摘要
目的构建携带小鼠信号转导子及转录激活子3(STAT3)的重组慢病毒载体,并检测STAT3的蛋白表达,进行慢病毒包装并鉴定。方法将小鼠成肌细胞总RNA通过STAT3引物逆转录为cDNA PCR扩增、回收后,同pLVX-IRES-ZsGreen1载体片段连接,酶切鉴定及测序,将pLVX-IRES-ZsGreen1-STAT3转染293T细胞,48h后收集细胞,Western blot检测STAT3表达量,通过瞬时转染法包装出病毒上清。结果测序结果证实STAT3成功插入pLVX-IRES-ZsGreen1慢病毒载体,成功构建了慢病毒载体pLVX-IRES-ZsGreen1-STAT3,共转染293T细胞48h,Western blot检测STAT3表达量明显增强。测定病毒滴度为8.4×107 TU/mL。结论成功构建了STAT3基因的重组慢病毒表达载体,为进一步应用奠定了基础。
Objective To construct recombinant lentivirus with the gene STAT3 of the Mus musculus,measure the expression of STAT3,and conduct lentivirus packing and identification.Methods The mRNA of mouse myoblast was extracted and transformed into STAT3 cDNA by the special primer.then,STAT3 cDNA was amplified and reclaimed and inseted into pLVX-IRES-ZsGreen1 vector.Cleavage map and sequencing analysis were used for identification of the recombinant lentivirus vector（pLVX-IRES-ZsGreen1-STAT3）.293 Tcells were transfected with main vector pLVX-IRES-ZsGreen1-STAT3.and 48 hlater,Western blott detected the expression of STAT3 protein.Lentiviral vectors were packaged and the titer was determined.ResultsThe lentiviral vector plasmid pLVX-IRES-ZsGreen1-STAT3 was identified correctly by cleavage map and Co-transfection of 293 T cells with 48 h,the expression of STAT3 was significantly enhanced by western blot.And DNA sequencing analysis confirmed that STAT3 gene sequencing was exactly the same with that reported by genbank.Conclusion Lentiviral vector carrying STAT3 was successfnlly constructed and could express STAT3 with high efficiency,and can be used in further study.
出处
《重庆医学》
CAS
北大核心
2015年第27期3803-3804,3807,共3页
Chongqing medicine
基金
重庆市科委自然科学基金资助项目(2012JJA10002)
重庆市卫生局基金资助项目(20122296)
关键词
信号转导子及转录激活子3
慢病毒载体
signal transducers and activators of transcription 3
lentivirus vector
