中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2010年
4期
267-270
,共4页
张桓虎%王丹琼%成军%赵婕%赵心恺%高虹
張桓虎%王丹瓊%成軍%趙婕%趙心愷%高虹
장환호%왕단경%성군%조첩%조심개%고홍
肝炎病毒,乙型%克隆,分子%抑制性消减杂交%PS1TP2BP1
肝炎病毒,乙型%剋隆,分子%抑製性消減雜交%PS1TP2BP1
간염병독,을형%극륭,분자%억제성소감잡교%PS1TP2BP1
Hepatitis B virus%Cloning,molecular%Suppression subtractive hybridization%PS1TP2BP1
目的 克隆HBV前S1蛋白反式激活蛋白2结合蛋白1(PS1TP2BP1)基因,并筛选与其相互作用的反式激活基因.方法 应用聚合酶链反应(PCR)扩增PS1TP2BP1基因,鉴定正确后再将其亚克隆到真核表达载体pcDNATM3.1/myc-His A上;以真核表达质粒pcDNATM3.1/mycHis A-PS1TP2BP1转染HepG2细胞,构建cDNA消减文库;进行测序及同源性分析.结果 从HepG2细胞来源的cDNA中扩增出PS1TP2BP1基因,并成功进行TA克隆,酶切、测序均正确后,成功构建真核表达重组体.消减文库扩增后得到35个阳性克隆,经菌落PCR分析,得到15个含200～1000 bp插入片段的菌落.对所得片段测序,并进行间源性分析,显示15种已知基因编码蛋白可能是PS1TP2BP1反式激活靶基因.结论 成功克隆PS1TP2BP1,并构建了PS1TP2BP1反式激活基因差异表达的cDNA消减文库,为今后进一步分析、研究病毒蛋白的致病机制奠定了基础.
目的 剋隆HBV前S1蛋白反式激活蛋白2結閤蛋白1(PS1TP2BP1)基因,併篩選與其相互作用的反式激活基因.方法 應用聚閤酶鏈反應(PCR)擴增PS1TP2BP1基因,鑒定正確後再將其亞剋隆到真覈錶達載體pcDNATM3.1/myc-His A上;以真覈錶達質粒pcDNATM3.1/mycHis A-PS1TP2BP1轉染HepG2細胞,構建cDNA消減文庫;進行測序及同源性分析.結果 從HepG2細胞來源的cDNA中擴增齣PS1TP2BP1基因,併成功進行TA剋隆,酶切、測序均正確後,成功構建真覈錶達重組體.消減文庫擴增後得到35箇暘性剋隆,經菌落PCR分析,得到15箇含200～1000 bp插入片段的菌落.對所得片段測序,併進行間源性分析,顯示15種已知基因編碼蛋白可能是PS1TP2BP1反式激活靶基因.結論 成功剋隆PS1TP2BP1,併構建瞭PS1TP2BP1反式激活基因差異錶達的cDNA消減文庫,為今後進一步分析、研究病毒蛋白的緻病機製奠定瞭基礎.
목적 극륭HBV전S1단백반식격활단백2결합단백1(PS1TP2BP1)기인,병사선여기상호작용적반식격활기인.방법 응용취합매련반응(PCR)확증PS1TP2BP1기인,감정정학후재장기아극륭도진핵표체재체pcDNATM3.1/myc-His A상;이진핵표체질립pcDNATM3.1/mycHis A-PS1TP2BP1전염HepG2세포,구건cDNA소감문고;진행측서급동원성분석.결과 종HepG2세포래원적cDNA중확증출PS1TP2BP1기인,병성공진행TA극륭,매절、측서균정학후,성공구건진핵표체중조체.소감문고확증후득도35개양성극륭,경균락PCR분석,득도15개함200～1000 bp삽입편단적균락.대소득편단측서,병진행간원성분석,현시15충이지기인편마단백가능시PS1TP2BP1반식격활파기인.결론 성공극륭PS1TP2BP1,병구건료PS1TP2BP1반식격활기인차이표체적cDNA소감문고,위금후진일보분석、연구병독단백적치병궤제전정료기출.
Objective To identify genes regulated by HBV preS1-transactivated protein 2 binding protein 1(PS1TP2BP1).Methods PS1TP2BP1 gene was amplified by polymerase chain reaction (PCR)technique and cloned into the eukaryotic expression vector pcDNATM3.1/my-c-His A.The mRNAs isolated from HepG2 cells transfected recombinant eukaryotic expression vector pcDNATM3.1/mycHisA-PS1TP2BP1 and pcDNATM3.1/myc-HisA empty vector were used to construct subtractive library.The differentially expressed genes were idenfied and analyzed.Results 35 differentially expressed clones were obtained.Colony PCR identified 15 clones with 200-1000 bp inserts.Sequence analysis identified 1 5 differentially expressed genes.Conclusion This study provides data for further characterize the function of PS1TP2BP1.
