恒河猴LFA3胞外段CD2结合域在毕赤酵母中的重组表达及活性分析

Recombinant Expression and Characterization of CD2-binding Domain of Macaca mulatta Lymphocyte Function-associated Antigen 3 in Pichia pastoris

人淋巴细胞功能相关抗原3（hLFA3）是一种重要的T细胞黏附分子,其与CD2结合后可促进T细胞活化。恒河猴被广泛用作人类免疫疾病动物模型。因免疫系统存在严格的种属特异性,有必要研究恒河猴LFA3的功能。本文通过PCR扩增获得mmLFA3Sh基因,将其与人IgG1Fc片段融合,插入毕赤酵母表达载体构建表达质粒pPIC9K-mmLFA3Sh-Ig。诱导表达后获得融合蛋白mmLFA3Sh-Ig,产量为3-4mg/L。mmLFA3Sh-Ig能够特异性与CD2阳性细胞结合,能够抑制Con A和同种抗原刺激的猴和人淋巴细胞增殖。这些实验结果表明,mmLFA3Sh-Ig可能作为一种新的生物制剂用于研究恒河猴免疫疾病发病机制和实验治疗。

Human lymphocyte function-associated antigen 3（hLFA3）has been identified as an important T cell accessory molecule.Rhesus monkeys（Macaca mulatta）have been widely used as animal models for human immune disorders.Due to the species-specificity of immune system,it is necessary to study M.mulatta LFA3（mmLFA3）.In this study,the gene encoding mmLFA3CD2-binding domain（mmLFA3Sh）was amplified by polymerase chain reaction（PCR）and genetically fused to human IgG1 Fc fragment in pPIC9 Kto construct the expression plasmid pPIC9KmmLFA3Sh-Ig.Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media,and mmLFA3Sh-Ig produced by the P.pastoris can bind to the CD2 positive cells,and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner.These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.