人白细胞介素10重组腺病毒载体的构建

Construction of recombinant adenovirus vector expressing the human IL-10

目的构建人白细胞介素 10重组腺病毒载体 ,为真核表达及其临床研究提供实验基础。方法自行设计一对引物 ,应用逆转录多聚酶链式反应 ( RT- PCR )技术 ,从活化的正常人外周血单个核细胞中扩增出 h IL - 10 c DNA,并将h IL - 10 c DNA克隆到腺病毒载体 p Ad CMV - L ink1中 ,构建 p Ad CMV/ h IL - 10表达质粒。将此表达质粒与腺病毒重组质粒p JM17共转染 2 93细胞 ,通过同源重组产生 h IL - 10重组腺病毒。结果扩增到的 c DNA片段经酶切鉴定、PCR扩增、DNA测序最终确定为 h IL- 10 c DNA;所得人白细胞介素 10重组腺病毒滴度为 1.9× 10 9pfu/ m L。结论本实验为今后研究 h IL-

ObjectiveTo construct the recombinant adenovirus vector expressing the human interleu kin 10 Methods A pair of DNA primers we re designed and synthesized(each 30 bases) A 562 bp fragment containing hIL 10 gene was successfully amplified by using RT PCR and then cloned into pAdCMV Link1 plasmid Recombinant adenovirus pAdhIL 10 was derived by co transfecting DNAs of pAdhIL 10 and pJM17 into 293 cells using lipos ome methods and the replication deficient hIL 10 recombinant adenovirus was generated efficiently by homologous recombination The hIL 10 recombinant ad enovirus was selected and sequenced Results Analysis of restriction enzyme digestion of recombinant adenovirus and the re sults of sequencing showed that the fragment cloned in pAdCMV Link1 was hI L 10 cDNA The human IL 10 recombinant adenovirus was obtained with the t iters of 1 9×10 9 pfu/mL Conclusion Success in constructing recombinant adenovirus pAdCMV Link1/hIL 10 will promote the further research in molecular biology of hIL 10 and the pAdCMV Link1/hIL 10 can be potentially used in inflammatory disease gene therapy