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Preparation method and application of brucellosis specific fusion protein antigen

A brucellosis and fusion protein technology, applied in the direction of fusion polypeptides, bacteria, hybrid peptides, etc., to achieve the effect of strong specificity, good repeatability and high sensitivity

Active Publication Date: 2016-08-31
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem of cross-reaction with other bacteria when the traditional ELISA method uses whole bacteria as the antigen to detect antibodies in serum, the present invention provides a brucellosis-specific fusion protein antigen

Method used

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  • Preparation method and application of brucellosis specific fusion protein antigen
  • Preparation method and application of brucellosis specific fusion protein antigen
  • Preparation method and application of brucellosis specific fusion protein antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 Design of multi-epitope fusion gene of Brucella and construction of plasmid

[0031] The amino acid sequence of the dominant outer membrane protein of Brucella was retrieved from the Pubmed database, and BepipredLinear Epitope Prediction, ABCpred prediction Server, COBEpro and other software were used to predict the antigenic epitope of Brucella, and the dominant antigenic epitope was selected. A linking peptide sequence is added between adjacent epitopes, and DNAStar is used to optimize the tandem sequence of antigenic epitopes and the selection of linking peptides, and the selected α-helix, less β-fold, hydrophilic and antigenic sequence is used as the target amino acid The sequence is used for the expression of the multi-epitope fusion protein of Brucella in the next step and the preparation of the ELISA detection kit for the diagnosis of Brucellosis.

[0032] Using DNAStar to predict the immunological parameters of the designed Brucella multi-epitope fu...

Embodiment 2

[0037] The optimization of the expression condition of embodiment 2 Brucella multi-epitope fusion protein

[0038] The expression host strain Escherichia coli BL21 (DE3) was preserved by the Department of Health Inspection, School of Public Health, Jilin University; the expression vector PET-28b plasmid containing Brucella multi-epitope fusion protein was constructed in Example 1; various molecular biology All reagents were purchased from Biological Reagent Company.

[0039] Transform the PET-28b plasmid obtained in Example 1 into Escherichia coli BL21 (DE3) expressing bacteria, heat shock at 42°C for 90s, let it stand on ice for 2 minutes, spread it on an LA plate (containing 30 μg / mL kanamycin), and culture at 37°C overnight. Pick a single colony of the expression strain Escherichia coli BL21 (DE3) and culture it overnight in LB medium (containing 30 μg / mL kanamycin) at 37°C and 220 r / min. The next day, the overnight culture was expanded with fresh LB medium (containing 30...

Embodiment 3

[0041] Example 3 Purification of Brucella multi-epitope fusion protein

[0042] Get the bacterial supernatant after the ultrasonic crushing of above-mentioned embodiment 2, contain Brucella soluble multi-epitope fusion protein in the described bacterial supernatant, adopt nickel agarose affinity chromatography and anion exchange chromatography to bacterial supernatant The Brucella soluble multi-epitope fusion protein in the solution was purified, and the purification solution and purification method were carried out according to the instructions of the kit. The collected purified protein solution was centrifuged and concentrated in an ultrafiltration centrifuge tube, and then placed at 4 ° C, - Store at 20°C and -80°C. The purified Brucella multi-epitope fusion protein was subjected to SDS-PAGE electrophoresis and Western Blot detection, the results are shown in the attached Figure 4 And attached Figure 5 As shown, the molecular weight of the expressed fusion protein is be...

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Abstract

The invention discloses a brucellosis specific fusion protein antigen. The brucellosis specific fusion protein antigen is a fusion protein expressed by Escherichia coli BL21 (DE3) after inserting a fusion protein gene constructed by serially connecting amino acid sequences on dominant antigen epitopes of Brucella outer membrane proteins BP26, OMP31, OMP16 and OMP2b to pET-28b, approaches a natural protein in conformation, keeps the antigenicity and the solubility of the natural protein, and can be used as a diagnosis antigen to produce a human brucellosis antibody ELISAS kit. The kit overcomes the disadvantages of long separate culture enrichment time, easy pollution to environment and easy human infection of traditional etiological diagnosis, and also improves the severe cross reaction of traditional immunological methods. The kit has the advantages of strong specificity, good repeatability and high sensitivity, provides means for rapid, accurate and specific diagnosis of brucellosis, and provides bases for early diagnosis and early treatment of brucellosis patients.

Description

technical field [0001] The invention relates to the fields of molecular biology technology and immunology. It specifically relates to a Brucella specific fusion protein antigen and its application in a brucellosis diagnosis kit. Background technique [0002] Brucellosis ( Brucellosis ), also known as undulating fever, is caused by Brucella ( Brucella ) invade the body and cause acute or chronic infectious diseases, which belong to zoonotic infectious diseases. At present, the prevalence of brucellosis has been found in more than 160 countries and regions around the world, and more than 500,000 new cases of brucellosis are reported every year. The "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" enacted by my country in 1989 listed brucellosis as a legal infectious disease. The rising trend has spread to 31 provinces, autonomous regions and municipalities directly under the central government, among which Shanxi, Jilin, Heilongj...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/68G01N33/569
CPCC07K14/23C07K2319/00C12N15/70C12N2800/101C12N2800/22G01N33/56911G01N33/68G01N2469/20
Inventor 李娟李丽徐坤宋秀玲殷德辉宋丹丹刘玉申鞠文王娟曲笑锋赵超魏佳
Owner JILIN UNIV
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