细粒棘球蚴GST-mu2基因的克隆、表达及重组蛋白反应原性研究

Cloning and Expression of GST-mu2 Gene of Echinococcus granulosus and Reactionogenicity of Recombinant Protein

为了研究细粒棘球蚴(Echinococcus granulosus,Eg)谷胱甘肽S-转移酶mu 2(GST-mu 2)蛋白的反应原性,根据GenBank中Eg GST-mu2基因cDNA序列设计特异性引物,以Eg头节总RNA为模板,进行RT-PCR扩增,将PCR产物克隆到pMD19-T载体,测序验证后,将GST-mu2基因亚克隆至表达载体pET-28a中,构建pET-GST-mu2原核表达载体,将其转化至大肠杆菌BL21(DE3)感受态细胞中,用IPTG进行诱导表达,并对表达蛋白进行反应原性分析。结果表明,GST-mu2cDNA全长660个核苷酸,编码219个氨基酸,该多肽含有2个N端酰基化位点,2个PKC磷酸化位点,3个CKⅡ磷酸化位点,1个TYR磷酸化位点,抗原表位区集中在28~70、147~219位;SDS-PAGE可以检测到32kDa的蛋白特异性条带;Western blot分析显示,表达的pET-GST-mu2重组蛋白能与Eg阳性血清发生特异性反应,具有较强的反应原性,为进一步利用GST-mu2蛋白作为Eg感染诊断的候选抗原奠定了基础。

In order to study the reactogenicity of glutathione s-transferase mu 2(GST-mu2),specific primer derived from Echinococcus granulosus(Eg)genome database in GenBank was designed and the open reading frame(ORF)sequence of GST-mu2 was cloned by RT-PCR from hydatid protoscolex.Then the amplified product was cloned into pMD19-T vector,sequenced and subcloned into the expression vector pET-28 a.The recombinant pET-GST-mu2 expression vector was generated successfully and then was transformed into E.coli competent cells of BL21(DE3),induced by IPTG.Then the reactogenicity of recombinant protein GST-mu2 was analyzed.The results showed that GST-mu2 gene had a length of 660 bp,encoding 219 amino acids containing two N-acylation sites two CKⅡphosphorylation sites;three PKC phosphorylation sites;one TYR phosphorylation site.The epitopes focus on the amino acid from 28 to 70 and 147 to 219.The recombinant protein with a molecular weight of 32 kDa was examined by SDS-PAGE and further confirmed by Western blot,which displaying stronger reactionogenicity.This study laid a preliminary foundation for further Eg.diagnostic antigen based on GST-mu2 as a candidate protein.