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Indirect ELISA kit for detecting salmonella abortus equi antibody, preparation method and application of indirect ELISA kit

A Salmonella and kit technology, applied in the biological field, can solve problems such as structural damage, instability, and complex structure, and achieve good specific effects

Pending Publication Date: 2020-07-28
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The flagellin component of Salmonella is mainly protein. The flagellar antigen contains different H antigens, which are divided into type I and type II, namely FliC and FljB proteins. Each flagella is composed of three subunits: matrix, hook and filament. The structure is extremely complex, with good shrinkage and antigenicity, but unstable, once it encounters alcohol or is exposed to high temperature, its structure will be destroyed

Method used

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  • Indirect ELISA kit for detecting salmonella abortus equi antibody, preparation method and application of indirect ELISA kit
  • Indirect ELISA kit for detecting salmonella abortus equi antibody, preparation method and application of indirect ELISA kit
  • Indirect ELISA kit for detecting salmonella abortus equi antibody, preparation method and application of indirect ELISA kit

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Embodiment 1

[0037] The establishment of embodiment 1 indirect ELISA method

[0038] 1 Materials and methods

[0039] 1.1 Plasmids and strains

[0040] PMD18T vector and Escherichia coli DH5α competent cells were purchased from Shanghai Kangwei Century Co., Ltd.; the virulent strain C77-1 of Salmonella equine abortus and the vaccine strain C355 were donated by Harbin Veken Biotechnology Co., Ltd. The vaccine strain C39 and other isolates were preserved In the Innovation Team of Equine Infectious Diseases and Lentivirus Research, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0041] 20180316.H.AES.G strain, classified as Salmonella abortus (Salmonella abortus), is preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the address is at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Microbiology Institute, the preservation number is CGMCC No.18341, and th...

Embodiment 2

[0096] Embodiment 2 is used for the assembly and the use method of the indirect ELISA kit that detects the antibody of Salmonella abortus equine

[0097] 1. Assembly of kit

[0098] Included in the described test kit:

[0099] 1) an ELISA plate coated with the FljB protein of Salmonella equine abortus, wherein the FljB protein of Salmonella equine abortus is encoded by the sequence shown in SEQ ID NO.2, and the coating antigen concentration of the Salmonella equine abortus FljB protein is 1 μg / ml .

[0100] 2) Enzyme-labeled secondary antibody: HRP-labeled goat anti-horse IgG secondary antibody

[0101] 3) Coating solution: phosphate buffer saline

[0102] 4) Blocking solution: 5% w / v skim milk

[0103] 5) Serum diluent: 5% w / v BSA solution

[0104] 6) Chromogenic solution: Chromogenic solution TMB

[0105] 7) Washing solution: PBST buffer

[0106] 8) Stop solution: 2M H 2 SO 4

[0107] 2. How to use

[0108] (1) The ELISA plate coated with the FljB protein of Salmo...

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Abstract

The invention discloses an indirect ELISA kit for detecting a salmonella abortus equi antibody, and a preparation method and application of the indirect ELISA kit. The kit comprises an elisa plate coated with a salmonella abortus equi FljB protein, wherein the salmonella abortus equi FljB protein is coded by a sequence shown in SEQ ID NO.2. According to the indirect ELISA kit and method of the invention, sequence analysis is carried out, the specific sequence of the salmonella abortus equi is amplified by using P3 / P4, is cloned to a prokaryotic expression vector pET-28a; a recombinant expression plasmid pET28a-FljB is constructed, and is transferred into Rosetta (DE3); iPTG is used for inducible expression; the purified FljB protein is used as a coating antigen to establish the indirect ELISA method of the salmonella abortus equi antibody; and results show that the indirect ELISA method established by using the protein as the coating antigen has good specificity, sensitivity and repeatability, and can be used for clinical detection of the salmonella abortus equi antibody.

Description

technical field [0001] The invention relates to an indirect ELISA kit and its preparation method and application, in particular to an indirect ELISA kit for detecting antibodies to Salmonella equine abortus and its preparation method and application. The invention belongs to the field of biotechnology. Background technique [0002] Equine abortus Salmonellosis is an equine animal infectious disease mainly characterized by abortion of pregnant horses caused by Salmonella or Salmonella typhimurium (S.typi), also known as equine paratyphoid fever ( Equi parathphoid) or Salmonella equi miscarriage. The disease is easy to infect primiparous dams. Most dams generally have no obvious clinical symptoms before abortion occurs. Most of the sudden abortions are stillbirths. A small number of surviving fetuses will also die a few days after birth. When pregnant mares abort, the germs are excreted with the aborted fetus, afterbirth, amniotic fluid and vaginal secretions, and sick male ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/535
CPCG01N33/56916G01N33/543G01N33/535G01N2333/255
Inventor 王晓钧胡哲
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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