减毒伤寒杆菌为载体的甲肝疫苗候选株的构建和免疫应答  

Construction of hepatitis A vaccine with attenuated Salmonella typhimurium as vector and its immune response

作　　者：胡玉琳[1] 牛俊奇[1] 苏秀芬[1] 

机构地区：[1]吉林大学第一医院传染科,吉林长春130021

出　　处：《吉林大学学报（医学版）》2004年第3期415-417,共3页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅资助课题 (95 35 4 3)

摘　　要：目的 :构建以减毒鼠伤寒杆菌为载体的甲肝疫苗候选株 ,并证明其免疫源性。方法 :将甲型肝炎病毒编码区 c DNA插入高效表达质粒 p BV2 2 1。转化大肠杆菌 DH5 α。用核酸探针和限制性内切酶筛选和鉴定阳性克隆。将阳性重组质粒用电转移法转化减毒鼠伤寒杆菌 BRD5 0 9。用抗生素平板筛选法筛选出转化成功的伤寒杆菌 ,用温度诱导法诱导目的基因在伤寒杆菌中表达。结果 :蛋白质印迹法 (Western blotting)和酶联免疫吸附测定(EL ISA)检测表明 ,表达蛋白可与人抗甲型肝炎 Ig G发生特异性反应。用此伤寒杆菌口服或腹腔注射免疫小鼠 ,小鼠血清检测到抗甲型肝炎病毒的抗体。结论 :以减毒鼠伤寒杆菌为载体的甲肝疫苗构建成功 ,并可在体内外产生免疫应答。Objective To construct hepatitis A virus vaccine with attenuated Salmonella typhimurium as vector, and study its immune response. Methods The plasmid pHAV5 containing hepatitis A virus genome cDNA was digested by restrict endonucleic acid enzyme, the 5′ non coding region was deleted, the remains were inserted into the high level expressive plasmid pBV221 and then were transformed into E coli DH5α. The positive clone was screened and identified by DNA probe and restrict endonucleic acid enzyme. The postive recombinant plasmid was used to transform attenuated Salmonella typhimurium BRD509 by electroporation. The transformed Salmonella typhimurium was screened in Amp control medium. The aim gene was expressed in Salmonella typhimurium by temperature induced method. Results The expressed proteins reacted specifically with HAVIgG antibody were demonstrated by ELISA and Western blotting. The transformed Salmonella typhimurium was administrated to mice by oral or abdominal injection, HAVIgG antibody was found in the mice sera. Conclusion Hepatitis A vaccine with attenuated Salmonella typhimurium as vector can induce immune response.

关 键 词：肝炎病毒 甲型 沙门菌 鼠伤寒 疫苗 减毒 甲肝疫苗 基因表这调控 病毒 

分 类 号：Q78[生物学—分子生物学]