丝状支原体丝状亚种SC型脂蛋白QN末端基因的表达、纯化与抗原性分析

Expression, purification and antigen activity analysis of the N-terminal domain of lipoprotein LppQ of Mycoplasma mycoide subsp. mycoides SC

为了表达丝状支原体丝状亚种SC型(MmmSC)中国分离株HVRIⅩ脂蛋白Q(LppQ)N末端基因,将该基因经PCR扩增后克隆至原核表达载体pET32a中,经酶切、PCR、测序证实获得了重组表达质粒,转化Escherichia coliBL21(DE3)菌,经IPTG诱导后获得可溶性融合蛋白,表达量占菌体总蛋白的53.7%,用Ni-NTAHis.Bind纯化试剂盒纯化后,蛋白纯度达95%以上。表达蛋白经Western blot检测其抗原活性,结果表明纯化蛋白可与CBPP标准阳性血清发生强烈的反应,而与阴性血清不发生反应。

The gene sequence coding the N-terminal domain of LppQ was amplified from Mycoplasrna mycoides subsp. mycoides SC （MmmSC） HVRI X strain by PCR using special primers and was cloned into the EcoR Ⅰ/Sal Ⅰ sites of pET32a vector to construct the expression recombinant plasmids. The recombinant plasmids were indentified by restriction digestion, PCR and sequence analysis. The gene was overexpressed in Escherichia coli BL21 （DE3）host cell and the soluble protein was purified with Ni-NTA His· Bind purification kits. The amount of recombinant protein reached 53.7 % of the total mass of bacterial protein. The purity of recombinant protein reached to over 95 %. The antigen activity of the purified protein was examined with Western blot analysis. The purified protein reacted strongly with the standard positive serum and didn＇t react with the negative sera of contagious bovine pleuropneumonia（CBPP）.