摘要
目的筛选并鉴定与乙型肝炎病毒(HBV)DNA聚合酶反式调节蛋白(HBVDNAPTP1)相互作用的蛋白。方法将HBVDNAPTP1基因片段插入酵母细胞表达载体pGBKT7中,转化AH109酵母菌株并获得表达,与含有白细胞文库质粒的Y187酵母菌株配合,利用营养缺陷型培养基筛选阳性克隆。提取单个阳性文库质粒进行酶切鉴定,测序后回复验证其在酵母中的相互作用。利用免疫共沉淀方法进一步验证HBVDNAPTP1与成对免疫球蛋白受体α(PILRα)在体外的相互作用。结果经RT-PCR扩增获得HBVDNAPTP1基因片段,成功构建HBVDNAPTP1基因的酵母细胞"诱饵"表达质粒,获得了与HBVDNAPTP1具有相互作用的已知功能蛋白4种,分别为PILRα、酶原颗粒蛋白16、羧酸脂酶1、β转导素样蛋白2。免疫共沉淀结果表明HBVDNAPTP1与PILRα在体外存在相互作用。结论获得了4种可与HBVDNAPTP1相互作用的候选蛋白,HBVDNAPTP1可能参与了肝细胞的增殖分化、信息传递和生长代谢。
Objective To screen and identify the proteins interacting with human novel gene HBVDNAPTP1 trans-activated by hepatitis B virus DNA polymerase. Methods DNA fragment of HBVDNAPTP1 was inserted into pGBKT7 to construct pGBKT7-HBVDNAPTP1 vector,and then transformed into AH109 yeast strains. Positive clones were screened by mating AH109 with Y187 containing leucocyte cDNA library plasmid and auxotrophic medium. The proteins interacting with HBVDNAPTP1 were obtained by blast contrasting and homology analysis. After identification by enzyme digestion and sequencing,the interactions in vitro between HBVDNAPTP1 and the proteins obtained above were tested further by co-immunoprecipitation in yeast cells. Results HBVDNAPTP1 yeast bait expression vector was constructed successfully. There were four kinds of hepatocyte proteins interacting with HBVDNAPTP1:paired immunoglobulin-like type 2 receptor alpha (PILRα),zymogen granule protein 16,carboxylesterase 1,and transducin-beta like protein 2. The result of co-immunoprecipitation showed that there existed interactions between PILRα and HBVDNAPTP1. Conclusions Four kinds of proteins interacting with HBVDNAPTP1 have been obtained by yeast two-hybrid system. HBVDNAPTP1 may participate in cell proliferation,differentiation,signal transduction,growth and metabolism in the hepatocytes.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期973-975,共3页
Medical Journal of Chinese People's Liberation Army