HCV非结构蛋白质5A基因全长和高免疫源区的表达及免疫活性检测

目的探讨HCV非结构蛋白质5A（NS5A）基因全长和高免疫源区的表达并比较其检测灵敏度。方法以含HCV全基因组的质粒pBR^tm／HCV-3011（1b型）为模板，采用PCR方法扩增出NS5A全长基因（以下用NS5AL代替）和高免疫源区基因（以下用NS5AS代替），与pET-32a载体相连接构建重组表达载体pET-NS5AL和pET-NS5AS，分别转化E.coli Rosetta（DE3）pLysS和BL21（DE3）感受态细胞，经异丙幕-β-D-硫代半乳糖苷（IPTG）诱导后，蛋白质印迹法检测其表达，镍螯合（Ni2^＋-NTA）琼脂糖亲和层析柱纯化重组NS5AL和NS5AS蛋白，最后通过ELISA法检测重组蛋白的免疫活性并对其榆测灵敏度进行比较。结果SDS—PAGE鉴定与蛋白质印迹验证结果表明，重组NS5AL和NS5AS蛋白均得到很好地表达；纯化的重组NS5AL和NS5AS蛋白浓度分别为0．146和0．426mg／ml，纯度均〉96％；ELISA检测结果表明，重组NS5AL和NS5AS蛋白均具有较好的免疫活性，且重组NS5AL蛋白的检测灵敏度明显高于NS5AS。结论成功地表达出重组NS5AL和NS5AS蛋白，均具有较高的免疫活性，将有助于提高HCV临床检测的灵敏度。

Expression and immune detection of full-length gene and immunodominant region of HCV nonstructural protein 5A

Objective To explore the expression of full-length gene and immunodominant region of HCV nonstructural protein 5A (NS5A) and the detection sensitivity of ELISA. Methods Full-length gene and immunodominant region of HCV NS5A (NS5AL and NS5AS) were amplified by PCR by using the plasmid pBRtm/HCV-3011 (type 1b) containing full-length HCV gene as a template, and then inserted into the plasmid pET-32a to construct recombinant plasmids pET-NS5AL and pET-NS5AS. The expression of the recombinant plasmids in competent E.coli cells Rosetta(DE3)-pLysS and BL21(DE3) were induced with IPTG and analyzed with SDS-PAGE and Western blotting. After being purified by Ni2+ affinity chromatography, the immunoactivity of the purified proteins were determined using ELISA. Results The SDS-PAGE and Western blotting showed expressions of NS5AL and NS5AS in the competent E.coli cells. The concentration of the purified fusion proteins NS5AL and NS5AS were 0.146 and 0.426 mg/ml respectively, the purity of the both antigens were higher than 96%. ELISA detected specific immunoactivity in both the fusion proteins NS5AL and NS5AS. The detection sensitivity of NS5AL is higher than that of NS5AS. Conclusion Both fusion protein NS5AL and NS5AS can be expressed with a high immunoactivity, which can be used to increase the detection sensitivity for clinical use.