猪D型产毒素多杀性巴氏杆菌毒素分段基因的原核表达

根据GenBank已发表的产毒素多杀性巴氏杆菌（T＋Pm）toxA基因序列（AF240778）设计1对引物,从猪源D型T＋Pm中扩增出toxA基因片段（3858 bp）,再根据toxA基因序列设计3对引物,分别扩增出ZQ（1084 bp）、ZH（1092 bp）、HM（906 bp）3个分段基因,将其克隆至原核表达载体pET32a,转入大肠杆菌BL21中进行融合表达。经SDS-PAGE和West-ern blotting检测分析,发现ZQ、HM基因的表达产物以包涵体形式存在,大小分别为75、50 kDa,ZH基因不表达。动物试验结果表明,HM重组蛋白具有良好的免疫原性、反应原性及一定的生物学活性,而ZQ重组蛋白没有免疫原性、反应原性及生物学活性。通过间接ELISA的方法检测抗毒素的阳性猪血清,结果表明,HM重组蛋白较天然蛋白具有更高的特异性。

Prokaryotic expression of sub-toxA gene for toxigenic Pasteurella Multocida toxin of swine serotype D

A pair of primers was designed using toxA gene sequence of toxigenic Pasteurella multocida（T＋Pm）（Gen-Bank）,and a 3858bp fragment was amplified from toxigenic Pasteurella multocida with swine serotype D using PCR.With the template of toxA gene sequence,three pairs of primers were designed which amplified 1084bp（ZQ）,1092bp（ZH） and 906bp（HM） gene fragments.The acquired PCR products were inserted into prokaryotic expression pET32a（＋）,and trans-formed into E.coli BL21（DE3） for expression induced by IPTG.The expressed recombinant protein was detected using SDS-PAGE and western blotting.It was found that the expression products of ZQ and HM existed as inclusion body with 75 and 50 kDa,while ZH gave no expression.Mouse test results showed that HM recombinant protein had good immunological reaction and biological activities,while ZQ protein was found without any activity.The antitoxic positive serum of swine was detected by indirect ELISA method,and the specificity of the recombinant protein HM was found to be higher than that of negative protein.This study provides useful data for further use of the Pasteurella multocida toxin（PMT）.