丙型肝炎病毒NS5A不同免疫源区及NS3的表达与检测评估
发布时间：2018-07-16 13:02
【摘要】： 丙型肝炎病毒(Hepatitis C Virus,简称HCV)的非结构蛋白3(non-structuralprotein 3 NS3)和非结构蛋白5A(non-structural protein 5A NS5A)分别为HCV诊断第二代、第三代酶联免疫吸附实验(enzyme-linked immunosorbent assay ELISA)所必需的片段,对于HCV临床诊断有重要意义。本研究通过基因工程方法得到丙型肝炎病毒NS5A的长短两片段与NS3的全长序列,并进行了初步的免疫学检测鉴定。 以含丙型肝炎病毒全基因的质粒pBR~(tm)/HCV-3011(1b型)为模板,经PCR方法扩增出编码丙型肝炎病毒的第三代抗-HCV酶联免疫吸附实验所需全长NS5A和高免疫源区NS5A蛋白(氨基酸2212-2313)的基因,分别命名为NS5AL和NS5AS,克隆入pET-32a载体中,成功构建重组表达载体pET-NS5AL和pET-NS5AS,将pET-NS5AL转化大肠杆菌Rosetta(DE3)pLsS、pET-NS5AS转化大肠杆菌BL21(DE3),成功构建工程菌,分别命名为Rosetta(DE3)pLysS- NS5AL、BL21(DE3)-NS5AS,经IPTG诱导,SDS-PAGE分析NS5AL出现一条约68kDa的条带,NS5AS出现一条约39kDa的条带,与预期融合蛋白的分子量相符,Western-blot显示诱导后的菌体在相应的位置出现特异性的杂交带,通过Ni-NTAAgarose纯化重组蛋白,得到了浓度分别为0.146mg/mL、0.426mg/mL,纯度超过96%的目的蛋白,选择5例确诊HCV感染者的阳性血清,通过ELISA对重组蛋白NS5AL与NS5AS的检测性进行鉴定,并比较两重组蛋白的检测灵敏度,ELISA结果表明:重组蛋白NS5AL与NS5AS都具有良好的免疫学检测效果,且重组蛋白NS5AL的检测灵敏度明显高于NS5AS。 将克隆有NS3基因的重组质粒pProEX-HTb-NS3转化BL21(DE3),成功构建工程菌BL21(DE3)-NS3,IPTG诱导表达NS3蛋白,进行SDS-PAGE与Western-Blot分析,NS3出现一条约为72kDa的条带,Ni-NTA Agarose纯化重组蛋白NS3,得到了浓度为0.346mg/mL、纯度超过93%的重组蛋白NS3,ELISA方法证明了纯化后的蛋白具有良好的免疫学检测效果。 本研究运用原核表达体系,成功地得到了NS5A蛋白的长短片段和全长的NS3蛋白。ELISA结果表明NS3具有很好的免疫学检测效果;NS5AL的检测灵敏度优于NSSAS,在将来临床应用中,长片段NS5AL能够弥补较短片断特异性较强,而灵敏度不足的缺陷,可以更好地应用于HCV的临床检测。同时也为研究HCV NS5A、NS3的其它生物学功能奠定基础。
[Abstract]:Hepatitis C virus (non-structuralprotein 3 NS3) and nonstructural protein 5A (non-structural protein 5A NS5A) are the necessary fragments for the second generation and the third generation enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay Elisa), respectively. In this study, the length of two fragments of hepatitis C virus NS5A and the full-length sequence of NS3 were obtained by genetic engineering. Using the plasmid pBR- (tm) / HCV-3011 (1b) as template, the full-length NS5A and NS5A (amino acid 2212-2313) genes encoding the third generation anti-HCV Enzyme-linked immunosorbent assay (Elisa) of hepatitis C virus (HCV) were amplified from the plasmid pBR- (tm) / HCV-3011 (type 1b). The recombinant expression vectors pET-NS5AL and pET-NS5ASwere successfully constructed and transformed into E. coli Rosetta (DE3) pLsSpET-NS5AS into E. coli BL21 (DE3). Rosetta (DE3) pLysS- NS5ALN BL21 (DE3) -NS5AS. NS5AL was induced by IPTG and SDS-PAGE was used to analyze that NS5AL had a treaty 68kDa band NS5AS and a treaty 39kDa band. Western-blot analysis showed that the induced bacteria had specific hybridization bands in the corresponding position, in line with the molecular weight of the expected fusion protein. The recombinant protein was purified by Ni-NTAAgarose, and the target protein with a concentration of 0.146 mg / mL of 0.426 mg / mL and purity of more than 96% was obtained. The positive sera of 5 patients with HCV infection were selected, and the detectability of the recombinant protein NS5AL and NS5AS were identified by Elisa. The results of Elisa showed that the recombinant protein NS5AL and NS5AS had good immunological effect, and the sensitivity of recombinant protein NS5AL was significantly higher than that of NS5AS. The recombinant plasmid pProEX-HTb-NS3 containing NS3 gene was transformed into BL21 (DE3). The recombinant strain BL21 (DE3) -NS3 was induced to express NS3 protein by IPTG. SDS-PAGE and Western-Blot analysis showed that NS3 had a 72kDa band of Ni-NTA Agarose to purify the recombinant protein NS3. The recombinant protein NS3 with a concentration of 0.346 mg / mL and purity of more than 93% was obtained by Elisa. In this study, the length and length of NS5A