小鼠热休克蛋白10(HSP10)基因腺病毒载体的构建及在卵泡中的表达

目的:构建小鼠热休克蛋白(heat shock protein 10,HSP10)基因重组腺病毒载体,为更好地研究HSP10蛋白在PCOS发病中的作用。方法:用RT-PCR的方法从小鼠卵巢组织中扩增HSP10基因全长,经T/A克隆后,亚克隆至AdEasy腺病毒穿梭质粒pAdTrack-CMV上,构建穿梭质粒pAdTrack-HSP10。PmeI酶切pAdtrack-HSP10,然后分别将腺病毒骨架质粒pAdEasy-1和穿梭质粒pAdtrack-HSP10转化至BJ-5183感受态细菌中进行同源重组。PacI酶切线性化重组质粒AdCMV-HSP10后转染AD-293细胞进行病毒包装和扩增。通过GFP报告基因观察重组病毒的产生。用获得的重组腺病毒感染小鼠卵泡,Western blotting方法检测HSP10基因的表达。结果:从小鼠卵巢组织中扩增出309bp的cDNA,PCR和测序分析证实为小鼠HSP10基因。构建的小鼠HSP10基因的重组腺病毒载体,PacI酶切重组质粒见30000bp和4500bp的特征性条带。制备出高滴度重组腺病毒,病毒上清PCR反应同样扩出309bp的目的条带。分离并培养小鼠卵泡,感染重组的腺病毒,Western blotting检测到显著的HSP10条带。结论:本文成功构建了小鼠HSP10基因重组腺病毒载体,并且在小鼠卵泡中有效表达。

Construction of the Recombinant Adeno-Virus Vector of Mouse Heat Shock Protein 10 (HSP10) Gene and Its Expression in Mouse Follicles

Objective： AdEasy system was used to construct recombinant adenovirus vector carring mouse HSP10 gene and to have a better understand of the mechanism of heat shock protein 10（HSP10） in the pathogenesis of PCOS. Methods： Mouse HSP10 cDNA was amplified by RT-PCR from mouse ovaries and was cloned into PGEM-T easy vector. HSP10 cDNA was then subcloned into the shuttle vector pAdTrack-CMV for the construction of pAdtrack-HSP10. The shuttle vector was linearized with Pme I and subsequently transformed into E.coli B J-5183 cells with adenovirus backbone plasmid pAdEasy-1 to achieve homologous recombination. After digestion with Pac I, it was transfected into AD-293 ceils for packaging and amplification. The appearance of recombinant adenovirus was determined by GFP expression. The mouse follicles were infected by the recombinant adenovirus and the expression of HSP10 was detected by Western blotting analyses. Results： A 309 bp cDNA of HSP10 was amplified by RT-PCR from mouse ovaries, and its PCR and sequence were analyzed to be correct as expected. The mouse HSP10 gene adenovirus vector was constructed, and the specific band of 4 500 bp and another band of 30 000 bp were observed after digested by Pac I. In addition, recombinant adenovirus vector AdCMV-HSP10 with high virus titter was successfully obtained, and the positive 309 bp band was also amplified by PCR products of the virus supernatant. What＇s more, the mouse follicles were isolated from the ovaries and were cultured individually, and HSP10 expression was detected by Western blotting in the infected mouse follicles. Conclusion： The recombinant adenovirus of HSP10 had been constructed successfully and also had been expressed in the mouse follicles efficiently.