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Gamma-interferon sandwich ELISA detection method based on recombinant fusion antigen protein

A technology for fusing antigens and antigenic proteins, applied in the field of medical testing and determination, can solve problems such as long detection cycle, poor sensitivity and specificity, and unsuitability for large-scale census

Active Publication Date: 2013-10-02
常州同泰生物药业科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to address the shortcomings of the existing detection technology for gamma-interferon in whole blood of cattle and cattle, such as long detection period, unsuitability for large-scale general survey, heavy workload, troublesome detection operation, poor sensitivity and specificity, etc., and specifically for buffalo The effective detection technology of γ-interferon is in a blank state, providing a γ-interferon sandwich ELISA detection method based on recombinant fusion antigen protein (CFP10, ESAT6, MPB70) and γ-interferon-specific monoclonal antibody

Method used

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  • Gamma-interferon sandwich ELISA detection method based on recombinant fusion antigen protein
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  • Gamma-interferon sandwich ELISA detection method based on recombinant fusion antigen protein

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Example 1 Expression of Recombinant Fusion Antigen Protein MPB70-CFP10-ESAT6

[0062] (1) Primer design

[0063] According to the three similar gene sequences in Mycobacterium bovis AF2122 / 97 in GenBank (accession number: NC_002945), 8 primers were designed: MPB70 and CFP10 both deleted the stop codon, and introduced linker (Gly4Ser1)3 and YAPQDP, respectively. A total of 45bp and 18bp bases (marked by lines). The primers were synthesized by Dalian Bao Biological Engineering Co., Ltd., and the primer sequences are shown in SEQ ID NO: 3-10 in Table 1.

[0064] Table 1 Amplifies the primer nucleotide sequence of the recombinant antigen gene MPB70-CFP10-ESAT-6

[0065]

[0066] (2) PCR amplification of recombinant gene

[0067] Using the genomic DNA of Mycobacterium bovis as a template, the primer pairs in Table 1 were used to amplify the CFP10, ESAT6, and MPB70 genes respectively. The specific reaction system was as follows:

[0068] 10×EX Taq Buffer 2.5μL

[0069...

Embodiment 2

[0107] Example 2 Preparation of gamma-interferon monoclonal antibody

[0108] The gamma-interferon recombinant protein of the present invention uses the buffalo genome as a cloning template and is obtained by a gene recombination expression method. The steps for cloning the gamma-interferon recombinant protein are as follows:

[0109] (1) After the buffalo peripheral blood lymphocytes were cultured for 17 hours, the lymphocytes and their culture suspension were collected by centrifugation, and RNA was extracted according to the instructions of the RNA extraction kit (purchased from Shanghai Bioengineering Company).

[0110] (2) Perform reverse transcription on the RNA obtained above, and the RT reaction system is as follows: Take 16 μL of the extracted RNA sample in a PCR tube, add 1.0 μL of IFN-γ2 (25 pmol / μL), 5.0 μL of 5×Buffer, dNTPs ( 2.5mmoL) 2.0μL, RNasin (40U / μL) 0.5μL, M-MLVRTase (200U / μL) 0.5μL, put in a PCR machine, 42°C for 60min, 94°C for 5min, cDNA was obtained a...

Embodiment 3

[0156] Example 3 Utilize recombinant fusion antigen protein and gamma-interferon monoclonal antibody to detect gamma-interferon in bovine serum

[0157] This detection method is based on the principle of the sandwich ELISA method, and the whole blood of common yellow cattle is randomly selected as the sample, and the number of samples is 6. The detection method is as follows:

[0158] (1) Sample pretreatment: high-speed centrifugation of the bovine serum sample to obtain the supernatant, mix the recombinant fusion antigen protein with the bovine blood supernatant at a volume ratio of 100ul:1000ul, incubate the mixture at 37°C for 20 hours, and collect by centrifugation supernatant.

[0159] (2) Coating: Coat the microtiter plate with γ-interferon monoclonal antibody, and the amount of γ-interferon monoclonal antibody added in each well is 5~7×10 -2 ug, after the addition, the microtiter plate was incubated at 37°C for 1.5h.

[0160] (3) Blocking: seal the coated ELISA plate ...

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Abstract

The present invention relates to a gamma-interferon sandwich ELISA detection method based on recombinant fusion antigen protein, further relates to a recombinant fusion antigen protein preparation method and a gamma-interferon monoclonal antibody preparation method, and belongs to the technical field of medical examination determination. According to the present invention, the recombinant fusion antigen protein is formed by linking secretory antigen protein MPB70, antigen protein CFP10 and antigen protein ESAT6 through peptide bonds, the gamma-interferon monoclonal antibody is prepared through immunization of animals, cell fusion, hybridoma cell screening, cloning and purification, and the recombinant fusion antigen protein and the gamma-interferon monoclonal antibody are adopted to establish a sandwich ELISA detection method; and the recombinant fusion antigen protein can rapidly stimulate a bovine blood sample to secret gamma-interferon, and the gamma-interferon monoclonal antibody can quickly and specifically identify bovine gamma-interferon, such that strong sensitivity and strong specificity are provided, and broad application prospects are provided in the field of buffalo tuberculosis diagnosis.

Description

technical field [0001] The invention belongs to the technical field of medical examination and determination, in particular to a γ-interferon sandwich ELISA detection method based on recombinant fusion antigen protein. Background technique [0002] Bovine tuberculosis (tuberculosis, TB) is a chronic wasting disease caused by Mycobacterium bovis, which is distributed worldwide. The World Organization for Animal Health (OIE) lists bovine tuberculosis as a B-type animal disease, and my country lists it as a B-type animal disease. For a long time, people used to think that bovine tuberculosis rarely occurs in buffaloes. However, from 1998 to 2000, bovine tuberculosis with buffaloes as natural hosts broke out in Kroger National Park in South Africa. The infection rate of buffaloes in the south of the park reached 42%. The infection rate was 21%, giving people a hint of the severity of tuberculosis infection in buffalo. In recent years, the rapid development of milk buffalo indu...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/68G01N33/577G01N33/543C12R1/32
Inventor 徐贤坤熊毅刘棋龙剑明冯淑萍胡巧云付薇马琳黄胜斌
Owner 常州同泰生物药业科技股份有限公司
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