CAJ | 학술논문

目的构建小鼠CD160胞外段的真核表达载体,并稳定转染CHO细胞进行真核表达.方法从C57BL/6小鼠脾脏中提取总RNA,经RT-PCR扩增CD160胞外段,然后将其克隆到真核表达载体pcDNA3.1和pEGFP-N1两种质粒中,构建重组质粒psCD160和pEGFP-sCD160.重组质粒经双酶切鉴定及测序正确后,用脂质体转染CHO细胞,并用RT-PCR和Western blot检测CD160胞外段的表达,流式细胞术检测重组psCD160与其配体的结合能力.结果获得长度约为520 bp的小鼠CD160胞外段基因,经测序证实其序列正确.用脂质体将含有CD160胞外段基因的重组质粒载体转染CHO细胞后.RT-PCR和Western blot分析证实转染的CHO细胞可表达重组的psCD160基因,并且表达的可溶性CD160可与其配体结合.结论成功构建小鼠CD160胞外段基因的真核表达载体,并转染CHO细胞后表达出相应蛋白,为进一步研究CD160胞外段的功能效应奠定实验基础.
목적 구건소서CD160포외단적진핵표체재체,병은정전염CHO세포진행진핵표체.방법 종C57BL/6소서비장중제취총RNA,경RT-PCR확증CD160포외단,연후장기극륭도진핵표체재체pcDNA3.1화pEGFP-N1량충질립중,구건중조질립psCD160화pEGFP-sCD160.중조질립경쌍매절감정급측서정학후,용지질체전염CHO세포,병용RT-PCR화Western blot검측CD160포외단적표체,류식세포술검측중조psCD160여기배체적결합능력.결과 획득장도약위520 bp적소서CD160포외단기인,경측서증실기서렬정학.용지질체장함유CD160포외단기인적중조질립재체전염CHO세포후.RT-PCR화Western blot분석증실전염적CHO세포가표체중조적psCD160기인,병차표체적가용성CD160가여기배체결합.결론 성공구건소서CD160포외단기인적진핵표체재체,병전염CHO세포후표체출상응단백,위진일보연구CD160포외단적공능효응전정실험기출.
Objective To construct eukaryotic expressing vector of mouse soluble CD160 and stably transfect into CHO cells for eukaryotic expression.Methods Recombinant soluble CD160(rsCD160) was constructed by gene recombination.Total RNA was extracted from the spleen of C57BL/6 mice.cDNA was amplified for the soluble form of CD160.Then,the PCR product was cloned tO pcDNA3.1 and pEGFP-N1.The recombinant plasmid was identified by restriction map and sequence analy-sis.The soluble CDl 60 expression in CHO cells transfected with recombinant psCDl 60 was verified by RT-PCR and Westernblot.The binding ability of psCD160 tO its ligand was detected by FACS.Results 520 bp mouse soluble CD160 gene was obtained.Recombinant mouse psCD160 was successfully constructed.After transfection,soluble CD160 expression in the culture supernatant of CHO cells was successfully detected.FACS analysis indicated that soluble CD160 could bind tO its ligand.Conclusion Recombinant mouse psCD160 is successfully constructed,which will benefit our further study on soluble CD160 for immune therapy against tumor in the future experiments.