小鼠HAX-1基因重组腺病毒载体的构建表达和鉴定

目的构建小鼠Hax-1基因的重组腺病毒载体，以期进一步进行系统性红斑狼疮模型BXSB小鼠的体内实验研究。方法用RT-PCR方法扩增小鼠全长Hax-1基因，经T／A克隆后，亚克隆至穿梭质粒pAdtrack-CMV上，在BJ5183细菌中和AdEasv-1进行同源重组，筛选阳性克隆，酶切、测序鉴定正确后，脂质体法转染AD-293细胞进行包装，扩增，氯化铯密度梯度离心纯化病毒并检测其滴度。RT-PCR鉴定重组腺病毒转染AD-293细胞后Hax-1的表达，同时进行野生型腺病毒的检测。结果经酶切及测序证实Hax-1基因重组腺病毒载体构建成功。PCR检测转染后AD-293细胞内Hax-1基因水平的表达，且无野生型腺病毒的产生。结论成功构建了含小鼠Hax-1基因的重组腺病毒载体，为探讨Hax-1的生物学功能奠定了基础。

Construction, expression, and identification of the recombinant adenovirus vector of HS1-associated protein X-1

Objective To construct a recombinant adenovirus encoding HS1-associated protein X-1(Hax-1)for further animal model experiments in vivo. Methods Full-length mouse-derived Hax-1 DNA was obtained by the method of RT-PCR. The Hax-1 DNA was cloned in pMD18-T, and then subcloned into pAdtrack-CMV shuttle plasmid. The product was linearized for homologous recombination with AdEasy-1 vector in BJ5183 bacteria. The positive clone was identified by restriction endonuclease digestion and confirmed by sequencing, and then the recombined adenovirus DNA was transfected into AD-293 cells for packaging and amplification of AdHax-1 virus. The virus was purified by CsCl density gradient centrifugation and the expression of Hax-1 in infected cells was monitored by EGFP fluorescence. Results Transfection of adenovirus DNA could caused cytopathic effects only in AD-293 cell but not in HeLa cell line, which proved that only the replication-defective adenovirus was produced. There was no evidence for production of wild type adenovirus. The specific expression of mouse Hax-1 was identified by PCR in AD-293 cell after infection with AdHax-1, but not in the control AD-293. Conclusion The recombinant adenovirus AdHax-1 is constructed successfully, which will be useful for investigating the role of T lymphocytes apoptosis in animal models of systemic lupus erythematosus in vivo.