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Prokaryotic Expression、Purification of Three Lipoproteins of Brucella and Immunogenicity of the Expressed Products

Author: MaChangSheng
Tutor: XieZhiJing
School: Shandong Agricultural University
Course: Clinical Veterinary Medicine
Keywords: Brucella outer membrane lipoprotein express purify immunogenicity
CLC: S852.6
Type: Master's thesis
Year: 2012
Downloads: 124
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Abstract


Brucellosis is a zoonotic disease which caused by the Brucella, and it is serious harm tothe development of animal husbandry and human health. Brucellosis is a worldwide publichealth problem in more than170countries around the world. In recent years, the monitoringresults showed that brucellosis was more and more serious in China. Sheep and cattlebrucellosis infection rates rose so fast that it became one of the fastest growing diseases thatincreased in the number of notifiable and reported infectious diseases.Outer membrane proteins (OMPs) play important roles in stabilizing the structure of theouter membrane, adapting to external and intracellular environments, and Brucella virulence.OMPs are located on the surface of the bacteria and can be easily recognized by host immunesystem, and involved in the interaction of the bacteria with the host immune system, stimulatethe immune response. Some of the outer membrane proteins of Brucella have immunogenicity,and some of them are protective immune antigen. Therefore, a lot of research about Brucellpathogenic mechanisms and immune response mechanisms are concentrated in the outermembrane protein research.In this study, we selected three Brucella outer membrane lipoprotein, constructed thecloning vectors and purified the three proteins, to analysis their immunogenicity, and toexplore the feasibility as diagnostic antigen and the type of subunit vaccine candidateproteins.According to the Brucella outer membrane lipoproteins gene sequences OMP10（GenBank：L27995.1）,OMP16（GenBank：JF918760.1）,OMP19（GenBank:L27997.1）publishedin GenBank, three pairs of specific primers were designed by using the DNASTAR Primer5software. BamHⅠrestriction sites in the upstreams and HindⅢ restriction sites in thedownstreams were introduced in the three pairs of primers. Brucella abortus vaccine strainS19genomic DNA was extracted by the method of boiling, and the three lipoprotein genefragments ere amplified by the polymerase chain reaction（PCR）with the total genomic DNAas template, the size is381bp，504bp，534bp, respectively. The three amplified gene fragmentswere cloned into PEASY-T3, then transformed into compETent cells E.coli DH5α to constructthree cloned carriers: pEASY-T3/OMP10, pEASY-T3/OMP16, pEASY-T3/OMP19. The plasmid were extracted after the carrier were tested to be right by double digestion andsequencing. The recombinant plasmid and the expression vector pET-32a were doubledigested with the restriction enzymes BamH Ⅰand Hind HindⅢ. Then the target genefragments were ligated into the expression vector through T4ligase. Then they weretransformed into DH5a, and constructed the recombinant expression plasmids: pET-32a/OMP10, pET-32a/OMP16, pET-32a/OMP19. After double digestion and sequencing, thethree restructuring expression plasmids were transformed into E.coli BL21(DE3) respectively.After inducible expressed by the final concentration of1mM/L IPTG, identified bySDS-PAGE and Western-blot and soluble analysis, we found that the recombinant proteinswere in the form of inclusion bodies. Then the recombinant proteins were purified by theNi-NTA Spin Kit. The purified pET-32a/OMP10was emulsified with complete Freundsadjuvant/incomplete Freunds adjuvant in the same volume, then immuned4-6weeks-oldKunming mice by subcutaneous multi-point injection. The Kunming mice were immuned forthree times at intervals of two weeks, then the heart blood was collected7days after the thirdimmunization. The antiserum was obtained at4℃centrifuge5000rpm/min, and centrifugedfor10min. Then the antiserum was analysised by Western blot.CONCLUSION: In this experiment, the cloning vectors and the expression vectorscontaining the three lipoprotein genes were successfully constructed, respectively. Severaltimes were tried to find the optimized expression conditions. The fusion proteins wereexpressed and purified, then obtained the high purity of the recombinant protein. The proteinslaid the foundation for further study of protein immunogenicity and anti-infection immunity.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, )
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