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目的研究Tn5细胞中单抗抗人重组质粒(B7-2)基因的表达及其活性。方法基于具备信号肽单抗抗人B7-2构建,构建系统Bac-to-Bac并含单抗抗人B7-2杆状重组病毒AcBacmid单抗B7-2,对Tn5细胞采用不同感染复数(MOI)实施感染,进行24、48、72h不同时点细胞蛋白活性的分泌表达,并分析人外周血中单个核细胞受Raji刺激呈增殖表达,予以单抗抑制的效果。结果应用不同MOI对Tn5细胞感染,达72h后进行细胞收集,予以聚丙烯酰氨凝胶电泳(PAGE-SDS)分析,其MOI为1~10表达目的蛋白的量,确定最佳MOI为5;MTT法下自动酶标仪波长570nm位置吸光度,其吸光值的大小同细胞PBMC增殖可呈正相关;各组吸光值显示细胞Raji刺激外周血单个核细胞(PBMC)呈增殖表达,单抗抗人B7-2可以抑制Raji细胞刺激PBMC出现的增殖效应。结论单抗B7-2抗原抗体相结合的活性,以及对PBMC产生的抑制效应均存在,但未见单抗B7-2效应明显表达,有待进行更进一步的试验研究。
Objective To study the expression and activity of anti-human recombinant plasmid (B7-2) gene in Tn5 cells. Methods Based on the construction of anti-human B7-2 with signal peptide monoclonal antibody, the constructed Bac-to-Bac system was used to construct the B7-2 recombinant baculovirus B7-2 with anti-human B7-2 baculovirus recombinant baculovirus. The Tn5 cells were infected with different multiplicity of infection (MOI ) For 24 h, 48 h, 72 h, respectively. The secretion of cell protein was detected at 24 h, 48 h and 72 h after transfection. The mononuclear cells in human peripheral blood were analyzed by Raji-stimulated cell proliferation. Results The Tn5 cells were infected with different MOI, and the cells were collected after 72 hours. The cells were analyzed by polyacrylamide gel electrophoresis (PAGE-SDS). The MOI was 1 ~ 10, and the optimal MOI was 5. Absorbance at 570 nm was detected by MTT assay. The absorbance value of the plate was positively correlated with the proliferation of PBMCs. The absorbance of each group showed that Raji stimulated the proliferation of peripheral blood mononuclear cells (PBMCs) -2 can inhibit the proliferation of Raji cells to stimulate PBMC proliferation. Conclusion The activity of B7-2 antigen antibody binding and the inhibitory effect on PBMC are both existed. However, no obvious effect of B7-2 monoclonal antibody was expressed, and further experimental studies are needed.