论文部分内容阅读
目的探讨幽门螺杆菌(Hp)热休克蛋白A(HspA)在食品级乳酸乳球菌NZ3900菌株中的克隆表达及免疫反应性。方法利用基因重组技术构建乳酸乳球菌重组子NZ3900/pNZ8110-hspA;绘制生长曲线,观察hs-pA插入对乳酸乳球菌重组子生长影响及乳酸链球菌素(Nisin)对重组子生长影响;采用钠十二烷基硫酸盐-聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测HspA在乳酸乳球菌中的表达;应用western-blot鉴定重组子表达HspA的免疫反应性。结果成功扩增出幽门螺杆菌河南分离株(MEL-Hp27)的hspA基因,构建了hspA基因的乳酸乳球菌原核表达系统(NICE);细菌生长曲线显示hspA的插入未对乳酸乳球菌重组子的生长产生影响,除10 ng/mL Nisin对重组子生长影响较小外,20～100 ng/mL nisin对重组子的生长均有明显抑制;SDS-PAGE和Tricine SDS-PAGE检测均未观察到HspA条带;western-blot鉴定结果显示乳酸乳球菌表达的HspA抗原蛋白具有良好免疫反应性。结论HspA在乳酸乳球菌中的少量表达影响重组菌生长。
Objective To investigate the cloning, expression and immunoreactivity of Helicobacter pylori (Hp) heat shock protein A (HspA) in food grade Lactococcus lactis strain NZ3900. Methods Recombinant Lactococcus lactis NZ3900 / pNZ8110-hspA was constructed by gene recombination technique. The growth curve was drawn to observe the effect of hs-pA insertion on the growth of Lactococcus lactis recombinant and the effect of Nisin on the recombinant growth. The expression of HspA in Lactococcus lactis was detected by SDS-PAGE, and the immunoreactivity of recombinant HspA was identified by western-blot. Results The hspA gene of Helicobacter pylori isolates from Henan (MEL-Hp27) was successfully amplified and the Lactococcus lactis prokaryotic expression system (NICE) of hspA gene was constructed. The bacterial growth curve showed that hspA insertion did not affect the expression of Lactococcus lactis recombinant The results showed that 20 ~ 100 ng / mL nisin inhibited the growth of recombinant cells except for 10 ng / mL Nisin, while no HspA was observed by SDS-PAGE and Tricine SDS-PAGE Western blot analysis showed that Lactococcus lactis expressed HspA antigen protein has good immunoreactivity. Conclusion The low expression of HspA in Lactococcus lactis affects the growth of recombinant bacteria.