摘要
目的 获得 9 1C3胞内抗体基因的真核表达载体 ,观察胞内抗体对 9 1C3分子表达的抑制作用 ,为进一步研究 9 1C3分子对NK细胞功能的影响打下基础。方法 设计引物 ,以 9 1C3ScFv基因为模板进行PCR扩增 ,引入蛋白质内质网定位所需的信号肽、KDEL以及作为表达检测标记的E tag序列。回收纯化PCR产物 ,酶切后连接到pUC19载体上 ,并进行序列测定。序列正确后切下胞内抗体基因片段 ,重组到真核表达载体pRc/CMV中 ,酶切鉴定后用DEAE dextran方法瞬时转染真核细胞COS7,用胞内染色和Westernblot方法检测胞内抗体的表达。接着用Lipofectamine把胞内抗体基因转入 9 1C3阳性的Jurkat细胞 ,用FCM观察胞内抗体对 9 1C3分子表达的影响。结果 DNA序列测定证明 ,通过PCR成功地为 9 1C3ScFv引入了内质网定位所需的信号肽、KDEL及E tag序列 ,成功地构建了胞内抗体真核表达载体 ,通过胞内染色方法和Westernblot证明胞内抗体在COS7中得到表达。转入Jurkat细胞后发现胞内抗体能下调 9 1C3分子的表达水平。结论 以 9 1C3ScFv基因为模板PCR扩增得到 9 1C3胞内抗体基因 ,并构建了真核表达载体。
Objective To obtain the eukaryotic expression vector of 9.1C3 intrabody gene and investigate the inhibitory effect of intrabody on the expression of 9.1C3 molecule. Methods The signal peptide and KDEL peptide required for protein anchoring in endoplasmic reticulum were introduced into 9.1C3 ScFv gene by PCR. For facilitating the detection of intrabody expression, a label of E tag was also added. The PCR product was cloned into pUC19 vector and then sequenced. The intrabody gene of 9.1C3 was inserted into eukaryotic expression vector pRc/CMV, and the recombinant vector was transfected into COS7 cells by DEAE dextran method. Expression of this vector was detected by RT PCR as well as intracellular staining and FCM analysis. The intrabody gene was transfected into Jurkat cells, a 9.1C3 positive cell line, by Lipofectamine, and the expression of 9.1C3 molecule on Jurkat cells was detected by FCM. Results The signal peptide, KDEL and E tag sequence was successfully introduced into 9.1C3 ScFv, and the intrabody gene was inserted into eukaryotic expression vector which can be expressed in COS7 cells. And the intrabody can down regulate the expression of 9.1C3 molecule on Jurkat cells. Conclusion The eukaryotic expression vector containing the intrabody gene can be expressed in COS7 cells.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第1期58-61,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目! (3970 0 133)
