bcr-abl基因免疫诱导特异性细胞毒性T淋巴细胞杀伤功能  

Induction of CTL by immunization of bcr abl fusion gene

作　　者：刘楠[1] 孙秉中[1] 冯琦[1] 高杰英[2] 罗振革[2] 彭虹[2] 刘永全[2] 

机构地区：[1]第四军医大学西京医院血液内科,陕西西安710033 [2]军事医学科学院微生物流行病学研究所免疫室,北京100850

出　　处：《第四军医大学学报》2001年第16期1478-1481,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金资助项目 (3 9770 3 3 6 ) ;陕西省科研基金资助项目 (98SM40 )

摘　　要：目的　研究 bcr- abl融合基因免疫在慢性髓系白血病(chronic myelogenous leukem ia CML )中的作用以及探讨CML 基因免疫治疗的可行性 .方法　设计两条引物扩增 bcr-abl融合位点两侧约 490 bp大小的 c DNA,经测序鉴定正确后 ,将其克隆至真核表达载体 pc DNA3.1,通过电穿孔法转染至 p815细胞 ,应用 G418筛选建立稳定的靶细胞 ,同时将重组真核表达载体肌肉免疫 BAL B/ c小鼠以获取特异的效应细胞 ,乳酸脱氢酶 (L DH)法检测特异性杀伤率 .结果　 1PCR扩增出可用于基因免疫的位于 bcr- abl融合基因位点两侧的490 bp大小的 c DNA,序列测定除第 45 2位碱基 C突变为 T外其余序列均正确 ;2构建了重组真核高效表达载体 ,命名为pc DNA/ bcr- abl;3G418梯度筛选建立了稳定表达该融合基因的细胞系 ,逆转录 -聚合酶链反应 (RT- PCR)检测到该细胞系有 bcr- abl m RNA水平的表达 ;4bcr- abl基因免疫后的小鼠能有效诱导出特异性细胞毒 T细胞 (cytotoxic T lymph-cyte,CTL) .结论　位于融合位点两侧的 bcr- abl基因肌肉免疫小鼠能够诱导特异性 CTL ,杀伤 bcr-AIM To investigate the effect of bcr abl gene immunization on the chronic myelogenous leukemia (CML) and explore the feasibility of this fusion gene immunization in CML gene therapy. METHODS Two primers were designed for the amplication of a 490 bp bcr abl cDNA coding sequence by PCR. After the PCR product was confirmed by the sequence analysis, it was inserted into the expression vector pcDNA3.1, which was instantly expressed in p815 cells by means of electroporation. The stable expression cell colonies were obtained as a target cells after the G418 selection. We also used the recombinant expression vector to immunize the BALB/c mice to get effector cells. The cytotoxicity of CTL was detected with LDH assay. RESULTS ① A 490 bp bcr abl cDNA was amplified by PCR, and sequence analysis showed the whole correct sequence except base C mutated for T in position 452. ② A recombinant expression vector, named pcDNA/ bcr abl was constructed. ③ The bcr abl positive, expression stable p815 cells were obtained by G418 selection, and RT PCR examination showed that bcr abl had been transcribed in all positive cell clones. ④ LDH assay showed CTL was induced by the bcr abl immunization. CONCLUSION Fragement of 490 bp bcr abl gene immunization was capable of inducing specific CTL to kill the target cells that have expressed the bcr abl fusion gene.

关 键 词：基因治疗 ABL 电穿孔 T淋巴细胞 细胞毒性 肿瘤 白血病 BCR-ABL 

分 类 号：R733.7[医药卫生—肿瘤] R730.54[医药卫生—临床医学]