibody secreted by the hybridoma cell line M88 of claim 4.

6. A kit for detecting Machupo virus (MACV), comprising the monoclonal antibody of claim 5.

7. The kit according to claim 6, for use in combination with a TMB color development kit, comprising the following components:

a well plate coated with the monoclonal antibody of claim 5, FBS blocking solution, washing solution, HRP coat anti-mouse secondary antibody;

the FBS sealing liquid is an aqueous solution with the FBS content of 5% by volume;

the washing solution was a PBS solution containing 0.05% Tween20 by volume.