protein and the full-length NS3 protein were successfully obtained by using prokaryotic expression system. Elisa results showed that NS5 AL had a good immunological detection effect and the sensitivity of NS5AL was better than that of NSSAS, and NS5 AL could be used in clinical application in the future. Long fragment NS5AL can make up for the defect of short fragment specificity and low sensitivity, so it can be better used in clinical detection of HCV. It also lays a foundation for the study of other biological functions of HCV NS5 Agnes NS3.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
本文编号：2126470
[Abstract]:Hepatitis C virus (non-structuralprotein 3 NS3) and nonstructural protein 5A (non-structural protein 5A NS5A) are the necessary fragments for the second generation and the third generation enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay Elisa), respectively. In this study, the length of two fragments of hepatitis C virus NS5A and the full-length sequence of NS3 were obtained by genetic engineering. Using the plasmid pBR- (tm) / HCV-3011 (1b) as template, the full-length NS5A and NS5A (amino acid 2212-2313) genes encoding the third generation anti-HCV Enzyme-linked immunosorbent assay (Elisa) of hepatitis C virus (HCV) were amplified from the plasmid pBR- (tm) / HCV-3011 (type 1b). The recombinant expression vectors pET-NS5AL and pET-NS5ASwere successfully constructed and transformed into E. coli Rosetta (DE3) pLsSpET-NS5AS into E. coli BL21 (DE3). Rosetta (DE3) pLysS- NS5ALN BL21 (DE3) -NS5AS. NS5AL was induced by IPTG and SDS-PAGE was used to analyze that NS5AL had a treaty 68kDa band NS5AS and a treaty 39kDa band. Western-blot analysis showed that the induced bacteria had specific hybridization bands in the corresponding position, in line with the molecular weight of the expected fusion protein. The recombinant protein was purified by Ni-NTAAgarose, and the target protein with a concentration of 0.146 mg / mL of 0.426 mg / mL and purity of more than 96% was obtained. The positive sera of 5 patients with HCV infection were selected, and the detectability of the recombinant protein NS5AL and NS5AS were identified by Elisa. The results of Elisa showed that the recombinant protein NS5AL and NS5AS had good immunological effect, and the sensitivity of recombinant protein NS5AL was significantly higher than that of NS5AS. The recombinant plasmid pProEX-HTb-NS3 containing NS3 gene was transformed into BL21 (DE3). The recombinant strain BL21 (DE3) -NS3 was induced to express NS3 protein by IPTG. SDS-PAGE and Western-Blot analysis showed that NS3 had a 72kDa band of Ni-NTA Agarose to purify the recombinant protein NS3. The recombinant protein NS3 with a concentration of 0.346 mg / mL and purity of more than 93% was obtained by Elisa. In this study, the length and length of NS5A protein and the full-length NS3 protein were successfully obtained by using prokaryotic expression system. Elisa results showed that NS5 AL had a good immunological detection effect and the sensitivity of NS5AL was better than that of NSSAS, and NS5 AL could be used in clinical application in the future. Long fragment NS5AL can make up for the defect of short fragment specificity and low sensitivity, so it can be better used in clinical detection of HCV. It also lays a foundation for the study of other biological functions of HCV NS5 Agnes NS3.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
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