乳糖诱导对幽门螺杆菌UreB、HpaA和大肠杆菌LTKA63、LTB重组蛋白表达的影响  

Effects of lactose inducing on expression of Helicobacter pylori rUreB and rHpaA,and Escherichia coli rLTKA63 and rLTB

作　　者：钊守凤[1] 严杰[1] 胡爱萍[1] 

机构地区：[1]浙江大学医学院病原生物学教研室,杭州浙江310006

出　　处：《浙江大学学报（医学版）》2004年第6期519-523,共5页Journal of Zhejiang University(Medical Sciences)

基　　金：国家教育部优秀年轻教师基金;浙江省科技厅科研基金 (0 0 1110 4 38)资助项目

摘　　要：目的 :探讨乳糖诱导幽门螺杆菌尿素酶 B亚单位 (ure B)、黏附因子 (hpa A)和大肠杆菌肠毒素 B亚单位(lt B)、A亚单位突变体 6 3(lt KA6 3)重组基因表达的效果及最佳表达条件。方法 :采用 BIO- RAD凝胶图象分析系统测定表达的目的重组蛋白产量。采用 SDS- PAGE检测不同乳糖浓度、诱导时间和温度对不同生长阶段工程菌重组蛋白表达量的影响 ,并与异丙基硫代半乳糖苷 (isopropyl- β- D- thiogalactoside,IPTG)比较。结果 :乳糖较 IPTG更为有效地诱导 r Hpa A、r Ure B、r LTB、r L TKA6 3的表达。 37℃诱导的目的重组蛋白表达量明显高于 2 8℃。r Hpa A表达的最佳条件为起始菌液 OD60 0 值 0 .8、5 0 g/L乳糖 ,诱导 4 h;r Ure B和 r LTB均为起始菌液 OD60 0 值 1.2、10 0 g/L乳糖 ,诱导 5 h;r LTKA6 3为起始菌液 OD60 0 值 0 .8、10 0 g/L乳糖 ,诱导 4 h。结论 :乳糖较 IPTG更有效地诱导上述重组基因表达目的蛋白 ,且无毒和廉价 ,为幽门螺杆菌基因工程疫苗工业化生产奠定了有利的基础。Objective: To determine the effects of lactose inducing on the expression of recombinant Helicobacter pylori rUreB and rhpaA,and Escherichia coli rLTB and rLTKA63. Methods: BIO RAD gel image analysis system was applied to detect the outputs of the recombinant proteins.SDS PAGE was performed to measure the target protein expression of recombinant genes at various periods of growth,different lactose concentrations,various inducing temperatures and times.The results of the target protein expression induced by lactose were compared to those by isopropyl β D thiogalactoside (IPTG). Results: Lactose showed higher efficiency to induce the expression of rHpaA,rUreB,rLTB and rLTKA63 than IPTG.The expression outputs of target recombinant proteins induced at 37℃ were remarkably higher than those at 28℃.The optimal expression parameters were 0.8 of OD 600 value,50 g/L of lactose,4 hours of inducing time for rHpaA,and 1.2 of OD 600 value,100 g/L of lactose,5 hours of inducing time for both the rUreB and rLtB,and 0.8 of OD 600 value,100 g/L of lactose,4 hours for rLTKA63. Conclusion: Lactose,a sugar with non toxicity and low cost,is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG. [

关 键 词：幽门螺杆菌 基因工程疫苗 乳糖 IPTG 诱导 表达 

分 类 号：R346[医药卫生—基础医学]