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Recombinant virus and application thereof in mumps virus neutralizing antibody detection

A mumps virus, recombinant virus technology, applied in the direction of virus/phage, application, virus, etc., can solve the problems of low detection efficiency, manual judgment, high professional requirements of experimenters, etc., and achieve the effect of accurate detection results.

Active Publication Date: 2022-02-01
BEIJING CELL FUSION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional micro-cytopathic method generally takes 7-10 days, and requires manual judgment of the results (for details, please refer to the third part of the Pharmacopoeia), which has the problems of low detection efficiency and high professional requirements for experimenters.

Method used

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  • Recombinant virus and application thereof in mumps virus neutralizing antibody detection
  • Recombinant virus and application thereof in mumps virus neutralizing antibody detection
  • Recombinant virus and application thereof in mumps virus neutralizing antibody detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: A kind of recombinant virus

[0042] This embodiment provides a recombinant virus, which uses mumps virus strain QS-F (preservation number is CCTCC No: V201948, recorded in the patent application text with publication number CN111019910A) as a living vector to express the green The gene of fluorescent protein (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), wherein the gene encoding green fluorescent protein is inserted into the NP segment and P / Between the V segments (recombinant virus genome architecture pattern see figure 1 ).

[0043] The preparation method of described recombinant virus comprises the steps:

[0044] 1. Construction of pAC-rMuV-eGFP (N-P) full-length clone

[0045] Synthesize DNA fragments F1, F2; use restriction enzyme P ml I andN ae I (purchased from Takara Company) digested the recombinant plasmid pAC-MuV (recorded in the patent application text with publication number ...

Embodiment 2

[0051] Embodiment 2: A kind of recombinant virus

[0052] This embodiment provides a recombinant virus, which uses mumps virus strain QS-F (preservation number is CCTCC No: V201948, recorded in the patent application text with publication number CN111019910) as a living vector to express the green The gene of fluorescent protein (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), wherein the gene encoding green fluorescent protein is inserted into the F segment and SH segment of the mumps virus genome Between (recombinant viral genome architecture pattern see figure 1 ).

[0053] The preparation method of described recombinant virus comprises the steps:

[0054] 1. Construction of pAC-rMuV-eGFP (F-SH) full-length clone

[0055] Synthesize DNA fragments F3, F4, and F5; use F3 and F4 as templates, and F3-GFP-F, F4-GFP-R as primers, perform fusion PCR to obtain fragments F3+F4; use restriction endonuclease X ma I and S a...

experiment example 1

[0061] Experimental Example 1: Performance Growth Verification of Recombinant Viruses

[0062] Vero cells (purchased from ATCC) were 8*10 5 The inoculum of each individual was plated in advance in a six-well plate containing 2000 μL / well DMEM medium (purchased from Gibco), and cultured at 37°C until the cells covered a monolayer; Influenza virus strain QS-F) was used as a control, and the recombinant virus pAC-rMuV-eGFP (N-P) and the recombinant virus pAC-rMuV-eGFP (F-SH) were inoculated to Vero In the cells, after adsorption and infection at 37°C for 1 hour, the DMEM medium was sucked off with a pipette gun, and the cells containing 2% (v / v) fetal bovine serum were added to the six-well plate at an addition amount of 2000 μL / well. The maintenance solution (the cell maintenance solution is DMEM medium purchased from Gibco Company) was cultured at 34°C, and the culture supernatant was collected on the 1d, 2d, 3d, 4d, and 5d of the virus inoculation, and the virus titer was det...

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Abstract

The invention relates to a recombinant virus and application thereof in mumps virus neutralizing antibody detection, and belongs to the technical field of biology. The invention provides a recombinant virus and a method for detecting a neutralizing antibody of a mumps virus by using the recombinant virus as a neutralizing virus. The recombinant virus takes the mumps virus as a live vector to express a marker gene; the traditional mumps virus neutralizing antibody detection method needs 7 days for final judgment of the result, while the neutralizing antibody detection method disclosed by the invention only needs 3 days, and the result conformity is extremely high; moreover, according to the neutralizing antibody detection method, the neutralizing titer of the antibody in the to-be-detected sample is calculated through fluorescence, the neutralizing antibody detection method can be used in a high-flux fluorescence detection instrument, data can be automatically and electronically stored, and meanwhile, the limitation of human factors in result judgment is shielded.

Description

technical field [0001] The invention relates to a recombinant virus and its application in the detection of mumps virus neutralizing antibodies, belonging to the field of biotechnology. Background technique [0002] Mumps is an acute respiratory infectious disease caused by mumps virus (MuV). The main clinical symptoms are fever, non-suppurative swelling and pain of the parotid gland; some pathologies may be secondary to aseptic meningitis, pancreatitis, orchitis, etc.; severe symptoms may lead to disability or death. Mumps is a Class C infectious disease under statutory management in my country. At present, vaccination is still an important and effective means of prevention and control of the disease, and the determination of neutralizing antibodies after prevention is an important means of judging the immune effect after vaccination. [0003] Neutralization test is a method of incubating a mixture of virus and specific antibody under appropriate conditions in vitro to ma...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/12G01N33/569
CPCC12N7/00C07K14/43595G01N33/56983C12N2760/18721G01N2333/12G01N2469/20
Inventor 安祺田大勇张楠张亚静朱凤才
Owner BEIJING CELL FUSION BIOTECHNOLOGY CO LTD
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