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Preparation method and application of Brucella multi-epitope fusion protein vaccine

A fusion protein, brucellosis technology, applied in the field of brucellosis multi-epitope fusion protein antigen, preparation of brucellosis vaccine, can solve problems such as poor immune effect and miscarriage

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

AI Technical Summary

Problems solved by technology

Due to the shortcomings of these vaccines such as animal abortion and poor immune effect, so far there is no ideal Brucella vaccine for the prevention and control of Brucellosis

Method used

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  • Preparation method and application of Brucella multi-epitope fusion protein vaccine
  • Preparation method and application of Brucella multi-epitope fusion protein vaccine
  • Preparation method and application of Brucella multi-epitope fusion protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Design of multi-epitope fusion gene of Brucella and construction of plasmid

[0022] The amino acid sequence of the dominant outer membrane protein of Brucella was retrieved from the Pubmed database, using BepiPred (http: / / www.cbs.dtu.dk / services / Bepipred / ), ABCpred (http: / / www.imtech.res .in / raghava / abcpred / ) and COBEpro (http: / / scratch.proteomics.ics.uci.edu / ) and other software to predict the B cell epitope of Brucella outer membrane protein, using IEDB (http: / / tools.iedb.org / main / tcell / ) The T cell epitope prediction tool on the website predicts T cell epitopes, uses BLASTPanalysis to analyze the similarity of antigenic epitopes, selects the dominant antigenic epitope, and selects the dominant antigenic epitope. Add a linking peptide sequence between them, use DNA Star to optimize the tandem sequence of antigenic epitopes and the type of linking peptide, and select a sequence with good immunogenicity as the amino acid sequence of the target fusion prot...

Embodiment 2

[0027] Example 2 Expression of Brucella rMEP

[0028] 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 the base sequence of Brucella rMEP vaccine was constructed in Example 1; various molecular Biological reagents were purchased from Biological Reagent Company.

[0029] Transform the pET-28b plasmid obtained in Example 1 into Escherichia coli BL21 (DE3) expression 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 expressed strain and culture it in 4mL LB medium (containing 30μg / mL kanamycin) at 37°C and 220r / min for 18-24h. Take 0.4mL of the culture and add it to 4mL of fresh LB medium (containing 30μg / mL kanamycin) to continue the culture. When the OD of the bacterial solution 600 When ...

Embodiment 3

[0031] Example 3 Purification of Brucella rMEP

[0032]Get the supernatant after the ultrasonic crushing of above-mentioned embodiment 2, contain Brucella soluble rMEP in the described bacterial supernatant, adopt nickel agarose affinity chromatography and anion exchange chromatography to the Brucella in the supernatant Bacteria-soluble rMEP was purified. The specific purification solution and purification method were prepared and operated according to the instructions of the relevant kit. After the eluted components of the collected purified protein liquid were detected by SDS-PAGE, the high-purity components were dialyzed into PBS. In buffer solution (containing 0.1% SKL, pH = 7.4), dialyze at 4°C overnight, filter and sterilize with a 0.45 μm filter membrane, aliquot and store in a -80°C refrigerator for long-term storage. SDS-PAGE electrophoresis and Western Blot detection were used to detect the purified fusion protein, and the results are shown in the attached Figure 4...

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Abstract

The invention discloses a Brucella multi-epitope fusion protein antigen. Amino acid sequences of dominant epitopes of Brucella outer membrane proteins BP26, OMP31, OMP16 and OMP2b are in series connection to construct a fusion protein gene to express the proteins, and a Brucella multi-epitope fusion protein antigen vaccine is prepared. Mice challenge tests show that the Brucella multi-epitope fusion protein antigen vaccine plays a role in immune protection in Brucella infection.

Description

technical field [0001] The invention relates to the fields of molecular biology technology and immunology. It specifically relates to a Brucella multi-epitope fusion protein antigen and its application in preparing Brucella vaccines. Background technique [0002] In recent years, with the rapid development of economic globalization, new infectious diseases continue to occur, and some old infectious diseases have begun to resurface, especially brucellosis, which is more harmful to humans, and its incidence has increased year by year in recent years This trend has brought huge public health problems and economic losses to countries all over the world. [0003] Brucellosis is a zoonotic infectious disease caused by Brucella invading the body. People infected with Brucella can cause systemic symptoms such as fever, sweating, fatigue, and migratory joint pain. In severe cases, hepatosplenomegaly, testicular enlargement, joint deformation, and eventually loss of working ability ...

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 徐坤李娟李丽殷德辉宋秀玲王娟刘玉申宋丹丹曲笑锋鞠文赵超庞博孟祥君张惠雯翟玥暴昊
Owner JILIN UNIV
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