鸡survivin重组腺病毒载体的构建和体外表达

根据GenBank中鸡survivin cDNA序列,设计引物,从鸡胚组织提取总RNA,利用RT-PCR扩增鸡survivin全长cDNA,经T-A克隆后插入腺病毒穿梭载体、骨架载体,构建腺病毒重组质粒,转染293E4pIX细胞,构建鸡survivin重组腺病毒.T-A克隆的测序结果与GenBank中鸡survivin cDNA完全一致.限制性内切酶分析和PCR表明腺病毒质粒携带survivin基因,Western blot证实重组腺病毒正确表达survivin,survivin基因已被成功重组到腺病毒基因组.

Construction and Expression in Vitro of Recombinant Chicken Survivin Adenoviral Vector

Total RNA was isolated from chicken embryos and subjected to RT-PCR for amplification of chicken survivin using primers according to it's cDNA sequence in GenBank.The amplified product was inserted into T-A clone vector and then subcloned into adenoviral shuttle vector and adenoviral backbone vector.The recombinant adenoviral plasmid was transfected into 293E4pIX cells to generate recombinant chicken survivin adenoviral vector.The sequence of chicken survivin was confirmed by sequencing to be identical with that in GenBank and the recombinant adenoviral plasmids were screened by restriction enzyme digestion.Further,PCR and Western blot analysis was used to confirm the expression of survivin in the transfected cells.The recombinant chicken survivin adenoviral vector was constructed successfully and could be further used in tumorimmunotheapy.