摘要
目的将钩端螺旋体外膜基因ｏｍｐＬ１克隆到大肠杆菌－卡介苗（Ｅ．ｃｏｌｉ－ＢＣＧ）穿梭质粒载体ｐＹ６００２中，将重组质粒导入ＢＣＧ并对重组ＢＣＧ的表达进行初步表达研究。方法采用ＰＣＲ技术直接从致病性钩端螺旋体（钩体）赖型０１７株基因组中扩增钩体外膜蛋白基因ｏｍｐＬ１，并克隆到Ｅ．ｃｏｌｉ－ＢＣＧ穿梭质粒载体ｐＹ６００２中，重组质粒经电转化导入ＢＣＧ。斑点杂交筛选重组ＢＣＧ，再通过免疫印迹对其表达进行初步研究。结果在所得６个重组ＢＣＧ中，有３个表达了ｏｍｐＬ１基因产物，其中一个表达较强。结论本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。
Leptospiral outer membrane antigen gene ompL1 was amplified from the genome of pathogenic leptospira serovar lai 017 by PCR,and cloned in E.coliBCG shuttle plasmid pY6002.Recombinant plasmids were isolated by dot blotting with Digoxigeninlabelled 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 transformants were recombinants,in which,the ompL1 gene has been integrated into the genome of BCG. By immunoblot with Leptospira serovar 017 infected rabbit antiserum,which was preabsorbed to remove antibody against E.coli 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
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1998年第2期98-102,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金
华西医科大学基础医学院青年基金
