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Preparation method of antigen protein for detecting rabies virus antibody, and kit

An antigen protein, rabies virus technology, applied in the field of medical supplies, can solve the problems of not accurately reflecting the true level of rabies virus neutralizing antibody, lack of post-translational modification function in prokaryotic expression system, unable to truly reflect the level of neutralizing antibody, etc. Avoid biosafety risks, high sensitivity, wide linear range effect

Pending Publication Date: 2019-05-31
WUHAN LIFE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some researchers tried to use the method of genetic engineering to successfully express the G protein using the prokaryotic expression system, which was used as the raw material of the antigenic protein for the detection of rabies virus antibody, and was prepared into a rabies virus antibody detection kit (enzyme-linked immunoassay) for use in rabies virus. And antibody detection, but the test results are compared with the gold standard method for rabies virus neutralizing antibody detection rapid fluorescent focus inhibition test (RFFIT), the former generally has obvious missed detection and false negative phenomena, resulting in inaccurate antibody detection results and cannot truly reflect Neutralizing antibody levels in vivo
This defect exists mainly because the target protein G protein is a glycoprotein, and in the process of its expression and secretion, sufficient glycosylation modification plays a crucial role in its stability, antigenicity and biological activity, while The prokaryotic expression system lacks a relatively complete post-translational modification function, and the expressed G protein conformation is quite different from the natural conformation. Therefore, using the expressed G protein as a diagnostic antigen raw material for detecting rabies virus neutralizing antibodies cannot accurately reflect the serum G protein. True level of rabies virus neutralizing antibody

Method used

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  • Preparation method of antigen protein for detecting rabies virus antibody, and kit
  • Preparation method of antigen protein for detecting rabies virus antibody, and kit
  • Preparation method of antigen protein for detecting rabies virus antibody, and kit

Examples

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Effect test

Embodiment 1

[0039] Such as figure 1 Shown a kind of preparation method for detecting the antigenic protein of rabies virus antibody, comprises the following steps:

[0040] S1: Infecting insect cells with the recombinant baculovirus to obtain infected insect cells;

[0041] S2: culture and proliferate the infected insect cells;

[0042] S3: the antigenic protein of the rabies virus antibody is extracted from the insect cells in S2.

[0043] In S1, the G protein gene fragment containing the rabies virus neutralizing antibody binding site is cloned into a baculovirus expression vector and infected with insect cells to obtain infected insect cells, and the insect cells are Sf9 cells.

[0044] The DNA coding sequence of the G protein gene fragment containing the rabies virus neutralizing antibody binding site is the sequence of SEQ ID NO:1.

[0045] Such as figure 2 As shown, S1 includes the following steps:

[0046] S11: the Sf9 cells were recovered by stationary culture in Kelvin flas...

Embodiment 2

[0057] According to the G protein gene sequence of rabies virus CVS-11 strain included in Genbank, primers were designed, and EcoRI and HindIII restriction sites were introduced into the upstream and downstream primers. The upstream primer was 5'-CCGGAATTCATGGTTCCTCAGGT-3'; the downstream primer was 5'- GGGTTCGAATTACAGTCTGATCT-3' (synthesized by Shanghai Sangon Bioengineering Co., Ltd.). Using the prokaryotic expression recombinant vector pET23a-G (constructed and preserved by Wuhan Life Science and Technology Co., Ltd.) containing the G protein DNA fragment of rabies virus CVS-11 strain (SEQ ID NO: 1) as a PCR template, the G protein DNA fragment was amplified by PCR. After double-enzyme digestion, it was cloned into the baculovirus expression system donor plasmid pFastBacHTB that was also subjected to the same double-enzyme digestion. After the correct expression frame was identified by sequencing, the recombinant donor plasmid pFastBacHTB-G was obtained. DH10Bac competent c...

Embodiment 3

[0061] The rabies virus antibody detection kit in the present embodiment comprises the following components:

[0062] 1) The above-mentioned G protein antigen;

[0063] 2) ELISA plate;

[0064] 3) Enzyme markers;

[0065] 4) Series calibrator;

[0066] 5) Sample diluent;

[0067] 6) concentrated washing liquid;

[0068] 6) Chromogenic solution A;

[0069] 7) Chromogenic solution B;

[0070] 8) Stop solution.

[0071] in,

[0072] The enzyme marker is mouse anti-human IgG labeled with horseradish peroxidase, and the preparation method is as follows: Weigh 5 mg of horseradish peroxidase (HRP) and dissolve it in 1 mL of double-distilled water, add 500 μL of newly prepared 0.06 mol / L NaIO4, placed at 4°C for 30 minutes, at this time the solution was early green, added 0.5mL ethylene glycol (0.16mol / L) and reacted in the dark for 30 minutes at room temperature. Then add 1 mL of 5 mg / mL mouse anti-human IgG, and dialyze in 0.05 mol / L pH9.5 carbonate buffer at 4°C overnight....

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Abstract

The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a preparation method of antigen protein for detecting a rabies virus antibody and a kit. The method comprises the steps: employing a recombinant baculovirus containing a rabies virus G protein expression cassette for infecting insect cells to obtain infected insect cells; culturing and propagating the infected insect cells; extracting antigen protein of the rabies virus antibody from insect cells; and preparing the kit through the antigen protein. The problem that rabies virus whole-virus particles are used as coating antigens in a traditional method due to a fact that the antibody detection result contains a neutralizing antibody and a non-neutralizing antibody is solved. The method can be usedfor large-scale production and detection of other infectious disease positive serum, has no cross reaction, has the advantages of strong specificity, high sensitivity, good repeatability and wide linear range, avoids biological safety risks, and has very strong creativity.

Description

technical field [0001] The invention relates to the technical field of medical supplies, in particular to a method for preparing an antigenic protein and a kit for detecting rabies virus antibodies. Background technique [0002] Rabies (also known as hydrophobia) is a zoonotic infectious disease caused by Rabies virus (RV). Once onset, the fatality rate is almost 100%, and it is the acute infectious disease with the highest fatality rate in humans. Rabies virus is bullet-shaped and is a single-stranded negative-sense RNA virus. The viral genome is about 12kb long, and five structural proteins of rabies virus, N, P, M, G, and L, are arranged in sequence from the 3' end to the 5' end, encoding nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and Large transcriptase protein. Glycoprotein is related to rabies virus infection and immunity, and it is the only protein among the above five proteins that can stimulate the body to produce neutralizing antibodies. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/535
Inventor 华俊清曾强黄晶王诺项雅丽吴边
Owner WUHAN LIFE TECH
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