Position: Home > Articles > Construction of TIG-Fc Eukaryotic Expression Vector and its Effect on the Function of Natural Killer Cells
Current Biotechnology
2020
(1)
90-96
TIG-Fc真核表达载体的构建及其对自然杀伤细胞功能的影响
作 者：
李国华;高锦伟;周希胜;王霞;程伟华
单 位：
陕西省宝鸡市中心医院肝胆胰脾外科;陕西省宝鸡市凤翔县医院;陕西省延安市延安职业技术学院;陕西省延安市安塞区中医医院
关键词：
TIGIT;融合蛋白;真核表达;自然杀伤细胞
摘 要：
T细胞免疫球蛋白和免疫受体酪氨酸抑制性基序结构域[T-cell immunoglobulin and immunoreceptor tyrosinebased inhibitory motif (ITIM) domain,TIGIT]是一种新型的免疫抑制性受体,在机体的免疫调节网络中扮演着重要角色。为了进一步探究TIGIT对自然杀伤(natural killer,NK)细胞的免疫调节功能,构建了融合蛋白TIGIT胞外段(即TIGIT第1～135位氨基酸,以保留功能结构域Ig V区,此胞外段简称为TIG)-人免疫球蛋白G3(immunoglobulin G3,Ig G3) Fc段的真核表达载体,并对TIG-Fc融合蛋白的表达及其对NK-92细胞功能的影响进行了初步研究。利用分子生物学方法,将携带人TIG与人Ig G3 Fc段的基因融合,然后插入真核表达载体pc DNA3.1(-)中,以构建重组质粒pc DNA3.1(-)-TIGFc。随后将重组质粒转染至人胚肾细胞HEK-293T中,通过流式细胞仪和Western blot检测TIG-Fc融合蛋白在293T细胞中的表达;再将重组质粒转染至NK-92细胞中,通过WST-1检测TIG-Fc融合蛋白对NK-92细胞增殖的影响,并利用ELISA法检测TIG-Fc融合蛋白对NK-92细胞分泌IFN-γ的水平的影响。结果成功构建了人TIG与人Ig G3 Fc融合表达的真核表达载体,且TIG-Fc融合蛋白的表达能够显著抑制NK-92细胞的增殖和分泌IFN-γ的能力(P<0.05),为后期TIGIT的功能研究奠定了重要基础。
译 名：
Construction of TIG-Fc Eukaryotic Expression Vector and its Effect on the Function of Natural Killer Cells
作 者：
LI Guohua;GAO Jinwei;ZHOU Xisheng;WANG Xia;CHENG Weihua;Yan'an Vocational and Technical College;Ansai District Hospital of Traditional Chinese Medicine;Fengxiang County Hospital;Department of Hepatobiliary Pancreatic and Spleen Surgery,Baoji Central Hospital;
关键词：
TIGIT;;fusion protein;;eukaryotic expression;;NK cell
摘 要：
T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif( ITIM) domain( TIGIT) is a novel immunosuppressive receptor,which plays an important role in the immune regulatory network of the body. In order to further explore the immunomodulatory function of TIGIT in natural killer( NK) cells,a eukaryotic expression vector for fusion protein composed by TIGIT extracellular domain( TIGIT amino acids 1 ～ 135,to retain the functional domain Ig V region,this extracellular segment is simply called TIG) and Fc segment of human immunoglobulin G3( IgG3) was constructed,and the expression of TIG-Fc fusion protein and its effect on NK-92 cell function were preliminarily studied. Using molecular biology method,the gene carrying human TIG and Fc segment of IgG3 were fused,and then inserted into eukaryotic expression vector pc DNA3.1(-) to construct recombinant plasmid pc DNA3.1(-)-TIG-Fc. Subsequently,the recombinant plasmid was transfected into human embryonic kidney( HEK) 293 T cells,and the expression of TIG-Fc fusion protein in 293 T cells was detected by flow cytometry and Western blot. Then the recombinant plasmid was transfected into NK-92 cells,and the effect of TIG-Fc fusion protein on NK-92 cell proliferation was detected by WST-1,and the effect of TIG-Fc fusion protein on IFN-γ secretion level of NK-92 cells was detected by ELISA. The results showed that the eukaryotic expression vector for human TIG and human IgG3 Fc fusion expression was successfully constructed,and the expression of TIG-Fc fusion protein could significantly inhibit the proliferation of NK-92 cells and their secretion level of IFN-γ( P< 0.05),which laid an important foundation for the functional research of TIGIT in the later stage.
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