CAJ | 학술논문

目的对HCV非结构蛋白2反式激活蛋白(NS2TP)进行原核表达,制备多克隆抗体,并探讨其在不同肝组织中的表达情况.方法 PCR法获得HCV NS2TP基因,将其克隆至原核表达载体pET-32a(+)上,并转化入E.coli BL21.在大肠埃希菌中诱导表达,表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,经免疫印迹分析验证后,大量表达并纯化该重组蛋白.将重组蛋白免疫家兔后获取多克降抗体,应用免疫组织化学技术检测健康人及慢性HCV患者的肝组织.结果成功获得丁重组目的蛋白(相对分子质量为33×103)及高滴度、高特异度的多克隆抗体.慢性HCV患者肝组织内NS2TP的表达高于健康肝组织,且以细胞核内分布为主.结论明确了慢性HCV患者肝组织内NS2TP表达量及细胞内定位的变化,为进一步研究NS2TP的生物学功能和HCV的致病机制奠定了基础.
목적 대HCV비결구단백2반식격활단백(NS2TP)진행원핵표체,제비다극륭항체,병탐토기재불동간조직중적표체정황.방법 PCR법획득HCV NS2TP기인,장기극륭지원핵표체재체pET-32a(+)상,병전화입E.coli BL21.재대장애희균중유도표체,표체산물진행십이완기류산납-취병희선알응효전영(SDS-PAGE)분석,경면역인적분석험증후,대량표체병순화해중조단백.장중조단백면역가토후획취다극강항체,응용면역조직화학기술검측건강인급만성HCV환자적간조직.결과 성공획득정중조목적 단백(상대분자질량위33×103)급고적도、고특이도적다극륭항체.만성HCV환자간조직내NS2TP적표체고우건강간조직,차이세포핵내분포위주.결론 명학료만성HCV환자간조직내NS2TP표체량급세포내정위적변화,위진일보연구NS2TP적생물학공능화HCV적치병궤제전정료기출.
Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.