论文部分内容阅读
CD4 0 /CD4 0L途径是除B7/CD2 8途径之外的重要的共刺激信号转导途径 ,在T细胞活化过程中扮演着十分关键的角色。因而 ,该途径可能在同种移植排斥的诱导和效应阶段的不同时期发挥关键作用。本研究旨在获得人CD4 0分子胞外区和人IgG 1Fc段的融合蛋白 ,以探索阻断CD4 0 /CD4 0L共刺激途径在免疫治疗中的潜在应用。首先 ,从人B淋巴瘤细胞系Daudi中提取细胞总RNA ,利用RT PCR技术扩增人CD4 0分子胞外区基因 ,将扩增产物连接入pGEMTEasy载体中构建克隆载体 pGE4 0 ,利用全自动DNA测序仪进行序列测定。将测序正确的胞外区片段插入至含人IgG 1类抗体重链基因组序列的 pIG 1载体中构建瞬时表达载体 ,命名为 pIG/ 4 0Ig。用DEAE Dextran/Chloroquine法转染COS 7细胞 ,夹心ELISA法和Western印迹分析鉴定培养上清中CD4 0 Ig融合蛋白的表达及免疫学活性。在重组载体 pIG/ 4 0Ig转染COS 7细胞后 ,ELISA法检测到细胞培养上清中有融合蛋白的表达 ;Western印迹鉴定发现在相对分子量 5 0kD左右有一特异条带 ,该分子可同时被抗人CD4 0单抗G2 8 5和抗人Igγ链的单抗识别。本研究成功地扩增人CD4 0基因并构建了人CD4 0 Ig融合基因表达载体 ,在C0S 7细胞中获得功能性表达 ,为进一步研究CD4 0 /CD4 0L途径在移植物抗宿主
The CD4 0 / CD4 0L pathway is an important costimulatory pathway other than the B7 / CD28 pathway and plays a crucial role in T cell activation. Thus, this pathway may play a key role at different stages of the induction and response stage of allograft rejection. The purpose of this study was to obtain a fusion protein between the extracellular domain of human CD4 molecule and the human IgG1Fc segment in order to explore the potential application of blocking the co-stimulatory pathway of CD4 0 / CD4 0L in immunotherapy. First, total cellular RNA was extracted from the human B lymphoma cell line Daudi. The human CD4 extracellular region gene was amplified by RT-PCR. The amplified product was ligated into pGEMTEasy vector to construct cloning vector pGE40. Sequencer sequenced. The correct ectodomain fragment was inserted into the pIG 1 vector containing the human IgG class 1 antibody heavy chain genomic sequence to construct the transient expression vector named pIG / 40Ig. COS 7 cells were transfected with DEAE Dextran / Chloroquine method. The expression and immunological activity of CD4 0 Ig fusion protein in culture supernatant were identified by sandwich ELISA and Western blot analysis. The fusion protein was detected in the cell culture supernatants by the recombinant plasmid pIG410Ig after transfection with COS7 cells. Western blot showed that there was a specific band about 50kD in molecular weight, Human CD4 0 monoclonal antibody G2 8 5 and anti-human Ig gamma chain monoclonal antibody recognition. This study successfully expanded human CD4 0 gene and constructed a human CD4 0 Ig fusion gene expression vector, obtained functional expression in C0S 7 cells, in order to further study the CD4 0 / CD4 0L pathway in graft-versus-host