牛Klf4基因重组反转录病毒载体的构建及产毒细胞株的建立

Construction of Recombinant Retroviral Vector with Bovine Klf4 and Establishment of a Virus Producing Cell Strain

为了克隆牛Klf4基因并构建重组反转录病毒载体,得到稳定的产毒细胞株,本研究设计了带有酶切位点的一对引物,以接触抑制生长状态的MDBK细胞为材料,用RT-PCR方法克隆出牛Klf4基因的开放阅读框序列,将之插入干细胞反转录病毒载体pMSCVneo构成真核表达载体pMSCV-Klf4;采用脂质体法将pMSCV-Klf4转染包装细胞PT67,通过G418筛选得到稳定的产毒细胞株,电镜观察培养液上清中的病毒颗粒,同时病毒感染NIH3T3细胞测定其病毒滴度。结果表明,本研究克隆的牛Klf4基因开放阅读框序列与已发表序列(NM-001105385)比对有99.9%的高同源性;构建的重组载体质粒可正常包装,电镜下可观察到典型的病毒颗粒,筛选出的产毒细胞株病毒滴度达7.16×107CFU·mL-1。本研究成功构建的真核表达载体pMSCV-Klf4和得到的产毒细胞株,为进一步开展有关牛iPS细胞的研究奠定了基础。

In order to construct recombinant retroviral vector containing bovine Klf4 gene cDNA and get a stable virus producing cell strain,the primers with restriction enzyme sites were designed and the ORF（open reading frame） sequence of bovine Klf4 gene was amplified by RT-PCR from MDBK cells.Subsequently bovine Klf4 gene cDNA was inserted into the restriction sites of retroviral vector pMSCVneo to construt recombinant retroviral expression vector pMSCV-Klf4.The pMSCV-Klf4 was transfected into packaging cell line（PT67） by LipofectamineTM2000 to gain infectious retroviral particles which can be found under electron microscope.Then NIH 3T3 cells infected with infectious retroviral particles was used to test viral titer.The results showed that the ORF sequence was highly homologous to the published sequence（NM-001105385）.RNA from the recombinant retroviral vector pMSCV-Klf4 could be packaged into infectious retroviral particles in PT67 cell line.Then we successfully got a stable virus producing cell strain whose viral titer was up to 7.16×107 CFU·mL-1.The recombinant retroviral vector pMSCV-Klf4 and the stable virus producing cell strain were gained in this study,which would be helpful to the further work about bovine pluripotent stem cells induced by defined factors.