霍乱毒素B亚单位在乳酸乳球菌食品级表达系统中的分泌性表达  

Secretion expression of cholera toxin B subunit in food-grading Lactococcus lactis expression system

作　　者：孙强正[1] 李振军[1] 李娟[1] 王艺婷[1] 金东[1] 郑霄[1] 张晓嫒[1] 熊衍文[1] 叶长芸[1] 徐建国[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《中华流行病学杂志》2009年第12期1288-1291,共4页Chinese Journal of Epidemiology

基　　金：基金项目：中国疾病预防控制中心青年科研基金课题（2009A103）

摘　　要：目的 在乳酸乳球菌（Lactococcus lactis）食品级分泌性表达系统L．lactis MBP71／pSQZ和L．lactisMBP71／pSQ中克隆表达霍乱毒素B亚单位（CTB），实现CTB的分泌性表达。方法采用PCR扩增霍乱弧菌569B菌株染色体中CTB蛋白编码基因片段，克隆到食品级分泌表达载体pSQZ和pSQ中，转化入宿主菌L．1actisMBP71，构建CTB的分泌性表达系统L．lactisMBP71／pSQz—ctB和LlactisMBP71／pSO—ctB；采用Western．blot检测重组菌株中CTB的表达及表达量。结果PCR扩增得到CTB编码基因片段ctB，酶切、连接到载体pSQZ和pSQ并转化到宿主菌L．lactisMBP71中，构建了分泌性表达系统L．1actisMBP71／pSQZ，ctB和L．lactisMBP71／pSQ．ctB。Western．blot检测发现，L．lactisMBP71／rISQ．ctB和L．1actisMBP71／pSQZ．ctB系统均能分泌性表达CTB，L．lactisMBP71／pSQ—ctB上清的表达量约为2μg/ml；L．lactisMBP71／pSQ—ctB的表达效率远远高于L．lactisMBP71／pSQZ—ctB。结论成功实现了CTB在食品级表达系统中的分泌性表达。Objective To clone and secretion express cholera toxin B subunit （CTB） in food-grading Lactococcus lactis expression systems. Methods ctB fragment that encoding CTB was amplified by polymerase chain reaction （PCR） using the genomic DNA of Vibrio cholera strain 569B as template and was inserted into two secretion expression vector pSQZ and pSQ to construct food-grading expression system L.lactis MBP71/pSQZ-ctB and Llactis MBP71/pSQ-ctB. The expressed CTB was detected by Western-blot assay. Results The ctB fragment was successfully amplified from Vibrio cholera strain 569B and inserted into two secretion expression vectors pSQZ and pSQ to construct food-grading expression system L. lactis MBP71/pSQZ-ctB and L. lactis MBP71/ pSQ-ctB. Western-blot assay demonstrated that CTB was secretion and expressed from L.lactis MBP71 harboring vectors pSQZ-ctB and pSQ-ctB, and the quantity of CTB secreted by L. lactis MBP71/pSQ-ctB was about 2μg/ml, higher than that of L. lactls MBP71/pSQZ-ctB. Conclusion CTB was successfully secreted and expressed by food-grading L. lactis expression systems.

关 键 词：乳酸乳球菌 霍乱毒素B亚单位 食品级载体 分泌性表达 

分 类 号：R392[医药卫生—免疫学]