Cloning, Expression Of The LAg42 Membrane Protein Gene Of Leptospira Lai And Its Effects On Pathogenicity And Immunogenicity

Leptospirosis, caused by infection with pathogenic Leptospira spp., is an important zoonotic disease that is distributed worldwide. Our country is badly endangered by Leptospirosis, which makes it impressive to prevent this disease. Through a lot of effort, we have made great progress in the thearapy of this disease, however, we still know little about the mechanism of it and existing vaccine only can produce short period and incomplete protection.There are more than 230 serovars among the pathogenic leptospires; this diversity of serovars among leptospires is attributed to differences in LPS carbohydrate composition. Leprospiral LPS is a protective immunogen that is generally specific for each serovar, but can't protect for other serovars. In contrast to LPS, however, protein extracts prepared from a pathogenic Leptospira isolate can induce protective immunity against challenge with a heterologous serovar in an experimental animal model. This result indicates that leptospiral proteins are potential candidates for a new vaccine that generates broad cross-protection beyond serovars. Identification of immunogenic proteins of leptospiral proteins, such as LipL41, OmpL1 and LipL32 (also known as Hapl), are found to be protective immunogens that are conserved among pathogenic leptospires.In 2003, by using convalescent sera from a animal model,a novel antigen of L.manilae UP-MMC strain, designated LAg42 was identified by Nobuo Koizumi. The results of its research suggest that LAg42 may be involved in virulence and may also be a target for protective immune response. In this study, it is aimed to research LAg42 in the Leptospira lai.First, Lag42 gene was amplified by PCR from genome of Leptospira lai 017 strain, 56601 strain and Patoc I , and was subcloned into vector pET-32 a( +). DNA sequence was performed subsequently. Later, the structure of Lag42 was analyzed by Dnaman, NCBI and SignalP, furthermore it was expressed in E.coil BL21. The LAg42 fused protein which had a 6-His tag was expressed after induction by IPTG. Then the LAg42 fusion protein was purified by His-affinity column, and the antibodiy was prepared by immunize the Newzeland rabbit. In the aspect of eukaryotic expression, Lag42 gene was subcloned into vector pcDNA3.1A~+. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme analysis, it was transfected into COS7 cell by liposome. Its expression was analysed by RT-PCR.In our experiment, a 1100bp fragment was amplified in various virulent Leptospira interrogans serovar lai, not in Leptospira bilexa Patoc I strain. The recombinant plasmid was constructed triumphantly. There is a high homology of DNA squence of the various leptospira lai. The fused protein was successfully expressed in E.coil BL21. SDS-PAGE showed that the molecular weight of the protein was about 42KDa and the fused protein LAg42 was soluble in E.Coli BL21 cytoplast mainly. The analysis of restrication enzyme indicated that the eukaryotic recombinet vector was correctly constructed. The result of RT-PCR showed that the cell transfected with recombinant plasmid got a specific amplified fragment, but the cell transfected with blank plasmid had not this band. It suggested that pcDNA3.1A~+-lag42 can express in mammalian cell.These results suggested that Lag42 gene may be involved in virulence and may also be a targe for protective immune response which maybe a candidate gene for DNA vaccine.