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目的构建pET32a-AgB8/1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扩增EgAgB8/1基因编码其分泌型多肽的片段,经测序后,以此基因片段为依据,人工合成合成EgAgB8/1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重组质粒进行双酶切,将获得的AgB8/1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性后,转化至E.coli BL21(DE3)Lys S,IPTG初步诱导表达pET32a-AgB8/1重组蛋白。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诱导后重组蛋白得到成功表达,在相对分子量约28 kDa处有表达条带。结论本研究成功构建了pET32a-AgB8/1原核表达质粒,并初步诱导表达出AgB8/1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 and express its recombinant protein in prokaryotic cells. Methods The total RNA was extracted from the protoscoleces of Echinococcus granulosus and the cDNA was reverse transcribed. CDNA of EgAgB8 / 1 gene was amplified by gene-specific primers using this cDNA as a template. After sequencing, The gene fragment was used as a basis to synthesize the EgAgB8 / 1 antigen-encoding nucleic acid sequence synthetically and cloned into the pUCm-T vector. The correctness of the sequence was confirmed by sequencing. The recombinant plasmid pUCm-T / AgB8 / 1 was double-digested. The obtained AgB8 / 1 antigen-encoding nucleic acid sequence was cloned into the prokaryotic expression plasmid pET32a using directional cloning technology. coli BL21 (DE3) Lys S and IPTG to induce the expression of pET32a-AgB8 / 1 recombinant protein. The expression level of the recombinant protein was identified by SDS-PAGE electrophoresis analysis. Results sequencing showed that the AgB8 / 1 antigen encoding nucleic acid sequence was inserted into the pET32a plasmid in the positive direction. SDS-PAGE electrophoresis analysis showed that the recombinant protein was successfully expressed after induced by IPTG and expressed at a molecular weight of about 28 kDa. Conclusion The prokaryotic expression plasmid pET32a-AgB8 / 1 was successfully constructed and the recombinant AgB8 / 1 protein was induced initially, which laid the foundation for further study on its immunological properties.