人源分子佐剂hC3d3促进人免疫活性细胞协同刺激分子表达及对hCGβ抗原的反应性

The molecular adjuvant hC3d3 improves expressions of co-stimulatory molecules in human inimuno-competent cells and their responsiveness to the hCGβantigen

目的探讨分子佐剂hC3d3与hCGβ的融合蛋白hCGβ-hC3d3对人外周血单个核细胞(PBMC)中的B细胞与T细胞表面协同刺激分子表达及对hCGβ抗原反应性的影响。方法从人肝细胞cDNA文库克隆hC3d基因片段;构建pCI-gs-signal-6His-hCGβ及pCI-gs-signal-6His-hCGβ-hC3d3真核表达质粒,表达和纯化重组蛋白抗原hCGβ及hCGβ-hC3d3融合蛋白。实验分组:将B细胞、B+T细胞组或PBMC,分别应用hCGβ、hCGβ-hC3d3或美洲商陆(PWM)等抗原进行体外处理48 h。用流式细胞术分析培养后的上述淋巴细胞表面的CD80、CD86、CD154、CD25等协同刺激分子的表达;用ELISA检测培养上清中IL-2的含量。结果本研究成功克隆了hC3d基因,构建了真核表达质粒pCI-gs-signal- 6His-hCGβ及pCI-gs-signal-6His-hCGβ-hC3d3;经转染CHO细胞,获得了目的蛋白高效分泌表达。经鉴定及纯化成功获得了较高纯度的目的蛋白hCGβ及hCGβ-hC3d3。hCGβ-hC3d3蛋白能够显著上调B细胞表面CD80、CD86分子的表达,尤其是CD86分子的表达(P<0．01);能明显增强T细胞表面CD154、CD25分子的表达(P<0．05)。hCGβ-hC3d3蛋白提高B细胞抗原提呈能力及T细胞活化后分泌IL-2的能力(P<0．05)。结论人源分子佐剂hC3d3明显促进人免疫活性细胞协同刺激分子表达及对hCGβ抗原的反应性。

Objective To find if human molecular adjuvant hC3d3 promotes expressions of co-stimulatory molecules in human immuno-competent cells and responsiveness to the hCGβ antigen when fused into the antigen. Methods Human C3d gene was cloned from human hepatic cDNA library, the eukaryotic expression plasraids of pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3 were constructed. CHO cells were transfected by the recombinant expression plasmids with aid of LipofectAMINE 2000 reagent. The recombinant proteins were expressed and purified using His. Bind cohann and Sephadex G150 column gel filtration chromatography. The isolated B cells, B cells ＋ T cells and PBMC were treated with 100 nmol/L hCGβ, hCGβ-hC3d3 or PWM for 48 h. The expression of CD80, CD86, CD154 and CD25 in the cells was analyzed by flow cytometry. The IL-2 production in the supematant was assayed by sandwich ELISA. Results The hC3d gene has been successfully cloned from human hepatic cDNA library, and the eukaryotic expression plasmids of pCI-gs-signal-6His-hCGβ and pCI-gs- signal-6His-hCGβ-hC3d3 were constructed. The correspondent recombinant proteins were secreted into the cultural supematant by CHO cells. The hCGβ-hC3d3 significantly enhanced the expressions of CD80 and CD86 molecule in B cell, especially CD86 （P 〈0.01）, and that of CD154 and CD25 molecule in T cells, hCGβ-hC3d3 promoted human PBMC producing more IL-2 than the hCGβ. Conclusion The human molecular adjuvant hC3d improves expressions of co-stimulatory molecules in human immuno-competent cells and their responsiveness to the hCGβ antigen.