Unlock instant, AI-driven research and patent intelligence for your innovation.

ELISA method and kit for detecting classical swine fever virus antibody

An expression vector and in vitro expression technology, applied in the field of molecular biology, can solve the problems of limited production, inability to be used as a differential diagnosis test, high cost, etc., and achieve the effects of safe use, cheap culture medium, and high expression

Active Publication Date: 2014-06-25
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used methods for detecting CSFV antibodies use CSFVE2 protein or concentrated and purified cell culture CSFV antigens. For most of the more successful labeled vaccines, these methods cannot be used as supporting differential diagnosis tests.
So far, E as a diagnostic antigen rns (E0) proteins are expressed by recombinant fowlpox virus, insect cells or directly in the form of synthetic peptides or prokaryotic expression systems 【1-7】 , the cost is high, and the output is also limited to a certain extent

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • ELISA method and kit for detecting classical swine fever virus antibody
  • ELISA method and kit for detecting classical swine fever virus antibody
  • ELISA method and kit for detecting classical swine fever virus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1E rnsProtein expression and activity detection in vitro

[0027] 1. Construction of recombinant expression vector

[0028] 1.1 The classical swine fever virus (CSFV) E rns The protein gene was modified, the codons were selectively optimized, and an EcoRI restriction site was added to the 5' end of the fragment, and a NotI restriction site was added to the 3' end of the fragment. Optimized E rns The full-length sequence is shown in SEQ ID No.1, and the sequence is further obtained through artificial synthesis.

[0029] 1.2 Connect the artificially synthesized SEQIDNo.1 and the pGM-T plasmid to establish a reaction system at a ratio of 5:1, then gently add 10 μL of the ligated product to DH5α competent cells, and gently rotate the EP tube to mix the contents , placed on ice for 30 minutes. After heat shock in a water bath at 42°C for 90 sec, quickly transfer to ice, place for 2-3 min, add 800 μL LB liquid medium, and shake at 200 rpm for 45-60 min to revive ...

Embodiment 2

[0047] The establishment of embodiment 2 swine fever virus antibody detection ELISA method

[0048] 1. Establishment of ELISA method for detection of classical swine fever virus antibody

[0049] Determination of Antigen E Using Square Array Titration rns The optimal antigen coating concentration and the dilution of the pig serum to be tested are determined, and the optimal reaction program is determined at the same time. Using the ratio of the OD values ​​of positive and negative serum wells, that is, the P / N value, as an index, a comparative test was carried out on items such as the concentration of the coated antigen, the dilution factor of the serum, the optimum working concentration of the enzyme conjugate, and the reaction time of each step to determine the optimal results. Suitable reaction conditions, the purified recombinant E rns The protein was diluted to an appropriate concentration with 0.025 mol / L carbonate buffer (pH 9.6), coated with a 96-well microtiter plat...

Embodiment 3

[0071] The composition of embodiment 3 test kits

[0072] ELISA plate: coated with E rns For recombinant protein, the coating concentration is 6.25 μg / mL, 50 μL per well;

[0073] Enzyme-labeled secondary antibody: horseradish peroxidase-labeled rabbit anti-pig IgG;

[0074] Chromogenic solution: 3′, 3′, 5′, 5′-tetramethylbenzidine (TMB) single-phase solution

[0075] Stop solution: 2MH 2 SO 4 the solution

[0076] Diluent: washing solution containing 5% skimmed milk

[0077] Washing solution: PBST

[0078] Positive standard: known swine fever positive serum

[0079] Negative standard: known swine fever negative serum

[0080] Note: Negative and positive standard products were purchased from the National Veterinary Microbiology Collection Center.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to an ELISA method for detecting classical swine fever virus antibody. The method comprises the following steps: optimizing full-length classical swine fever virus (CSFV)Erns protein gene sequence to establish an eukaryotic expression system of classical swine fever virus Erns protein-pichia pastoris expression system to realize the efficient expression of the Erns protein; optimizing to establish an ELISA serological diagnosis method for large-scale screening swine fever specific antibody through the ELISA condition by using the expressed Erns protein as the antigen. The method further provides an ELISA kit for the above detection; the kit is good in specificity, sensibility and repeatability, and capable of fast and effectively detecting the classical swine fever virus antibody. The development of the swine fever serum antibody monitoring of the country is largely promoted, and the use and the popularization of the swine fever molecular marker vaccine are promoted, the export trade of live pig and fresh pork of the country are promoted, and the good economic benefit and social effect are realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an ELISA method for detecting swine fever virus antibody, and also relates to an ELISA detection kit used for the detection. Background technique [0002] Langedijk et al. found that in the E of CSFV rns There is an antigenic epitope on the protein, which has the function of differential diagnosis and is located in E rns 191-227 amino acids at the C-terminus of the protein, it can not only distinguish CSFV, BVDV, and BDV, but also can distinguish vaccine-immunized pigs from wild virus-infected pigs in the application of new vaccines (with E2 as the target protein) in the future , so it has a good application prospect. Pigs infected with classical swine fever virus can induce the production of E rns Antibodies against glycoproteins. E. rns Molecular analysis of glycoproteins can determine the antibodies induced by virus infection, which can help design effective diagnostic an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/81G01N33/569
Inventor 孙惠玲刘彦白佳桦许晓玲冯涛李桂萍张平
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES