摘要
目的:HBeAg被认为与HBV引起免疫耐受、免疫系统功能障碍有关。筛选并克隆人肝细胞cDNA文库中与乙型肝炎病毒e抗原(HBeAg)相互作用蛋白的基因,明确其具体作用机制。方法:应用酵母双杂交系统3,将多聚酶链反应(PCR)法扩增的HBeAg基因连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和X-α-半乳糖(x-α-gal)上进行双重筛选阳性菌落,提取阳性酵母菌落的质粒转化大肠杆菌,接种在氨苄青霉素-LB平板上选择并测序,结果在GenBank中进行生物信息学分析。结果:成功克隆出HBeAg基因并在酵母细胞中表达,与肝cDNA文库配合后选出既能在四缺(SD/-Trp-Leu-Ade-His)培养基又能在铺有X-α-gal的四缺培养基上生长,并变成蓝色的真阳性菌落39个,其中有5个未知基因,3个含金属硫蛋白2A基因的菌落,8个补体8α基因,1个补体因子H,1个补体1q,1个维甲酸受体应答元件2基因、2个细胞色素b基因、3个铁蛋白轻链,2个NAD(P)H脱_氢酶亚基,1个3-羟基类固醇表位酶,1个3-羟基,3-甲基戊二酰辅酶A合成酶,1个双-特异性酪氨酸-Y-磷酸化调节激酶1A转录子突变体,1个Syndecans-1,3个2胺氧化酶,4个醛缩酶B,1个CD81,1个ATP合成酶6基因。结论:成功克隆出乙型肝炎病毒e抗原的结合蛋白,为进一步研究HBeAg在病毒装配、分泌、影响宿主免疫系统功能等方面的具体作用提供了新线索。
AIM:To screen hepatic proteins interacting with hepatitis B virus e antigen (HBeAg) with yeast-two hybrid technique for investigating the function of HBeAg protein. METHODS:HBeAg bait plasmid was constructed by ligating the HBeAg gene with plasmid pGBKT7, then transformed into yeast α-AH109. The transformed yeast cells were amplified and mated with yeast cells α-Y187 containing liver cDNA library plasmid pCAT2 in 2×YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selecting twice and screening. After extracting plasmid from blue colonies, plasmid was transformed into competence E. coli and analyzed by DNA sequencing. Twenty genes were obtained from 39 positive colonies, which included five new genes. RESULTS:Twenty genes in thirty nine positive colonies were obtained including three metallothionein 2 A, eight complement component 8 alpha polypeptide, one complement component 1q, one complement factor H, one retinoic acid receptor responder (tazarotene induced), two cytochrome b, three D-amino-acid oxidase, three ferritin light polypeptide, two NAD(P)H dehydrogenase β, four aldolase B, one CD81, one syndecan 1, one 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2, one dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A transcript variant 1, one 3-hydroxysteroid epimerase, one ATP synthase 6 and five new genes. CONCLUSION:Genes encoding HBeAg interacting proteins in hepatocytes were successfully doned and the results provided some new clues for studying the biological functions of HBeAg and its associated proteins.
出处
《世界华人消化杂志》
CAS
2003年第4期422-425,共4页
World Chinese Journal of Digestology
基金
军队回国留学人员启动基金
No.98H038
