猪链球菌2型烯醇化酶的分子克隆与免疫学特性

Molecular cloning and immunological characterization of enolase from Streptococcus suis 2

摘要:

对新近测定的猪链球菌2型(S. suis 2) 05ZYH33全基因序列进行生物信息学分析, 并与相关家族蛋白进行同源性比较, 设计合成引物, PCR法扩增出约1.3 kb的烯醇化酶编码基因 (enolase, eno), 将其克隆入pMD-18T载体中, 进一步亚克隆入表达载体pET32a。将重组表达质粒pET32a::eno转化E. coli BL21 (DE3), 经IPTG诱导表达后, SDS-PAGE初步检测到分子量约为75kD的蛋白带。通过His-Tag亲和层析纯化, 获得融合蛋白His-ENO。Western-blot表明该表达产物具有免疫原性。基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S. suis 2 05ZYH33细菌的表面。这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用。

Abstract:

To understand the enolase (eno) gene and its product in Streptococcus suis serotype 2 (S. suis 2), bioinformatics was adopted to analyze the whole genome sequence of the Chinese strain 05ZYH33 of S. suis 2. A highly homologous eno gene was unveiled by the genome-wide mining. A pair of specific primers was designed for the eno, and the target DNA fragment of 1.3 kb was successfully amplified using the genomic template of 05ZYH33. Subsequently, eno gene was inserted into pMD18-T vector, and then subcloned into prokaryotic expression vector pET32a, generating a recombinant expression plasmid pET32a::eno. The resulting plasmid was confirmed by direct DNA sequencing and transformed into E. coli BL21 (DE3) competent cells. Protein expression analysis showed that a 75 kD protein can be observed in 12% SDS-PAGE, indicating that the recombinant 6His-fused ENO protein can be produced in E. coli under the induction of IPTG. Western-blot experiment demonstrated clearly it shares strong specific antigenicity. Moreover, ELISA result suggested that ENO can occur on the surface of 05ZYH33 strain. Together, our data supported that ENO can function as a novel antigen, and may play pivotal roles in the severe infection of S. suis 2.