丙型肝炎病毒高变区1融合蛋白的表达、纯化及其初步应用

Expression and purification of the fusion hepatitis C virus hypervariable region 1 protein and it's preliminary application

目的 克隆并表达中国不同地区不同基因型的丙型肝炎病毒高变区 1(HCVHVR1)蛋白 ,并用表达纯化产物检测慢性丙型肝炎患者血清中的相应抗体 ,分析其临床意义。方法 根据 31株HCVHVR1序列分析及免疫原性预测结果 ,选择 4株克隆 (1b型 3株 ,2a型 1株 ) ,从 pGEMT E2克隆中扩增得到 4个HVR1片段 ,将其分别克隆到原核表达载体 pQE4 0中 ,表达产物经纯化后用以检测慢性丙型肝炎患者血清中的HVR1抗体。结果 构建的HVR1原核表达载体在大肠埃希菌中成功表达了 4种相对分子质量约为 2 80 0 0的二氢叶酸还原酶 (DHFR) HVR1融合蛋白 ,纯化蛋白的获得率约为 32 0～ 80 0 μg/ 10 0ml培养液。这 4种融合蛋白 (SH1b、BJ1b、SD1b、SD2a)与慢性丙型肝炎患者的血清结合率分别为 72 .8% (5 1/ 70 )、6 0 % (4 2 / 70 )、4 8.6 % (34/ 70 )和 5 8.6 % (4 1/ 70 )。在 2 0例用干扰素治疗的丙型肝炎患者中 ,5 7% (4 / 7)的干扰素治疗无应答者治疗前血清能与之反应 ,而应答者血清中只有 15 .3% (2 / 13)能与之反应。干扰素治疗应答者血清与 4种融合蛋白反应的A值高于治疗无应答者 (P <0 .0 5 )。结论 选择的 4株HCVHVR1片段在大肠埃希菌中获得成功表达 ,所得

Objective To clone and express hypervariable region 1 (HVR1) fragments of different genotypes of HCV strains from different cities of China.Then detect the reactivity of the expressed HVR1 fusion proteins with sera of chronic he-patitis C patients to analyzing it's signification in clinical utilization. Methods HVR1 genes of four HCV strains (genotype 1b and 2a) were amplified from pGEMT-E2 plasmids and cloned into pQE40 vectors, respectively to construct four recombinant prokaryotic expression plasmids which expressed HVR1 as fusion proteins with DHFR in Escherichia coli strain TG1. Then we used the purified DHFR-HVR1 proteins to detect the anti-HVR1 antibodies in 70 serum samples of chronic hepatitis C patients. Results Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320～ 800 μg fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8%(51/70), 60%(42/70), 48.6%(34/70), and 58.6%(41/30) of the anti-HCV positive patients' sera, respectively by ELISA. 57% (4/7) of the non responders' sera taken before therapy reacted with all four HVR1 fusion proteins, while only 15.3% (2/13) of the sera from responders reacted with all of them. The A values of sera from IFN therapy responders were significantly higher than non responders (P <0.05). Conclusions The selected HVR1 fragments were successfully expressed in E.coli. The abtained HVR1 fusion proteins can broadly react with HCV-infected patients' sera.