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目的构建pET32a-AgB8/1-AgB8/2原核表达载体,并对其重组蛋白进行原核细胞表达。方法从细粒棘球绦虫原头蚴中提取总RNA,反转录生成cDNA,以此cDNA为模板,用基因特异性引物分别扩增EgAgB8/1和EgAgB8/2基因编码其分泌型多肽的片段,经测序后,以此两条基因片段为依据,人工合成EgAgB8/1-EgAgB8/2嵌合抗原编码核酸序列,将其克隆至pUCm-T载体,测序鉴定其正确性。通过对pUCm-T/AgB8/1-AgB8/2重组质粒进行双酶切,将获得的AgB8/1-AgB8/2嵌合抗原编码核酸序列用定向克隆技术克隆至原核表达质粒pET32a上,测序鉴定插入片段正确后,转化至E.coliBL21(DE3)Lys S,IPTG初步诱导表达pET32a-AgB8/1-AgB8/2重组嵌合蛋白。用SDS-PAGE电泳分析鉴定重组蛋白的表达水平。结果测序表明,AgB8/1-AgB8/2嵌合抗原编码核酸序列正方向插入至pET32a质粒。SDS-PAGE电泳分析显示,IPTG诱导后重组嵌合蛋白得到成功表达,在相对分子量约38 kD处有表达条带。结论成功构建了pET32a-AgB8/1-AgB8/2原核表达质粒,并初步诱导表达出AgB8/1-AgB8/2嵌合重组蛋白,为进一步研究其免疫学特性奠定了基础。
Objective To construct the prokaryotic expression vector pET32a-AgB8 / 1-AgB8 / 2 and express the recombinant protein in prokaryotic cells. Methods The total RNA was extracted from the protoscoleces of Echinococcus granulosus and the cDNA was reverse transcribed. Using this cDNA as a template, fragments encoding the secreted polypeptides of EgAgB8 / 1 and EgAgB8 / 2 genes were amplified by gene-specific primers After sequencing, based on these two gene fragments, the coding sequence of EgAgB8 / 1-EgAgB8 / 2 chimeric antigen was cloned into pUCm-T vector and sequenced to confirm its correctness. The double-digested recombinant plasmid pUCm-T / AgB8 / 1-AgB8 / 2 was used to clone the AgB8 / 1-AgB8 / 2 chimeric antigen-encoding nucleic acid sequence into the prokaryotic expression plasmid pET32a by sequencing. The recombinant plasmid pET32a-AgB8 / 1-AgB8 / 2 was induced by IPTG after being inserted into E.coli BL21 (DE3) Lys S correctly. The expression level of the recombinant protein was identified by SDS-PAGE electrophoresis analysis. Results Sequencing showed that the coding sequence of AgB8 / 1-AgB8 / 2 chimeric antigen was inserted into the pET32a plasmid in the forward direction. SDS-PAGE electrophoresis analysis showed that the recombinant chimeric protein was successfully expressed after induced by IPTG and expressed at a relative molecular weight of about 38 kD. Conclusion The prokaryotic expression plasmid pET32a-AgB8 / 1-AgB8 / 2 was successfully constructed and the chimeric recombinant protein AgB8 / 1-AgB8 / 2 was induced initially, which laid the foundation for further study on its immunological properties.