钩端螺旋体外膜抗原基因OMPL1在卡介苗中的克隆和初步表达

Cloning and Expression of Leptospiral Protective Antigen Gene OmpL1 in BCG

采用ＰＣＲ技术直接从致病性钩端螺旋体（简称钩体）赖型０１７菌株基因组中扩增钩体外膜蛋白基因ＯｍｐＬ１，并克隆到大肠杆菌—卡介苗穿梭质粒载体ｐＹ６００２中，重组质粒经电转化导入卡介苗。斑点杂交筛选重组卡介苗，再通过免疫印迹对其表达进行初步研究。在所得６个重组卡介苗中，有３个表达了ＯｍｐＬ１基因产物，其中一个表达较强。该研究为发展新一代高效广谱的钩体基因工程疫苗打下了基础。

This study was intended to produce a new living vaccine against leptospirosis using BCG as vector. Leptospiral outer envelop antigen gene OmpL1 was amplified from the genome of pathogenic leptopira serova Lai 017 by PCR, and cloned in E.coliBCG shuttle plasmid pY6002. Recombinant plasmids were isolated by dot blotting with Digoxigeninlabeled OmpL1 gene. After transforming the recombinant plasmids in BCG (Shanghai strain) by electroporation, the genomic DNA of all 21 transformants were prepared and hybridized with OmpL1. It showed that 6 of the 21 transfomants were recombinants in which the OmpL1 gene had been integrated into the genome of BCG.By immunoblotting with OmpL1 infected rabbit antiserum, which was preabsorbed to remove antibody against E. coli and SPAHRP, three recombinants, pLI1, pLI2 and pLI3, were detected to express OmpL1 protein. The ability of expression is in the order of pLI2>pLI1>>plI3. These studies provide the possibility of further research on the development of highly efficient recombinant vaccines against leptospirosis.