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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 intracellular antibody gene and to observe the inhibitory effect of intracellular antibody on the expression of 9 1C3 gene, so as to lay the foundation for further study on the effect of 9 1C3 molecule on NK cell function. Methods Primers were designed and 9 1C3ScFv gene was used as a template for PCR amplification. The signal peptide, KDEL and E tag sequences, which were used for the expression detection, were introduced into the protein endoplasmic reticulum. The purified PCR product was recovered, ligated to pUC19 vector and sequenced. After sequencing, the intracellular antibody gene fragment was cut out and recombined into the eukaryotic expression vector pRc / CMV. After restriction enzyme digestion, the recombinant plasmid was transiently transfected into COS7 cells by DEAE dextran method. Intracellular staining and Western blotting were used to detect the intracellular antibody expression. Then, the intracellular antibody gene was transferred into 9 1C3 positive Jurkat cells by Lipofectamine, and the effect of the intracellular antibody on 9 1C3 molecule expression was observed by FCM. Results DNA sequence analysis demonstrated that the signal peptide, KDEL and E tag sequences required for endoplasmic reticulum mapping were successfully introduced into 9 1C3ScFv by PCR, and the eukaryotic expression vector for intracellular antibody was successfully constructed. By means of intracellular staining and Western blot It was demonstrated that intrabodies were expressed in COS7. Into Jurkat cells found that intracellular antibodies can down 9 1C3 molecule expression levels. Conclusion The 9 1C3 intracellular antibody gene was amplified by PCR using the 9 1C3ScFv gene as template, and the eukaryotic expression vector was constructed.