人IL-1RⅡ基因腺病毒载体的构建及在子宫内膜异位症细胞中的表达

目的：利用AdEasy XL系统，构建并鉴定IL-1RⅡ基因重组腺病毒载体，并在子宫内膜异位症（EM）细胞中表达。方法-PCR扩增含有IL-1RⅡ全长cDNA的片段，亚克隆到pShuttle—CMV穿梭质粒，经酶切和测序验证无误后，再经电转化与pAdEasy-1质粒在大肠杆菌BJ5183中进行同源重组产生腺病毒载体质粒。经过抗性筛选、酶切鉴定以及再次测序验证无误后得到阳性的重组质粒，经PacⅠ酶切线性化再在293细胞中进行包装扩增，用ELISA检测IL-1RⅡ蛋白的表达。利用Adeasy XL系统的对照载体pShuttle-CMV-LacZ同上操作作为对照。收集的重组腺病毒感染原代培养的EM基质细胞，并以免疫组化法鉴定IL-1RⅡ表达。结果：测序证实连接后IL-1RⅡ序列完全正确；抗性筛选及酶切鉴定均表明重组腺病毒载体构建成功；pShuttle-CMV-LacZ转染293细胞3d后x-gal染色阳性，回收病毒可以重复感染293细胞，ELISA鉴定表达IL-1RⅡ可溶性蛋白，证明病毒包装成功。重组的腺病毒感染EM基质细胞后，免疫组化法证实IL-1RⅡ表达。结论：成功地构建了IL-1RⅡ基因重组腺病毒载体，并且在EM基质细胞中表达，为进一步研究IL-1RⅡ基因在EM中的作用乃至生物治疗都奠定了基础。

Construction of the recombinant adenovirus vector of interleukin-1 receptor Ⅱ and its expression in eutopic stromal cells of endometriosis

AIM: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).METHODS: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).RESULTS:It was confirmed by sequencing that the two lingand 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.CONCLUSION: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.