中文摘要：

目的克隆人类乙型肝炎病毒（HBV）前-S1蛋白（pre-S1）反式激活新型靶基因PSlTP5。构建其原核表达载体，并诱导其在大肠埃希菌中表达。方法以HBV前-s1表达质粒peDNA3．1（-）-preSl转染HepG2细胞，空载体pcDNA3-1（-）作为平行对照，提取mRNA进行抑制性消减杂交（suppression subtraetive hybridization，SSH）技术分析，结合生物信息学（bioinformaties）技术筛选得到人类新基因PSlTP5。应用逆转录聚合酶链反应（RT-PCR）技术，以提取的HepG2细胞mRNA为模板，扩增获得PSlTP5基因片段，选用pGEM-T载体进行TA克隆，通过PCR、限制性酶切分析及测序进行鉴定，再将其亚克隆到原核表达载体pET-32a（＋）中，转化进BL21（DE3）宿主菌，经IPTG诱导获得了PSlTP5重组蛋白的表达，表达产物进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，考马斯亮蓝染色，以抗-His的单克隆抗体进行Western blotting免疫印迹分析证实表达蛋白的特异性。结果成功扩增出PSITP5基因。并将其插入pET-32a（＋）原核表达载体，经BL21（DE3）受体菌转化、IPTG诱导、SDS-PAGE分析获得了PSITP5重组蛋白的表达，Western blotting证实了重组蛋白表达的特异性。结论应用SSH、RT-PCR与生物信息学技术筛选克隆出PSlTP5基因，构建了pET-32a（＋）-PSlTP5原核表达载体，并利用大肠埃希菌原核表达系统成功获得了pET-32a（＋）-PSlTP5重组蛋白的表达。为进一步研究PSlTP5免疫原性及慢性乙型肝炎发病机制创造了条件。

英文摘要：

Objective To clone and express hepatitis B virus pre-S1 protein activating gene PS1TP5 in Escherichia coli. Methods The mRNA from HepG2 cells transfected with pcDNA 3. 1 （ - ）-preS1 and pcDNA 3. 1 （ - ） empty vector was isolated respectively. SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. The novel coding gene transactivated by pre-S1 protein of hepatitis B virus was screened by bioinformat- ics method. Reverse transcription-polymerase chain reaction （RT-PCR） was used to amplify PS1TP5 from HepG2 eDNA template. The novel DNA fragment was ligated into pGEM-T cloning vector by TA cloning. After digestion with restric- tive enzyme and sequencing, the correct DNA fragment was inserted into inducible proeukaryotic expression vector pET- 32a（ ＋ ）. The competent BL21 （DE3） E. coli was transformed, then cultured and induced with IPTG. The expressed PS1TP5 was analyzed and confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting hybridization. Results The DNA fragment of PS1TP5 was successfully amplified by RT-PCR, and its expression vector was constructed. After transformation with pET-32a（ ＋ ）-PS1TP5 and induction with IPTG, recombinant PS1TP5 was expressed and confirmed by SDS-PAGE and Western blotting. Conclusions Human novel gene PS1TP5 was cloned by the application of SSH, RT-PCR and bioinformatics technique. And its recombinant prokaryotic expression vector pET-32a（ ＋ ）-PS1TP5 was constructed. The recombinant PS1TP5 gene can be expressed in prokaryotic expression system of E. coli. These results will certainly bring some new clues to study immunogenicity of PS1TP5 and pathogenesis of the chronic hepatitis B.