慢性粒细胞白血病bcr-abl融合基因克隆与真核细胞表达的研究  

Molecular cloning and eukaryotic expression of chronic myelogenous leukemia bcr-abl fusion gene

作　　者：姜扬文[1] 季明春[2] 钱莉[2] 龚卫娟[2] 万兵[2] 

机构地区：[1]扬州大学附属医院血液科,225001 [2]扬州大学医学院微生物学及免疫学教研室

出　　处：《江苏医药》2003年第12期899-901,共3页Jiangsu Medical Journal

基　　金：江苏省高校自然科学研究基金 (K0 10 913 8);江苏省应用基础研究基金 (BJ95 12 2 )

摘　　要：目的　为研究慢性粒细胞白血病 (CML)的肿瘤基因疫苗 ,克隆和表达CML的bcr abl融合基因片段。方法　根据bcr abl(b3a2 )融合基因序列自行设计一对寡核苷酸引物 ,以CMLK5 6 2细胞株的总RNA为模板 ,通过RT PCR扩增包含bcr abl融合位点周围 4 5 0bp的基因片段 ,并将其克隆进pGEM Teasy载体。经序列测定证实其阅读框架正确后 ,将bcr abl融合基因片段正向插入真核细胞表达载体 pcDNA3 1。以脂质体介导重组质粒pcDNAbcr abl转染中国仓鼠卵巢 (CHO)细胞 ,用间接免疫荧光法 (IFA)检测基因表达情况。结果　成功构建了包含融合位点基因片段的bcr abl融合基因真核细胞表达载体 pcDNAbcr abl,pcDNAbcr abl转染的CHO细胞的免疫荧光强度高于K5 6 2阳性对照细胞。结论　bcr abl融合基因片段在真核细胞中能高效表达 ,pcDNAbcr abl真核细胞表达载体的构建为进一步研究bcr abl融合基因在CML防治中的作用奠定了基础。Objective To provide the target gene and target antigen for the development of new vaccine against chronic myelogenous leukemia(CML).Methods According to the gene sequence encoding bcr abl(b3a2)fusion gene from CML K562 cell line,we designed a pair of oligonucleotide primers,obtained the 472bp gene fragment of bcr abl by using reverse transcription polymerase chain reaction(RT PCR),and inserted the gene into plasmid pcDNA3 1 via pGEM T easy vector to construct recombinant plasmid.The recombinant plasmid was transfected into CHO cell.The expression product of the gene fragment was analyzed by using indirect fluorescence immunoassay(IFA).Results The bcr abl fusion gene fragment encoding bcr abl of CML was amplified by using RT PCR.A recombinant eukaryotic expression plasmid was successfully constructed.The recombinant plasmid stably expressed the fragment of bcr abl fusion gene in CHO cell cytoplasm transfected with liposome through IFA.Conclusion Bcr abl fusion gene was expressed in CHO cells.The results of this investigation provided a basis for further research on the development of bcr abl fusion gene vaccine of CML.

关 键 词：慢性粒细胞白血病 BCR-ABL融合基因 克隆 真核细胞 肿瘤基因疫苗 脂质体 间接免疫荧光法 

分 类 号：R733.72[医药卫生—肿瘤]