Hou Zhen, Zhou Jing, Gao Li-ying, Hu Yan-qiu, Liu Jia-yin
Center of Clinical Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1102-5.
To develop a gene therapy vector of interleukin-1 receptor II(IL-1RII) with recombinant adenovirus and express IL-1RII in the eutopic stromal cells of endometriosis(EM).
Get full length of cDNA with IL-1RII gene was obtained by PCR. The gene was then subcloned into the pShuttle-CMV shuttle vector. The resultant plasmid (pShuttle-CMV-RII) was cotransduced into E.coli BJ5183 cells with pAdEasy-1 plasmid to undergo homologous recombination by electroporation. The linearized recombinant plasmid (pAd-RII) was transfected into 293 cells. The recombinant adenovirus was detected by examining the expression of IL-1RII while the recombinant adenovirus of LacZ gene was constructed as control. The stromal cells of EM were infected by the recombinant adenovirus and the expression of IL-1RII was detected by immunohistochemistry(IHC).
It was confirmed by sequencing that the two ligand products had no mutation of IL-1RII. Restriction endonuclease analysis confirmed the successful cloning of the gene into the pShuttle-CMV and the recombinants(pAd-RII) were selected for kanamycin resistance. Presence of the recombinant adenovirus was confirmed by the X-gal stain of LacZ and the expression of soluble IL-1RII was detected by ELISA. IL-1RII was expressed in the stromal cells of EM by IHC.
The recombinant adenovirus of IL-1RII has been successfully constructed and expressed in the stromal cells of EM, which provides a basis for the research into the role of IL-1RII and the effect of biological treatment in EM.
构建携带白细胞介素1受体Ⅱ（IL-1RⅡ）基因的重组腺病毒基因治疗载体，并使其在内异症（EM）在位内膜间质细胞中表达IL-1RⅡ。
采用PCR技术获取含IL-1RⅡ基因的全长cDNA，将其亚克隆至pShuttle-CMV穿梭载体。将所得质粒（pShuttle-CMV-RⅡ）与pAdEasy-1质粒共转化至大肠杆菌BJ5183细胞，通过电穿孔进行同源重组。将线性化的重组质粒（pAd-RⅡ）转染至293细胞。通过检测IL-1RⅡ的表达来检测重组腺病毒，同时构建LacZ基因的重组腺病毒作为对照。用重组腺病毒感染EM的间质细胞，通过免疫组织化学（IHC）检测IL-1RⅡ的表达。
测序证实两种配体产物的IL-1RⅡ无突变。限制性内切酶分析证实该基因成功克隆至pShuttle-CMV，筛选出对卡那霉素耐药的重组体（pAd-RⅡ）。通过LacZ的X-gal染色证实重组腺病毒的存在，通过ELISA检测可溶性IL-1RⅡ的表达。通过IHC检测到IL-1RⅡ在EM的间质细胞中表达。
成功构建了携带IL-1RⅡ的重组腺病毒，并在EM的间质细胞中表达，为研究IL-1RⅡ的作用及生物治疗在EM中的效果奠定了基础。
