摘要
目的构建含乙型肝炎病毒(HBV)PreS1基因的原核表达载体,获得高纯度、有生物活性的PreS1重组蛋白。方法采用PCR法扩增PreS1及其N末端和C末端部分基因序列,克隆入原核表达载体pGST-MOLUC。重组质粒经IPTG诱导表达后进行SDS-PAGE电泳分析和Western blot检测。用谷胱甘肽-Sepharose 4B凝胶亲和层析纯化的重组融合蛋白免疫新西兰家兔,制备GST-PreS1融合蛋白的抗血清。进一步用ELISA分析融合蛋白特异抑制HBV病毒与抗体结合的能力。结果重组质粒转化宿主菌后成功诱导出Mr39000、31000和32000的GST-PreS1、GST-PreS1N及GST-PreS1C融合蛋白,均与预期相对分子质量相符。Western blot检测获得特异的杂交条带。采用谷胱甘肽-Sepharose 4B凝胶纯化的融合蛋白纯度约为90%左右。融合蛋白免疫家兔后抗体滴度达到10-7。病毒捕获ELISA实验表明,融合蛋白能特异抑制病毒与家兔免疫血清结合。结论GST-PreS1融合蛋白能够在大肠杆菌中高效表达,其纯化产物是研究PreS1基因在HBV感染过程中作用的有用工具。
Objective To purify the recombinant preSl peptide of hepatitis B surface antigen in E. Coli and to investigate its physiochemical characters and immunogenicity. Methods The N and C terminal sequences of PreS1 and the PreS1 gene amplified by PCR, and then inserted into prokaryotic expression vector pGST-MOLUC, respectively. The recombinant plasmids were confirmed by sequence analysis and named pGST-PreS1, pGST-PreS1N, and pGST-PreS1C. The expression of recombinant proteins were induced with IFrG, and then the expressional products were confimed by Western blot analysis and further purified by affinity chromatography methods. For competitive binding assay, different amount of purified fusion proteins was added to the immunized sera prior to virus capture. Results Recombinant proteins of GST-PreS1, GST-PreS1N, and GST-PreS1C displayed bands of Mr 39 000, 31 000, and 32 000 on SDS-PAGE gel, respectively. The purity of the fusion protein was over 90% after purification by affinity chromatography method. The specific antibody titer could reach 10- 7 in GSr-PreS1 immunized rabbit. As for virus capture assay, all fusion proteins strongly inhibited the binding capacity of HBV virion to the pelyclonal anti- body. Conclusion PreS1 regions synthesized in E.coli can be achieved by fusion with GST tag. Purified fusion proteins laid a foundation for better understanding of the mechanism of HBV PreS1 protein in viral endocytosis and were helpful for seeking the PreSl-related protein.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第1期5-8,12,共5页
Immunological Journal
基金
国家自然科学基金资助项目(30400374)
