Research On Gene Clone And Fusion Protein Immunogenicity Of L7/L12 And OMP31 From Brucella

Brucellosis is a kind of disease which caused by brucella infect both human and animals. Brucellosis primarily leads to undulant fever and chronic infection, which cause abortion and infertile. It is prevalent in about 170 countries and regions at present. It has brought huge damage to people's health and development of animals breeding. Prevention, control and even eliminate brucellosis has become the most pressing matter of the moment.The attenuated vaccine (S19 and Rev.1) have played a certain action currently. But it has been disturbing all of the brucellosis researchers because of its insecurity, hard to identify and inconvenient to vaccine.Along with the advance of the molecular biology technique, accomplish of the brucella's cOmplete genome sequencing.The biological characteristics of several protective antigens and virulence-associated genes was revealed.Such as the ribosomal protein L7/L12, OMP31, virulence-associated genes Pmm,WboA,pgm and so on. At present the investigators use the genetic engineering vaccine constructing DNA vaccine and recombinant subunit vaccine. It is very hopeful to develop a new kind of safe and effective vaccine.In this study we worked on according to the published gene sequence. We chose the protective antigens L7/L12 and OMP31 which were both extremely conserved sequences between species and amplify with PCR technology. We used the restriction sites designed on the primer carrying on gene recombination, cloning the recombination gene to the express vector which had been constructed. Through the PCR detect and the digestion of restriction enzyme, we know the sequence was correct, and we constructed the effective express vector PME290-SDLOmp.We transformed competence (PAK/2PFS)with constructed vector. After bringing up the bacteria we got the expression product which was identified correct by the SDS-PAGE and Western Blot. We analyzed the recombination protein with DNASTAR 7.1 and proved that the protein was well hydrophilic. After animal was immunized by fusion protein, we found that the protein has good immunogenicity. At the same time, we cloned the gene L7/L12 to the vector pSTV29, transformed competence BL21, which was induced expression by IPTG. It provided detection antigen for identifying fusion protein. Our study had established the further develop of brucella subunit vaccine and the brucella detection technique.