CAJ | 학술논문

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-Zs-Green1载体片段连接,酶切鉴定及测序,将 pLVX-IRES-ZsGreen1-STAT3转染293 T 细胞,48 h 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293 T 细胞48 h,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。
목적：구건휴대소서신호전도자급전록격활자3(STAT3)적중조만병독재체,병검측 STAT3적단백표체,진행만병독포장병감정。방법장소서성기세포총 RNA 통과 STAT3인물역전록위 cDNA PCR 확증、회수후,동 pLVX-IRES-Zs-Green1재체편단련접,매절감정급측서,장 pLVX-IRES-ZsGreen1-STAT3전염293 T 세포,48 h 후수집세포,Western blot 검측STAT3표체량,통과순시전염법포장출병독상청。결과측서결과증실 STAT3성공삽입 pLVX-IRES-ZsGreen1만병독재체,성공구건료만병독재체 pLVX-IRES-ZsGreen1-STAT3,공전염293 T 세포48 h,Western blot 검측 STAT3표체량명현증강。측정병독적도위8.4×107 TU/mL。결론성공구건료 STAT3기인적중조만병독표체재체,위진일보응용전정료기출。
Objective To construct recombinant lentivirus with the gene STAT3 of the Mus musculus,measure the expres-sion 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 T cells were transfected with main vector pLVX-IRES-ZsGreen1-STAT3.and 48 h later, Western blott detected the expression of STAT3 protein.Lentiviral vectors were packaged and the titer was determined.Results The 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.