细粒棘球蚴EG19抗原基因的克隆表达及重组蛋白反应原性研究

Cloning and expression of EG19 gene of Echinococcus granulosus and reactionogenicity of recombinant protein

为研究细粒棘球蚴（Echinococcus granulosus,Eg）中的原头蚴特异性抗原Eg19蛋白的反应原性,根据Gen Bank中Eg19抗原基因c DNA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p MD19-T载体,测序验证后,将Eg19抗原基因亚克隆至表达载体p ET-32a中,构建p ET-Eg19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,并对表达的蛋白进行纯化及反应原性分析。结果 Eg19 c DNA全长534个核苷酸,编码177个氨基酸,等电点为4.54。该多肽含有2个N端酰基化位点,1个PKC磷酸化位点,1个CKⅡ磷酸化位点,1个ATP/GTP结合位点基序A（P-loop）;抗原表位区集中在20~93、96~146位;为亲水性蛋白。SDS-PAGE可以检测到35 k Da的蛋白特异性条带;Western blot结果显示,表达的重组蛋白Eg19抗原能与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Eg19抗原蛋白作为Eg感染诊断中的候选抗原奠定了前期基础。

In order to study the reactionogenicity of Eg19 gene,primers derived from the Echinococcus granulosus（ Eg） genome database in Gen Bank were designed and the open reading frame（ ORF） sequence of Eg19 was amplified by RT-PCR from hydatid protoscolex. Then the amplified products were cloned into the p MD19-T vector,sequenced and subcloned into the expression vector p ET-32 a. The recombinant p ET-Eg19 expression vector was generated successfully and then was transformed into the E. coli competent cells of BL21（ DE3）,induced by IPTG. Finally,the reactionogenicity of the recombinant protein Eg19 was analyzed. The results showed that Eg19 had a length of 534 bp,encoding 177 amino acids that contained two N-myristoylation sites,one CKⅡ phosphorylation site,one PKC phosphorylation site and one ATP/GTP-binding site motif A（ P-loop）. The epitope regions are concentrated on the amino acids from 20 to 93 and 96 to 146. The recombinant protein with a molecular weight of 35 k Da could be identified by SDS-PAGE and was confirmed by Western blot,which displayed strong reactionogenicity. This study laid a preliminary foundation for diagnosis of E. g. infection using Eg19 as a candidate antigen.