2型猪链球菌烯醇化酶的原核表达及其对体外血脑屏障模型通透性影响的检测

Prokaryotic expression of enolase from Streptococcus suis 2 and its effect on blood brain barrier permeability

通过原核表达的方式获得能在多种病原菌表面表达的烯醇化酶(Enolase,Eno),探索Eno在脑膜炎致病过程中的作用。根据GenBank上公布的基因序列设计Eno特异性引物,通过PCR技术扩增2型猪链球菌(Streptococcus suis serotype 2,SS2)的Eno基因序列,克隆到pMD18-T载体,进一步亚克隆到原核表达载体pET28a(+),构建重组表达质粒pET28a-Eno,转化入大肠杆菌BL21Codon Plus(DE3)感受态中诱导表达,经镍离子螯合柱纯化后,检测其对体外血脑屏障模型通透性的影响。结果显示,经1mmol/L IPTG诱导5h为最佳诱导条件,目的蛋白约54 000,与预期蛋白大小相符合;纯化的蛋白Western blotting分析表明,其具有较好的免疫原性;Eno可致体外血脑屏障模型通透性增加。结果表明,Eno作为SS2潜在的毒力因子在SS2引起脑膜炎的致病过程中起到重要的作用。

Enolase（Eno） had a strong affinity for plasminogen that was expressed on the surface of several pathogens,and played an important role in host invasion and metastasis. In order to explore the function of Eno in the pathogenesis of meningitis caused by Streptococcus suis serotype 2,we expressed Eno fused protein by prokaryotic expression. A pair of specific primers was designed for the Enolase,and the target DNA fragment was amplified by PCR. The amplified fragments were inserted into pMD18-T vector,and then subcloned into the expression vector pET28a （＋）, constructing a recombinant expression vector pET28a-Eno. The pET28a-Eno was transformed into E. coli BL21（DE3） and induced by IPTG. The fused protein were purified by using Ni2＋-NTA chelating column. The effect of Eno on permeability of BBB was observed. The results of SDS-PAGE indicated that a 54 000 protein clould be expressed and reach the maximum amount under the induction of 1mmol/L IPTG for 5 hours. Western blotting demonstrated that the purified protein displayed a great immunobiological activity. Eno can increase the permeability of BBB in vitro. In a conclusion, Enolase as a potential virulence factor may play a role pathogenesis of meningitis caused by Streptococcus suis serotype 2.