肠出血性大肠杆菌O157:H7溶血素保护性片段(HlyAN436)克隆、表达、初步纯化与生物学活性研究  

Cloning,expression,primary purification and biological activities of HlyAN436 gene of enterohaemorrhagic Escherichia coli O157:H7

作　　者：程建平[1] 邹全明[2] 易勇[2] 毛旭虎[2] 马颖[2] 王庆旭[2] 丁红雷[2] 

机构地区：[1]淮安市第二人民医院检验科,江苏223002 [2]第三军医大学临床微生物及免疫学教研室,重庆400038

出　　处：《中国免疫学杂志》2007年第5期407-410,414,共5页Chinese Journal of Immunology

基　　金：军队杰出人才基金项目(04J009)

摘　　要：目的获取重组表达肠出血性大肠杆菌O157的溶血素(Ehx)保护性片段,并研究其基本的生物学及免疫学特性。方法PCR法从EHECO157-SMR2菌株克隆O157:H7的HlyA亚单位的N端片段(436个氨基酸),T-A克隆后构建表达载体pET-28a(+)-HlyAN436,测序鉴定后转化到E.coliBL21(DE3),IPTG诱导表达,初步纯化后免疫动物。结果Hly-AN436成功地在大肠杆菌表达系统表达,动物实验证实其具有良好的免疫原性和反应性以及一定的免疫保护性。结论Hly-AN436蛋白有可能用于肠出血性大肠杆菌O157基因工程疫苗的研究。Objective To obtain the recombinant protective segment of Enterohemorrhagic E. coli toxin （HlyAN436） and to analyze the biological and immunological characteristics of the protein. Methods Amplifing gene HlyAN436 by PCR, the target gene was cloned into pET-28a（ ＋ ） plasmid after T-A cloning. Then the recombinant plasmid was transformed into E. coli BL21 （DE3） and the target protein induced by IPTG. After primary purification, HlyA436 was used to immunize 2 rabbits and 12 mice. After immunizing, oral challenge these mice with EHEC O157-SMR2（streptomycin-resistant）. Results HlyA436 was successfully expressed in prokaryotie expression system, have elicited high titer antiserum and has certain protective efficacy. Conclusion The results suggested that HlyAN436 recombinant protein may be used for development of Enterohemorrhagic genetic engineering vaccine.

关 键 词：肠出血性大肠杆菌 溶血素 基因克隆 表达 纯化 免疫保护 

分 类 号：R378.21[医药卫生—病原生物学]