YWHAZ重组腺病毒及逆转录病毒的制备

目的制备YWHAZ重组腺病毒及逆转录病毒,在293T细胞中验证基因表达情况。方法将YWHAZ基因分别克隆至腺病毒穿梭质粒pacAd5CMV-IRES-GFP及逆转录病毒质粒pLXSN-IRES-GFP,并用酶切鉴定、PCR及测序验证后分别转染至HEK 293T及Ampho293细胞,构建重组腺病毒及逆转录病毒。重组病毒分别感染293T细胞,观察荧光验证病毒侵染效果,RT-PCR检测YWHAZ基因表达情况。结果 PCR、酶切鉴定及测序分析,重组腺病毒及逆转录病毒构建正确,感染293T细胞后可使其高效过表达YWHAZ mRNA。结论成功构建了YWHAZ基因重组腺病毒载体及逆转录病毒载体,为体内外进一步研究YWHAZ基因及其相关信号通路在肿瘤发生发展中的作用奠定了基础。

ConstructionofRecombinantYWHAZAdenovirusandRetrovirus

Objective To construct the recombinant Adenoviral and retroviral vectors of human tyrosine 3-monooxy- genase/tryptophan 5-monooxygenase activation protein zeta polypeptide（YWHAZ）and detect the expression of these two viral vectors in 293T cell. Methods YWHAZ gene was cloned to adenoviral plasmid （pacAd5 CMV-IRES-GFP） and retroviral plasmid （pLXSN-IRES-GFP）, then confirmed by enzyme digestion,PCR and gene sequencing. YWHAZ adenovirus was made by transfecting the recombinant adenoviral plasmid and backbone plasmid to 293T. Recombinant YWHAZ retrovirus was made by transfecting the recombinant retroviral plasmid to Ampho293. The transfection effi- ciency was observed by fluorescence microscope,and the expression of YWHAZ gene was tested by RT-PCR. Results YWHAZ adenovirus and retrovirus were successfully constructed. They can infect target cells effectively and highly ex- press YWHAZ. Conclusion We successfully constructed YWHAZ adenovirus and retrovirus. They can be used as ide- al tools for further study of YWHAZ function in tumorigenesis＇and progression.