携带人胰岛素样生长因子-1重组腺病毒的构建和鉴定

Construction and Identification of Adenovirus with Human Insulin-like Growth Factor-1 Gene

[目的]采用Cre-LoxP同源重组系统构建并鉴定携带人胰岛素样生长因子-1(human insulin-likegrowth factor-1,hIGF-1)目的基因的复制缺陷重组腺病毒,为椎间盘退变的hIGF-1基因治疗奠定基础。[方法]PCR合成hIGF-1基因,用pMD18-T载体克隆hIGF-1目的基因,酶切、测序并进行NCBI BLAST相似性在线分析。将pMD18-T-hIGF-1和pDNR-1r质粒分别行EcoR Ⅰ和BamH Ⅰ双酶切,DNA连接酶连接双酶切产物,转化感受态大肠杆菌DH5α,合成中间载体pDNR-hIGF-1,酶切鉴定。Cre-loxP系统介导pDNR-hIGF-1和pInt.AV1.SpaT同源重组形成pInt.AV1.SpaT-hIGF-1重组表达质粒,行EcoR Ⅰ酶切鉴定。复苏293细胞,将pInt.AV1.SpaT-hIGF-1和pInt.B.B质粒在293细胞内进行同源重组成含hIGF-1基因的复制缺陷重组腺病毒。倒置显微镜观察293细胞病变样效(cytopathogenic effect,CPE);透射电镜观察293细胞中的病毒颗粒;提取细胞裂解液中病毒DNA,PCR鉴定hIGF-1目的基因的存在;Western blot检测hIGF-1蛋白表达。用半数组织培养感染量(tissue culture infectious dose 50,TCID50)方法测定重组腺病毒的滴度。[结果]克隆的hIGF-1目的基因经NCBI BLAST相似性在线分析证实与Homo sapiens IGF-1(GI:19923111)完全相同。pInt.AV1.SpaT-hIGF-1酶切鉴定,出现5.4

[Objective] To construct and identify replication defective adenoviral vectors with human insulin-like growth factor-1 gene of interest for hIGF-1 gene therapy of intervertebral disk degeneration by Cre-LoxP homologous recombination system, in order to lay a basis for hIGF-1 gene thorapy of intervertebral disk degeneration. [Methods] hIGF-1 gene of interest was synthesized by polymerase chain reaction(PCR) and cloned with pMD18-T vector. The cloned hIGF-1 cDNA fragments were se-quenced and similar analysis was done with NCBI B1AST technique in the internet. The pMD18-T -hIGF-1 and empty plasmid pDNR-1r were digested by EcoR I and BamH I, ligated and transformed competence E. coli DH5α. Positive recombinants were identified by EcoR I and BamHI digestion. pInt. AV1. SpaT-hIGF-1 was synthesized by homologous recombination of pDNR-hIGF-1 and pInt. AV1. SpaT with Cre-loxP system and identified by EcoR I digestion. Replication defective aden-ovirus with hIGF-1 gene was synthesized by cotransfection of pInt. AV1. SpaT-hIGF-1 and pInt. B. B plasmids into 293 cells. Cytopathogenic effects were observed through invert microscope. Adenovirus inclusions were observed through transmission electron microscope. Adenovirus DNA was extracted from cell lysis, hIGF-1 gene was identified with PCR technique. hIGF-1 protein expression was indentified by Western blot technique. The second generation adenovirus titers were tested by tissue culture infection dose 50(TCID50) method. [Results] The synthesized hIGF-1 gene of interest had the same sequence as that of Homo sapiens (GI: 19923111) . Both fragments of 5 400 and 1 000 base pairs were seen by restriction endonuclease digestion of pInt. AV1. SpaT-hIGF-1 as expected. Cytopathogenic effects including cell swelling and shedding were observed in most of 293 cells 12-14 days after transfection. hIGF-1 gene existed in the cell lysis. Hexahedron adenovirus inclusions were found in the cytopathic 293 cellular nuclei. hIGF-1 gene existed at recombinant adenovirus. Recombi-nant adenovirus expressed hIGF-1 protein. The second generation adenovirus tilers were 80×106 PFU/mL. [Conclusion] Replication defective adenovirus with hIGF-1 gene of interesl has been constructed successfully.