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.lactis MBP71/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.lactis MBP71,构建CTB的分泌性表达系统L.lactis MBP71/pSQZ-ctB和L.lactis MBP71/pSQ-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.lactis MBP71中,构建了分泌性表达系统L.lactis MBP71/pSQZ-ctB和L.lactis MBP71/pSQ-ctB.Western-blot检测发现,L.lactis MBP71/pSQ-ctB和L.lactis MBP71/pSQZ-ctB系统均能分泌性表达CTB,L.lactis MBP71/pSQ-ctB上清的表达量约为2 μg/ml;L.lactis MBP71/pSQ-ctB的表达效率远远高于L.lactis MBP71/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.

abstractsObjective 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 L.lactis 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 2 μg/ml,higher than that of L.lactis MBP71/pSQZ-ctB.Conclusion CTB was successfully secreted and expressed by food-grading L.lactis expression systems.