【摘要】 目的:通过人工合成HIV-1整合酶(integrase,IN)基因的编码序列,提高目的蛋白的表达。方法:根据IN的氨基酸序列,选择大肠杆菌偏好的密码子,设计并合成了877碱基,共编码288个氨基酸,克隆入pMD18-T载体,经测序证实后,构建原核表达载体pET-His-p31,转化大肠杆菌BL21(DE3),经IPTG诱导表达后,进行SDS-PAGE和Western blot分析。结果:酶切及测序结果表明表达载体构建成功,工程菌诱导表达后的电泳图谱在相对分子质量为32kDa的位置出现明显目的蛋白条带,表达产物主要以包涵体形式存在,占菌体总蛋白的65.9%,Western blot结果表明表达的聚合酶蛋白能与HIV-1阳性血清发生特异性反应。结论:已获得高效表达工程菌pET-His-p31,为下一步研究HIV诊断试剂奠定基础。更多还原

【Abstract】 Objective:To synthesize the gene encoding HIV-1 integrase and improved the expression of the target protein. Methods:DNA sequence of HIV-1 integrase was synthesized by the preferred condons of E.coli. The complete length of the synthesis HIV-1 integrase gene was 877bp,which encoded 288 amino acids. Clone the synthetic gene fragment into vector pMD18-T.Identify the cloned gene fragment by sequencing and insert into plasmid pET-His.Transform the constructed recombinant plasmid pET-His-p31 to E. coli BL21(DE3) The expression of the HIV-1 integrase was detected by SDS-PAGE and Western blot after IPTG induction. Results:Restriction enzyme digestion analysis and sequencing proved that the prokaryotic expression vector pET-His-p31 was successfully constructed. The expressed product in a form of inclusion body showed a relative molecular mass of about 32kDa,contained about 65.9% of the total protein,recognized specifically by HIV-1 positive serum. Conclusion:High expression vector pET-His-p31 was obtained,which laid a foundation for the developmentofHIV diagnosis kits.更多还原