LTβR-Ig融合蛋白的真核表达及其体外生物学功能的研究  

Expression of LTβR-Ig fusion protein by eukaryocytes and evaluation of its bioactivity in vitro

作　　者：周倩[1] 崔淑芳[2] 樊克兴[3] 吴桐[3] 钱卫珠[3] 郭亚军[3] 

机构地区：[1]同济大学附属第一妇婴保健院科教科,上海200040 [2]第二军医大学实验动物中心,上海200433 [3]第二军医大学肿瘤研究所,上海200433

出　　处：《同济大学学报（医学版）》2007年第3期11-15,共5页Journal of Tongji University(Medical Science)

基　　金：国家自然科学基金资助项目(30100172)

摘　　要：目的为了研究LTβR-Ig的生物学功能,构建重组的LTβR-Ig/pcDNA3.1(+)真核表达载体,在CHO细胞中表达,无血清扩大培养,并初步研究了融合蛋白LTβR-Ig的体外生物学活性。方法将鼠LTβR胞外段cDNA与人IgG1 Fc段cDNA共同克隆至真核表达载体pcDNA3.1(+),转染CHO-K细胞表达,阳性克隆进行无血清培养,采用PtoteinA亲和层析的方法进行纯化;3[H]TdR参入法检测对细胞增殖的影响。结果经PCR和限制性酶切鉴定以及DNA测序,结果表明克隆了正确序列的LTβR胞外段序列,成功构建了重组表达质粒LTβR-Ig/pcDNA3.1,并在CHO细胞中稳定表达了LTβR-Ig的融合蛋白,证明LTβR-Ig融合蛋白能抑制混合淋巴细胞反应。结论本研究建立了能稳定表达有生物活性的LTβR-Ig融合蛋白的真核表达系统,为今后研究LTβR在移植排斥中的作用机制,及开展基因重组药物进行疾病的生物治疗奠定了基础。Objective To express recombinant murine LTβR-Ig in CHO cells and evaluate its activities in suppressing T cell proliferation in vitro. Methods Insert LTβR outer domain cDNA and human IgG1 Fc cDNA into the expression vector pcDNA3.1（ ＋ ）, then transfect CHO-K cell line. Using Sepharose-protein A affinity column to purify the recombinant protein, and using^3[H] TdR incorporation method to measure T cell proliferation. Results The recombinant vector, LTβR-Ig/pcDNA3.1（ ＋ ） was constructed and verified by PCR amplification, restriction endonucleases digestion and following sequencing. LTβR-Ig recombinant protein was sta-bly and consistently expressed in CHO cells and the purified protein can effectively suppression T cell proliferation. Conclusion LTβR-Ig recombinant protein with biological activity was expressed in the eukaryotic system. It will provide a clue for study of LTβR in transplant rejection and develop a method by using recombinant gene drugs to treat diseases.

关 键 词：鼠LTβR 基因表达 CHO细胞株 LTβR—Ig融合蛋白 

分 类 号：R318.01[医药卫生—生物医学工程]