论文部分内容阅读
目的 构建幽门螺杆菌粘附素 (hpaA)和霍乱毒素B亚单位 (ctxB)融合基因的原核表达载体 ,诱导表达并进行纯化。方法 用PCR扩增hpaA和ctxB两个目的基因片段 ,克隆至同一pQE 30表达载体中 ,构建含双基因的表达质粒pQE hct,转化E .coliDH5α ,经IPTG诱导表达融合蛋白HCT ;经Westernblot分析其免疫原性 ,采用镍离子柱进行纯化。结果 经测序HCT融合基因片段由 116 1bp组成 ,为编码 387个氨基酸残基的多肽。经SDS PAGE分析相对分子质量约为 4 0 0 0 0。可溶性蛋白占菌体总蛋白的 2 5 %以上 ,经亲和层析后可获得纯度为 92 %以上的重组融合蛋白。经Westernblot检测可被Hp全菌抗血清和CT抗血清识别。结论 已成功构建融合蛋白HCT的原核表达载体并能高效表达 ,为研制Hp口服疫苗提供依据
Objective To construct prokaryotic expression vector of hpaA and cholera toxin B subunit (ctxB) fusion gene, and to induce expression and purification. Methods Two target genes of hpaA and ctxB were amplified by PCR and cloned into the same expression vector pQE30. The recombinant plasmid pQE hct was constructed and transformed into E.coli DH5α. The expression of fusion protein HCT was induced by IPTG. Western Blot analysis Immunogenicity, purified by nickel ion column. Results The sequenced HCT fusion gene consisted of 116 1bp and encoded 387 amino acid residues. SDS PAGE analysis of the relative molecular mass of about 40,000. Soluble protein accounted for more than 25% of the total bacterial proteins, and the recombinant fusion protein with the purity of over 92% can be obtained by affinity chromatography. Western blot can be Hp bacterial antiserum and CT antiserum identification. Conclusion Prokaryotic expression vector of fusion protein HCT has been successfully constructed and can be highly expressed, providing the basis for the development of Hp oral vaccine