棘阿米巴HSP20蛋白的原核表达及免疫原性鉴定

Prokaryotic expression and determination of the immunogenicity of Acanthamoeba HSP20

目的重组表达棘阿米巴热休克蛋白20(Heat shock protein 20,HSP20),并对HSP20作为疫苗候选分子的免疫活性进行验证。方法以棘阿米巴滋养体cDNA为模板,PCR扩增HSP20基因,构建pET-22b-HSP20原核表达载体,并将其转化大肠杆菌感受态细胞BL21;采用1mmol/L IPTG诱导重组HSP20蛋白(Recombinant heat shock protein 20,rHSP20)表达,并进行可溶性分析。采用His亲和层析法纯化r-HSP20,并免疫家兔制备兔多克隆抗体;采用ELISA试剂盒检测多克隆抗体IgG1及IgG2a分型;Western blot鉴定r-HSP20的免疫反应性。结果经PCR扩增得到579bp的目的基因片段,与预期大小一致;经菌落PCR及双酶切证明成功构建pET22b-HSP20原核表达载体;经可溶性分析r-HSP20主要存在于重组菌超声破碎上清中;经SDS-PAGE鉴定柱层析300mmol/L咪唑洗脱的蛋白为高纯度r-HSP20;用该蛋白免疫获得的兔多克隆抗体效价为1∶12800,其抗体亚型IgG1及IgG2a含量分别为183.8mg/ml和21.4mg/ml;Western blot鉴定r-HSP20能被兔多克隆抗体识别。结论成功构建pET-22b-HSP20原核表达载体,获得高纯度r-HSP20蛋白。抗体分型检测证明r-HSP20具有高效诱导体液免疫应答和细胞免疫应答的免疫原性,并具有良好的免疫反应性。

Objective To recombinantly express Acanthamoeba heat shock protein20(Heat shock protein 20,HSP20)and to verify the immune activity of HSP20 as a vaccine candidate molecule.Methods The gene fragment coding the HSP20 was amplified using PCR with cDNA of Acanthamoeba trophozoite as a template.The prokaryotic expression vector pET-22 b-HSP20 was constructed and transformed into E.coli competent cell BL21.Expression of the recombinant heat shock protein 20(rHSP20)was induced with 1 mmol/L IPTG,and a solubility analysis was performed.r-HSP20 was purified using chromatography,and immunized rabbits were used to prepare rabbit polyclonal antibodies.The polyclonal antibodies IgG1 and IgG2 awere detected with ELISA.Western blotting was used to identify the immunoreactivity of r-HSP20.Results Amplification with PCR resulted in a gene fragment 579 bp in length,which was consistent with the expected size.PCR and double digestion indicated that the prokaryotic expression vector pET22 b-HSP20 was successfully constructed.r-HSP20 was mainly present in the supernatant of ultrasonically disrupted recombinant bacteria according to the soluble analysis.The target protein was successfully eluted with 300 mmol imidazole according to SDS-PAGE and highly pure r-HSP20 was obtained.The titer of rabbit polyclonal antibody obtained as a result of immunization with the recombinant protein was 1:12800,and the content of antibody subtypes IgG1 and IgG2 awas 183.8 mg/mL and 21.4 mg/mL,respectively.Western blotting confirmed that r-HSP20 is recognized by the rabbit polyclonal antibody.Conclusion The prokaryotic expression vector pET-22 b-hsp20 was successfully constructed and highly pure HSP20 protein was obtained.Antibody typing indicated that r-HSP20 has high immunogenicity to induce a humoral immune response and cellular immune response and that it has good immunoreactivity.