摘要
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
CD40/CD40L, besides B7/CD28, is an alternative important costimulation signal transduction pathway. It plays a pivotal role in T cell activation. Moreover, it may play a critical role at many levels of sensitization and effector phases of allograft rejection. In order to get the fusion protein of human CD40 extracelluar region and IgG?1 Fc fragment, and investigate the potential role of blocking CD40/CD40L costimulation pathway in immunotherapy, total RNA was extracted from human lymphoma cell line Daudi, and CD40 gene extracelluar region was amplified by RT-PCR. The PCR products were inserted into pGEM T Easy vector, and the cloning vector pGE40 was obtained. The DNA sequence was analyzed by automatic DNA sequencer. After sequencing, the transient expressing vector was constructed by inserting correct fragment into pIG vector, which contains the genomic human IgG1 Fc(hinge, CH2 and CH3) gene. Hence the recombinant fusion expression vector was constructed successfully, and named after pIG/40 Ig. Then, COS-7 cells were transfected through DEAE-Dextran/chloroquine method. The CD40-Ig fusion protein expressed in COS-7 cell culture supernatant was identified by sandwich ELISA and Western blot. Result showed that the CD40-Ig fusion protein can be detected by sandwich ELISA in the cell culture supernatant. Western blot analysis also showed that it could react with McAbs of mouse anti-human CD40 G28-5 and mouse anti-human Ig γ chain. There is only one obvious band at the position of relative molecular weight 50?kD, and it is equivalent to the expected value. Above all, the recombinant fusion expression vector pIG/40 Ig was constructed, and CD40-Ig fusion protein gene was expressed in COS-7 cells successfully. It could be laid a foundation to investigate the potential role of CD40/CD40L pathway as the target of GVHD prevention and therapy.
出处
《中国实验血液学杂志》
CAS
CSCD
2000年第1期14-19,共6页
Journal of Experimental Hematology
