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目的 将人工合成的丙型肝炎病毒 (HCV)E1区的抗原表位基因 ,构建到一种新的抗原表位呈递系统的高效表达载体pET RNA2中进行高效表达。方法 运用基因工程技术将人工合成的HCVE1区的抗原表位基因 ,构建到一种新的抗原表位呈递系统的高效表达载体 pET RNA2中 ,构成一个嵌合基因进行高效表达。在该载体适当的位置插入外源抗原表位可使其呈现一定的空间构象 ,使抗原表位位于重组蛋白的表面并有望改善其免疫原性。结果 分别在载体的 10 6、15 3、3 0 5位氨基酸处插入外源抗原表位基因 ,成功构建了重组质粒 pET RNA HCV/A1、pET RNA HCV/A2和pET RNA HCV/A3 ,并分别在大肠杆菌中进行高效表达。表达产物相对分子质量约为 4 5 0 0 0。初步研究结果表明 ,在特定条件下不需要异丙基 β D硫代半乳糖苷 (IPTG)诱导就可使之获得高效表达 ,表达的嵌合蛋白占菌体总蛋白的 4 0 %以上。结论 HCV抗原表位基因成功构建到一种新的抗原呈递系统中并获得高效表达 ,为深入研究HCV抗原表位的免疫学和生物学特性奠定了基础 ,并对今后设计诊断试剂及抗原表位特异性疫苗有一定意义。
OBJECTIVE: To construct a highly efficient expression vector pET RNA2 of human hepatitis C virus (HCV) E1 region and to construct a new epitope expression system for high expression. Methods The gene of epitope of HCVE1 was synthesized by gene engineering into a highly efficient expression vector pET RNA2 of antigen epitope presentation system to form a chimeric gene for high expression. Inserting foreign epitopes into the proper position of the vector allows it to assume a certain spatial conformation so that the epitopes are located on the surface of the recombinant protein and the immunogenicity is expected to be improved. Results The exogenous epitopes were inserted into the 10 6,153,05 amino acids of the vector respectively and the recombinant plasmids pET RNA HCV / A1, pET RNA HCV / A2 and pET RNA HCV / A3 were constructed successfully Efficient expression in E. coli. The relative molecular mass of the expressed product was about 45,000. Preliminary results showed that under the specific conditions, isopropyl β D-thiogalactoside (IPTG) was not required to induce high expression, the expression of the chimeric protein accounted for more than 40% of total bacterial proteins. Conclusion The HCV epitopes have been successfully constructed into a new antigen presenting system and have been highly expressed. This study laid the foundation for the further study on the immunological and biological characteristics of HCV epitopes. The design of diagnostic reagents and epitopes Specific vaccines have some significance.