CAJ | 학술논문

目的构建家蝇幼虫抗菌肽的真核表达载体,并检测它在中国仓鼠卵巢(CHO)细胞中的表达情况.方法以pUCm-T/Defensin为模板,设计带His标签特异引物,扩增C-末端融合6×His标签的Defensin基因的cDNA全长编码区序列,将其克隆至pcDNA3.1(+)真核表达载体.经酶切及测序鉴定后,以阳离子脂质体Lipofect AmineTM 2000转染CHO细胞,G418抗性筛选稳定转染的阳性克隆细胞,以RT-PCR分析其mRNA的转录情况,通过Ni-NTA琼脂糖柱层析纯化目的蛋白并以Western免疫印迹鉴定蛋白质表达情况.结果构建了pcDNA3.1 (+)-Defensin-His表达质粒,建立了稳定转染的CHO细胞系.RT- PCR从阳性克隆CHO细胞系中扩增出320 bp大小的目的片段,从转录水平证实pcDNA3.1(+)-Defensin-His重组细胞株表达了Defensin基因.Western免疫印迹检测可见相对分子质量为10000的阳性条带,表明Defensin-His在该细胞系中成功表达.结论成功构建了质粒pcDNA3.1(+)-Defensin-His,并获得了稳定表达的CHO-Defensin细胞株,有利于进一步研究家蝇幼虫抗菌肽Defensin基因的生物学功能.
목적 구건가승유충항균태적진핵표체재체,병검측타재중국창서란소(CHO)세포중적표체정황.방법 이pUCm-T/Defensin위모판,설계대His표첨특이인물,확증C-말단융합6×His표첨적Defensin기인적cDNA전장편마구서렬,장기극륭지pcDNA3.1(+)진핵표체재체.경매절급측서감정후,이양리자지질체Lipofect AmineTM 2000전염CHO세포,G418항성사선은정전염적양성극륭세포,이RT-PCR분석기mRNA적전록정황,통과Ni-NTA경지당주층석순화목적단백병이Western면역인적감정단백질표체정황.결과 구건료pcDNA3.1 (+)-Defensin-His표체질립,건립료은정전염적CHO세포계.RT- PCR종양성극륭CHO세포계중확증출320 bp대소적목적편단,종전록수평증실pcDNA3.1(+)-Defensin-His중조세포주표체료Defensin기인.Western면역인적검측가견상대분자질량위10000적양성조대,표명Defensin-His재해세포계중성공표체.결론 성공구건료질립pcDNA3.1(+)-Defensin-His,병획득료은정표체적CHO-Defensin세포주,유리우진일보연구가승유충항균태Defensin기인적생물학공능.
Objective To construct eukaryotic vector expressing antibacterial Defensin (pcDNA3.1 (+)-Defensin-His) in housefly larva and detect its expression in Chinese hamster ovary (CHO) cells.Methods The whole coding sequence of Defensin gene cDNA with 6×His fusion tag at C-terminal was amplified using the Histagged specific primer with pUCm-T/Defensin as a model and was cloned into eukaryotic expression vectors pcDNA3.1 (+).After identification using restriction enzyme and sequencing technique,the recombinant pcDNA3.1 (+)-Defensin-His was transfected into CHO cells via Lipofect AmineTM 2000.The stably transfected positive cloning cells were screened with G418 site.Transcription of mRNA was analyzed using RT-PCR,and the protein expressed was purified adopting Ni-NAT agar column and detected by Western blotting.Results The eukaryotic expression plasmid of pcDNA3.1 (+) -Defensin-His was constructed and the CHO cell lines with stable transfection were established.The targeted 320 bp cDNA fragment was amplified from positive CHO cloning cells.Expression of Defensin gene in recombinant pcDNA3.1 (+)-Defensin-His cell lines was proved by RT-PCR at the level of transcription.Western blotting showed a 10 000 positive band,indicating successful expression in Defensin-His cell lines.Conclusion Recombinant pcDAN3.1 (+)-Defensin-His expression plasmid is successfully constructed and CHO-Defensin cell lines with stable expression are obtained,which benefit further researches on the biological activities of Defensin gene in housefly larva.