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

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.