丙型肝炎病毒NS5A不同免疫源區(qū)及NS3的表達與檢測評估
發(fā)布時間：2018-07-16 13:02
【摘要】： 丙型肝炎病毒(Hepatitis C Virus,簡稱HCV)的非結構蛋白3(non-structuralprotein 3 NS3)和非結構蛋白5A(non-structural protein 5A NS5A)分別為HCV診斷第二代、第三代酶聯免疫吸附實驗(enzyme-linked immunosorbent assay ELISA)所必需的片段,對于HCV臨床診斷有重要意義。本研究通過基因工程方法得到丙型肝炎病毒NS5A的長短兩片段與NS3的全長序列,并進行了初步的免疫學檢測鑒定。 以含丙型肝炎病毒全基因的質粒pBR~(tm)/HCV-3011(1b型)為模板,經PCR方法擴增出編碼丙型肝炎病毒的第三代抗-HCV酶聯免疫吸附實驗所需全長NS5A和高免疫源區(qū)NS5A蛋白(氨基酸2212-2313)的基因,分別命名為NS5AL和NS5AS,克隆入pET-32a載體中,成功構建重組表達載體pET-NS5AL和pET-NS5AS,將pET-NS5AL轉化大腸桿菌Rosetta(DE3)pLsS、pET-NS5AS轉化大腸桿菌BL21(DE3),成功構建工程菌,分別命名為Rosetta(DE3)pLysS- NS5AL、BL21(DE3)-NS5AS,經IPTG誘導,SDS-PAGE分析NS5AL出現一條約68kDa的條帶,NS5AS出現一條約39kDa的條帶,與預期融合蛋白的分子量相符,Western-blot顯示誘導后的菌體在相應的位置出現特異性的雜交帶,通過Ni-NTAAgarose純化重組蛋白,得到了濃度分別為0.146mg/mL、0.426mg/mL,純度超過96%的目的蛋白,選擇5例確診HCV感染者的陽性血清,通過ELISA對重組蛋白NS5AL與NS5AS的檢測性進行鑒定,并比較兩重組蛋白的檢測靈敏度,ELISA結果表明:重組蛋白NS5AL與NS5AS都具有良好的免疫學檢測效果,且重組蛋白NS5AL的檢測靈敏度明顯高于NS5AS。 將克隆有NS3基因的重組質粒pProEX-HTb-NS3轉化BL21(DE3),成功構建工程菌BL21(DE3)-NS3,IPTG誘導表達NS3蛋白,進行SDS-PAGE與Western-Blot分析,NS3出現一條約為72kDa的條帶,Ni-NTA Agarose純化重組蛋白NS3,得到了濃度為0.346mg/mL、純度超過93%的重組蛋白NS3,ELISA方法證明了純化后的蛋白具有良好的免疫學檢測效果。 本研究運用原核表達體系,成功地得到了NS5A蛋白的長短片段和全長的NS3蛋白。ELISA結果表明NS3具有很好的免疫學檢測效果;NS5AL的檢測靈敏度優(yōu)于NSSAS,在將來臨床應用中,長片段NS5AL能夠彌補較短片斷特異性較強,而靈敏度不足的缺陷,可以更好地應用于HCV的臨床檢測。同時也為研究HCV NS5A、NS3的其它生物學功能奠定基礎。
[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.
【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R373
本文編號：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.
【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R373
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