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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。
OBJECTIVE: To construct and identify the recombinant adenovirus vector of IL-1RII gene by using AdEasy XL system and express in endometriosis (EM) cells. Methods: The full-length cDNA fragment containing IL-1RII was amplified by PCR and subcloned into shuttle plasmid pShuttle-CMV. After being verified by restriction enzyme digestion and sequencing, the fragment was subcloned into pAdEasy-1 plasmid for homologous analysis in E. coli BJ5183 Recombinant adenovirus vector plasmid. After resistance screening, restriction enzyme digestion and sequencing, the positive recombinant plasmids were obtained. The recombinant plasmids were linearized with PacI and packaged in 293 cells. The expression of IL-1RII protein was detected by ELISA. Control vector pShuttle-CMV-LacZ, using the Adeasy XL system, was used as a control. Primary cultured EM stromal cells were infected with the collected recombinant adenovirus and the expression of IL-1RII was identified by immunohistochemistry. Results: Sequencing confirmed that the sequence of IL-1RII was completely correct. The results of resistance screening and restriction enzyme digestion showed that the recombinant adenovirus vector was constructed successfully. X-gal staining of 293 cells transfected with pShuttle-CMV-LacZ was positive and virus recovery could be repeated Infected 293 cells, ELISA identification of IL-1RII expression of soluble protein, virus packaging proved successful. After the recombinant adenovirus infected EM stromal cells, the expression of IL-1RII was confirmed by immunohistochemistry. Conclusion: The recombinant adenovirus vector of IL-1RII gene was successfully constructed and expressed in EM stromal cells, which laid the foundation for the further study on the role of IL-1RII gene in EM and even biological therapy.