幽门螺杆菌1pp20基因在大肠杆菌TB1中诱导表达方法的研究

目的 :研究影响幽门螺杆菌lpp2 0基因在大肠杆菌TB1中表达的因素 ,探索高效表达的条件 ,为幽门螺杆菌疫苗抗原的纯化和生产奠定基础。方法 :用PCR方法从幽门螺杆菌的染色体DNA上扩增出lpp2 0基因片段 ,将目的基因插入的表达载体pMAL c2X中 ,用重组质粒转化大肠杆菌 (E .coliTB1)。采用不同的诱导时间、IPTG浓度和诱导温度进行诱导表达 ,应用SDS PAGE方法对表达产物进行分析。结果 :在诱导 1～ 4h之间 ,融合蛋白的表达量变化较为显著 ,而 4～ 8h之间则无显著变化 ;IPTG终浓度低于 0 .2mmol/L或高于3 .2mmol/L均能显著减少融合蛋白的表达量 (P <0 .0 5 ) ,而IPTG终浓度在 0 .4～ 2 .4mmol/L之间时 ,对融合蛋白的表达量并无显著影响 ;诱导温度在 3 0℃～ 40℃之间变化对融合蛋白的表达量无显著影响 ,超出此范围可使表达量显著减少 (P <0 .0 5 ) ;在优化诱导条件后 ,融合蛋白的表达量可达全菌总蛋白的 3 4%。结论 :通过调整诱导时间、IPTG浓度和诱导温度 ,lpp2 0基因与载体pMAL c2X的重组质粒在E .coliTB1中能够高效表达。

Study on the methods for expression of the lpp20 gene of helicobacter pylori in escherichia coli TB1

Objective: To investigate the factors influencing expression level of the lpp20 gene of helicobacter pylori(Hp)in escherichia coli TB1 and the conditions under which the gene can be expressed most efficiently in order to construct a basis for purification and production of the antigen as an expected vaccine.Methods: The lpp20 gene of Hp was amplified from Hp chromosomal DNA by PCR techniques. The PCR product was inserted into the expression vector pMAL c2X. The recombinant vector was introduced into E. coli TB1, where the lpp20 gene was expressed at a variety of temperature, density of IPTG, and induction time. The expression products were analyzed using the SDS PAGE method.Results: During the induction time from 1?h to 4?h, the expression level kept changing markedly, while no remarkable change was found from 4?h to 8?h.No prominent difference had been found in expression of lpp20 gene using IPTG of 0.1～4.0 mmol/L. Using IPTG with a density of less than 0.2 mmol/L or more than?3.2?mmol/L,?expression level could? be? decreased? markedly; No?significant?difference hadbeen observed when the induction temperature was changed between 30℃～40℃ ;Under reasonable conditions for induction ,amount of the recombinant gene expression products could reach as high as 34% total proteins of the host.Conclusion :The recombinant vector constructed with pMAL-c2X and lpp20 gene could be expressed at a high level by adjustment of the induction conditions ,namely temper ature ,IPTG and induction time .