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目的 构建p3.1 /CTLA4Ig- IRES- OX4 0Ig真核表达载体,共同表达CTLA4Ig和OX4 0Ig,通过体外实验初步研究其生物学特性。方法 通过特异引物扩增OX4 0胞外区的cDNA ,经酶切、亚克隆、拼接构建了真核表达载体p3.1/ CTLA4Ig IRES- OX4- 0Ig,并使之在体外进行表达,用RT- PCR、SDS- PAGE和Westernblot鉴定目的基因的表达,ELISA法测定目的基因的表达水平和混合淋巴细胞反应研究其抑制免疫应答的效应。结果 测序证实所扩增的PCR产物是OX4 0胞外区的cDNA ,其序列与GenBank中的相一致;成功构建了p3.1 -CTLA4Ig IRES- OX4 0Ig真核表达载体;RT -PCR、SDS -PAGE和Westernblot结果显示,表达的蛋白为CTLA4Ig和OX4 0Ig ,ELISA结果提示,该两种蛋白均得到了高效表达,混合淋巴细胞反应结果证实转染了p3.1/ CTLA4Ig- IRES- OX4 0Ig的CHO细胞培养上清可以有效地抑制异基因淋巴细胞的刺激作用,其抑制效果强于转染p3.1 /CTLA4Ig和p3.1 /OX4 0Ig的CHO细胞培养上清。结论 成功构建了含CTLA4Ig和OX4 0Ig的真核表达载体,并在体外能够同时得到表达;体外实验证实该两种分子协同作用可以有效抑制免疫应答,并且优于该两种分子单独作用时的抑制效果。
Objective To construct the eukaryotic expression vector p3.1 / CTLA4Ig-IRES-OX4 0Ig to express CTLA4Ig and OX4 0Ig, and to study its biological characteristics in vitro. Methods The cDNA of OX4 0 extracellular region was amplified by specific primers. The eukaryotic expression vector p3.1 / CTLA4Ig IRES-OX4- 0Ig was constructed by restriction endonuclease, subcloning and splicing. PCR, SDS-PAGE and Western blot to identify the expression of the target gene, ELISA method to determine the expression level of the target gene and mixed lymphocyte reaction to study its inhibitory effect of immune response. Results The PCR product was confirmed to be OX4 0 extracellular domain by sequencing. The sequence was consistent with that in GenBank. The eukaryotic expression vector p3.1-CTLA4Ig IRES-OX4 0Ig was constructed successfully. RT-PCR and SDS- PAGE and Western blot results showed that the expressed protein was CTLA4Ig and OX4 0Ig, ELISA results suggest that the two proteins were highly expressed, mixed lymphocyte reaction confirmed the transfected p3.1 / CTLA4Ig- IRES-OX4 0Ig CHO Cell culture supernatant can effectively inhibit the stimulation of allogeneic lymphocytes, and its inhibitory effect is stronger than the CHO cell culture supernatant transfected with p3.1 / CTLA4Ig and p3.1 / OX4 0Ig. Conclusion The eukaryotic expression vector containing CTLA4Ig and OX4OIg was successfully constructed and expressed simultaneously in vitro. In vitro experiments show that the synergistic effect of the two molecules can effectively suppress the immune response and is superior to the inhibition of the two molecules alone effect.