EB病毒BARF1基因真核表达载体的构建

Construction eukaryotic vector for BARF1 gene of epstein-barr virus

①目的构建携带EB病毒(Epstein-Barr virus,EBV)编码BARF1基因的真核表达载体。②方法根据GenBan提供的BARF1基因的cDNA序列,设计特异性引物,并在两端添加酶切位点;常规培养B95-8细胞,提取细胞RNA,通过RT-PCR扩增BARF1基因,将其克隆到pUM-T载体,转化E.coli DH5α,经氨苄青霉素抗性筛选阳性克隆,提取质粒并用EcoRI和XbaI双酶切与测序鉴定,证实BARF1基因已克隆到pUM-T载体。凝胶回收双酶切产物BARF1片段,再将其连接到真核表达载体pcDNA3.1(+)-his,转化E.coli DH5α,通过氨苄青霉素抗性筛选阳性克隆,提取质粒并用EcoRI和XbaI双酶切分析,确认插入方向。③结果以B95-8细胞提取RNA进行RT-PCR扩增的cDNA为模板扩增BARF1,得到与预期片段大小相符的666bp的片段;对所提质粒进行鉴定后,证实目的基因BARF1片段已插入pUM-T载体,经双酶切和测序分析,确认BARF1基因已正确连接到pcDNA3.1(+)-his真核表达载体。④结论成功地构建真核表达载体。pcDNA3.1(+)/BARF1-his。

Objective To construct eukaryotic expression vector pcDNAS. 1（ ＋ ）/BARF1 - his with recombinant of BARF1 gene encoded by EBV and pcDNA3.1 （ ＋ ） - his. Methods The specific primers were designed according to the BARF1 gene cDNA sequence in GenBank provided and restriction sites added at both ends;B95 -8 ceUs was cultured, the cell RNA was extracted, BARF1 gene was amplified by RT - PCR. The PCR products of BARF1 were cloned into pUM - T vector and transformed into E. coli DH5α The positive clones were screened by ampicillin resistance, identified by restriction enzyme digestion of EcoRI and XbaI. Confirmation the BARF1 gene has been cloned into pUM - T vector. BARF1 fragments were recovered by Gel double - digestion product, and then inserted into the eukaryotic expression vector pcDNAS. 1 （ ＋ ） - his, transformed into E. coli DH5α. The positive clones were selected by ampicillin resistance ,and insertion direction was confirmed by double digestion and sequencing analysis. Resuits 666bp products of BARF1 were obtained with cDNA by RT - PCR of B95 - 8 cell RNA. BARF1 gene fragment was inserted into pUM - T vector by identification of double - digestion. BARF1 gene has been properly connected to the pcDNA3. 1 （ ＋ ） - his eukaryotic expression vector through sequencing analysis. Conclusion The eukaryotic expression vector pcDNA3.1 （ ＋ ）/BARF1 - his was constructed successfully.