酵母双杂交技术筛选白细胞中HCVNS3蛋白结合蛋白基因

Screening of hepatitis C virus NS3 protein-interacting proteins in leukocytes by yeast-two hybrid technique

目的:用酵母双杂交技术筛选白细胞中与丙型肝炎病毒(HCV)NS3蛋白结合蛋白的编码基因.方法:用多聚酶链反应(PCR)法扩增 NS3基因,连接入酵母表达载体 pGBKT-7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人白细胞文库质粒的酵母细胞 Y187进行配合,在营养缺陷型培养基上进行双重筛选阳性菌落,增菌后提出质粒,转化入大肠杆菌(DH5α),提取质粒并测序,进行生物信息学分析.结果:成功克隆出 NS3基因并在酵母细胞中表达,配合后选出在四缺(SD/-Trp-Leu-Ade-His)培养基和在铺有 X-α-半乳糖(x-α-gal)的四缺培养基上均能生长并变成蓝色的真阳性菌落18个,其中4个真核细胞翻译延伸因子2;2个免疫球蛋白λ轻链;1个肌动蛋白β;1个铁蛋白轻多肽;1个(丝裂原)活化蛋白激酶3;2个肌球蛋白因子1;1个白介素2受体β;1个富含精氨酸/丝氨酸剪切因子6;1个组织蛋白酶 S;1个2’-5’寡腺甘酸合成酶类似物;1个缺失精子缺乏相关蛋白2;1个 CNN2;1个新基因.结论:成功克隆出 HCV NS3的结合蛋白,为进一步研究HCV 的作用提供了新线索.

AIM:To investigate the biological function of hepatitis C virus NS3 protein(HCV NS3),we performed yeast-two hybrid to screen proteins in leukocyts interacting with HCV NS3. METHODS:HCV NS3 gene was amplified by polymerase chain reaction(PCR)and HCV NS3 bait plasmid was constructed by using yeast-two hybrid system 3,then the constructed vector was transformed into yeast AH109.The transformed yeast mated with yeast Y187 containing leukocyts cDNA library plasmid in 2×YPDA medium.Diploid yeast was 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 two times and screening.After extracting and sequencing of plasmid from blue colonies,we underwent analysis by bioinformatics. RESULTS:Eighteen colonies were sequenced.Among them, four colonies were eukaryotic translation elongation factor 2(EEF2),two colonies were immunoglobulin lambda light chain and myosin IF(MYOIF),and one colony was actin, beta(ACTB),ferritin,light polypeptide(FTL),mitogen- activated protein kinase kinase 3(MAPKK3),interleukin 2 receptor,beta(IL2RB),splicing factor,arginine/serine-rich 6 (SFRS6),cathepsin S(CTSS),2'-5'-oligoadenylate syn- thetase-like(OASL),DAZ(deleted in azoospermia)associ- ated protein 2(DAZAP2),calponin 2(CNN2),and one new gene without known function. CONCLUSION:Genes of HCV NS3 interacting proteins in leukocyts are successfully cloned and the results bring some new clues for studying the biological functions of HCV NS3 and associated proteins.