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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。
OBJECTIVE: To construct a recombinant adenovirus vector of mouse heat shock protein 10 (HSP10) gene in order to better study the role of HSP10 in the pathogenesis of PCOS. Methods: The full length of HSP10 gene was amplified by RT-PCR from mouse ovary tissue. After T / A cloning, the full length of HSP10 gene was subcloned into shuttle plasmid pAdTrack-CMV of AdEasy adenovirus to construct shuttle plasmid pAdTrack-HSP10. PAdI was used to digest pAdtrack-HSP10. The adenovirus backbone plasmid pAdEasy-1 and shuttle plasmid pAdtrack-HSP10 were respectively transformed into BJ-5183 competent cells for homologous recombination. PacI linearized the recombinant plasmid AdCMV-HSP10 and then transfected AD-293 cells for virus packaging and amplification. Recombinant virus production was observed by GFP reporter gene. Mouse ovarian follicles were infected with the obtained recombinant adenovirus and the expression of HSP10 gene was detected by Western blotting. Results: The cDNA of 309bp was amplified from the mouse ovary tissue. PCR and sequencing analysis confirmed the mouse HSP10 gene. The recombinant mouse adenoviral vector containing mouse HSP10 gene was constructed. The recombinant plasmids were digested with PacI and found to have characteristic bands of 30000 bp and 4500 bp. The high titer recombinant adenovirus was prepared, and the PCR reaction of the virus supernatant also expanded the target band of 309 bp. Mouse follicles were isolated and cultured and infected with recombinant adenovirus. Significant HSP10 bands were detected by Western blotting. Conclusion: The mouse HSP10 gene recombinant adenovirus vector was successfully constructed and expressed efficiently in mouse follicles.