摘要
目的 研究不同截短片段丙型肝炎病毒(HCV)的核心蛋白(CORE)在HCV持续感染和肝癌发病机制中的作用,构建并表达不同HCV病毒株及不同截短片断CORE原核表达质粒。 方法 用PCR扩增基因型为HCV 1 b型的7个不同截短片段CORE基因:氨基酸(aa)长度分别为癌中心株(BT):B:1-172 aa、1-126 aa、1-58 aa、59-126 aa、127-172 aa;癌旁株(BNT):1-172 aa及C191(HCV-J6):1-172 aa,扩增产物用BamH Ⅰ及EcoR Ⅰ双酶切后将其插入到原核表达质粒pGEX-4T-1中,阳性克隆转染到BL21中,IPTG诱导表达GST-CORE融合蛋白,纯化定量,并用western blot加以验证。 结果 7个不同片段CORE在体外都得到相应表达,但表达量存在一定的差异,较长片段表达量相对较低,其中BT及BNT的1-172 aa截短片段CORE的表达高于同基因型等长度片段的C191,而BT以59-126 aa片段的表达量最高。BT、BNT及C191三株HCV基因1b型病毒株部分截短片段可形成二聚体。 结论 构建不同HCV病毒株及不同截短片段的CORE原核表达质粒获得成功,为研究HCV不同病毒株及不同区域CORE功能奠定基础;不同截短片段的CORE表达量不等,可能与不同片段具有不同的疏水基团、细胞毒性及在HCV结构和在HCV致病性中所起作用不同有关;HCV基因1b型的CORE二聚体的形成依耐于59-126 aa区域。
Objective To study the role of different truncated core proteins (CORE) of hepatitis C virus (HCV) played in the pathogenesis of HCV persistent infection and hepatocellular carcinoma, and to construct seven different truncated prokaryotic expression plasmids of HCV CORE. Methods The gene sequences of different truncated HCV genotype 1b CORE were amplified from plasmids containing CORE sequences derived from tumor and non-tumor tissues of a patient infected with HCV. Amino acid (aa) lengths of HCV BT (from tumor tissue, patient B) were: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; of BNT (non-tumor tissue, patient B) were: 1-172 aa and HCV C191 (HCV-J6): 1-172 aa. PCR products were cleaved with restriction enzymes BamH I and EcoR I and cloned into pGEX-4T-l. Positive clones were transformed into BL21 and glutathione S-transferase(GST)-CORE fusion proteins were expressed with isopropylthio- p -D-galactoside (IPTG) induction, purified and verified by Western blot. Results Different truncated GST-CORE fusion proteins were expressed with different quantities. Except the fragment of 59-126 aa, the longer the fragment, the less its expression. The levels of truncated expression of CORE of BT and BNT were higher than that of C191, even though they all contained 1-172 aa. Some of truncated CORE of HCV genotype 1b could form dimmers. Conclusions Successful construction of truncated GST-CORE expression plasmids lays a basis for future study of the function of different domains of CORE of different HCV strains; different expression levels of HCV COREs might be related to their different hydrophobicity, cytotoxicity and their functions in HCV structure and their roles played in the pathogenesis; the domain of 59-126 aa is responsible for the HCV genotype 1b CORE dimmer formation.
出处
《中华肝脏病杂志》
CAS
CSCD
2004年第11期643-647,共5页
Chinese Journal of Hepatology
基金
"十五"国家科技攻关项目(2001BA705806)