1.Construction and Expression of Eukaryotic Expressing Vector of Mouse Soluble CD160
Lingfei HAN ; Wei WANG ; Shujie LIAO ; Chunxiao LI ; Maitituoheti MAYINUER ; Jia LIU ; Xi XIA ; Ronghua LIU ; Ding MA
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong 2009;38(6):748-751
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.
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