产气荚膜梭菌α毒素基因的克隆表达和生物学活性的鉴定  

Cloning and expression of α toxin gene of Clostridium perfringens and its biological activity

作　　者：梁光军[1] 田银芳 文其乙[1] 张小荣[1] 潘志明[1] 高玉[3] 高崧[1] 焦新安[1] 刘秀梵[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,扬州225009 [2]扬州大学实验农场 [3]苏北人民医院

出　　处：《中国人兽共患病杂志》2004年第7期573-576,共4页Chinese Journal of Zoonoses

摘　　要：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?Aim To express the functional α toxin in the prokaryotic expression system,a pair of primers was designed specific for applification of α toxin gene of Clostridium perfringens ,and the gene of α toxin was amplified by polymerase chain reaction (PCR).The PCR product was recovered and inserted into the inducible expression vector pGEX6p-1.The recombinant plasmid pGEX6p-1-cpα was transformed into E.coli BL21.After induction with IPTG,the expressed product was revealed by SDS-PAGE from recombinant E.coli BL21 evaluated its activities of the RBC(red blood cells) lysis,phospholipase C,and its toxicity to mice after intraperitoneal injection.The results showed China that the α toxin gene was successfully inserted into pGEM(r) T easy and expression plasmid pGEX6p-1 was identified by nucleotide sequence and endonuclease digestion.The result of SDS-PAGE showed a new protein was expressed,which has biological activities such as the activities of lysing RBC function,phospholipase C and lethal attack on mice.In conclusion,the recombinant bacterium BL21(pGEX6p-1-cpα) has expressed the α toxin in an active form.

关 键 词：产气荚膜梭菌 Α毒素基因 克隆表达 生物学活性 鉴定 聚合酶链式反应 

分 类 号：R378.8[医药卫生—病原生物学]