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目的 利用原核表达系统表达具有生物学活性的产气荚膜梭菌α毒素。方法 设计针对α毒素基因的一对引物 ,用聚合酶链式反应 (PCR)技术扩增出α毒素的全基因。经测序确认正确后 ,回收的α毒素目的条带与 pGEX6 p - 1表达载体经限制性内切酶XhoⅠ和EcoRⅠ双酶切后 ,进行连接 ,转化大肠杆菌BL2 1。诱导表达后 ,做SDS -PAGE电泳 ,出现特异的表达产物条带。表达产物做溶血活性、卵磷脂水解活性和腹腔接种小鼠实验。结果 构建的重组质粒经内切酶XhoⅠ和EcoRⅠ双酶切后 ,发现特异的目的基因条带。SDS -PAGE电泳后 ,发现重组菌株有特异的表达条带。实验证明表达产物具有溶血活性、卵磷脂水解活性和腹腔接种后致死小鼠特性。结论 重组菌株BL2 1(pGEX6p - 1-cpα)表达了具有活性的α毒素蛋白。目的 利用原核表达系统表达具有生物学活性的产气荚膜梭菌α毒素。方法 设计针对α毒素基因的一对引物 ,用聚合酶链式反应 (PCR)技术扩增出α毒素的全基因。经测序确认正确后 ,回收的α毒素目的条带与 pGEX6 p - 1表达载体经限制性内切酶XhoⅠ和EcoRⅠ双酶切后 ,进行连接 ,转化大肠杆菌BL2 1。诱导表达后 ,做SDS -PAGE电泳 ,出现特异的表达产物条带。表达产物做溶血活性、卵磷脂水解活性和腹腔接种小鼠实验。结果 构?
Objective To express the biologically active Clostridium perfringens alpha toxin using prokaryotic expression system. Methods A pair of primers targeting α toxin gene was designed and the whole gene of α toxin was amplified by polymerase chain reaction (PCR). After confirmed by sequencing, the target fragment of α - toxin recovered was digested with restriction endonucleases Xho Ⅰ and EcoR Ⅰ by pGEX6 p - 1 expression vector and ligated into E. coli BL21. After induction of expression, SDS-PAGE electrophoresis was performed and a specific band of the expression product appeared. The expression products were tested for hemolytic activity, lecithin-hydrolyzing activity and intraperitoneal inoculation in mice. Results The constructed recombinant plasmids were digested with endonucleases Xho Ⅰ and EcoR Ⅰ to find the specific gene band. SDS-PAGE electrophoresis, recombinant strains were found to have specific expression bands. Experiments show that the expression product has hemolysis activity, lecithin hydrolysis activity and lethality after inoculation in mice. Conclusion The recombinant strain BL2 1 (pGEX6p - 1 - cpα) expressed the active α - toxin protein. Objective To express the biologically active Clostridium perfringens alpha toxin using prokaryotic expression system. Methods A pair of primers targeting α toxin gene was designed and the whole gene of α toxin was amplified by polymerase chain reaction (PCR). After confirmed by sequencing, the target fragment of α - toxin recovered was digested with restriction endonucleases Xho Ⅰ and EcoR Ⅰ by pGEX6 p - 1 expression vector and ligated into E. coli BL21. After induction of expression, SDS-PAGE electrophoresis was performed and a specific band of the expression product appeared. The expression products were tested for hemolytic activity, lecithin-hydrolyzing activity and intraperitoneal inoculation in mice. Results structure?