柯萨奇病毒A组16型VP1蛋白的原核表达及其免疫原性

Prokaryotic expression and immunogenicity of coxsackievirus group A type 16 VP1

目的原核表达柯萨奇病毒A组16型(Coxsackievirus group A type 16,CA16)VP1蛋白,并检测其免疫原性。方法通过RT-PCR法从CA16病毒青岛株中扩增VP1基因,克隆至原核表达载体pET43.1a(+)中,构建重组表达质粒pET43.1a-VP1,转化感受态E.coli Rossatte(DE3),IPTG诱导表达。表达的重组CA16 VP1蛋白通过Ni柱亲和层析纯化后,采用不同剂量(5、10、20、40μg)免疫BALB/c小鼠,ELISA法检测血清中特异性IgG、IgG1、IgG2a、IgG2b、IgG3抗体效价,微量细胞病变抑制法检测血清中和抗体效价。结果重组表达质粒pET43.1a-VP1经双酶切及测序证实构建正确;表达的重组CA16 VP1蛋白相对分子质量约为34 000,主要以包涵体形式存在,表达量占菌体总蛋白的15%;纯化的重组CA16 VP1蛋白纯度可达95%以上,可与猴CA16抗血清反应;不同剂量的重组CA16 VP1蛋白免疫BALB/c小鼠,可诱导产生CA16特异性抗体,血清中总IgG、IgG1、IgG2a、IgG2b、IgG3抗体效价均明显高于对照组,且抗体效价与免疫剂量存在一定的量效关系;各剂量CA16 VP1组免疫小鼠血清中和抗体效价均小于1∶8。结论已成功在大肠杆菌中表达了重组CA16 VP1蛋白,纯化的重组蛋白可诱导小鼠特异性体液免疫应答,为进一步研究CA16的结构、功能及相关疫苗的研制奠定了基础。

Objective To express coxsackievirus group A type 16 （CA16） in prokaryotic cells and determine its im- munogenicity. Methods VP1 gene was amplified from Qingdao strain of CA16 by RT-PCR and cloned into prokaryotic expression vector pET-43, la（＋）. The constructed recombinant plasmid pET43, la-VP1 was transformed to E. coli Rossatte （DE3） and induced with IPTG. The expressed recombinant VP1 protein was purified by nickel ion affinity chromatography. BALB/c mice were immunized with the purified VP1 protein at various dosages （5, 10, 20 and 40 μg） and determined for specific IgG, IgG1, IgG2a, IgG2b and IgG3 titers in sera by ELISA, and neutralizing antibody titer by micro-cytopathic effect inhibition assay. Results Both restriction analysis and sequencing proved that recombinant plas- mid pET43, la-VPI was constructed correctly. The expressed CA16 VP1 protein, with a relative molecular mass of about 34 000, mainly existed in a form of inclusion body and contained 15% of total somatic protein. The purified VP1 protein reached a purity of more than 95% and showed specific reaction with monkey antiserum against CA16. The recombinant VP1 at various dosages induced specific antibody against CA16 in BALB/c mice. The titers of total IgG, IgG1, IgG2a, IgG2b and IgG3 titers in sera of immunized mice were significantly higher than those in control group, which were dose- dependent at a certain degree. However, all the neutralizing antibody titers in sera of mice immunized with VP1 at vari- ous dosages were less than 1 ： 8. Conclusion The CA16 VP1 protein was successfully expressed in E. coli, and induced specific humoral immune response in mice after purification, which laid a foundation of further study on structure and function of CA16 and development of the relevant vaccines.