GM-CSF与BZLF1融合基因修饰的重组BCG的构建与表达  

Construction and expression of recombinant BCG encoding GM-CSF and BZLF1 fusion gene

作　　者：薛庆节[1] 杨媛媛[1] 胡文洁[1] 吕厚东[1] 李士根[1] 陈廷[1] 

机构地区：[1]济宁医学院病原生物学教研室,医学免疫学山东省重点学科,山东济宁272067

出　　处：《中国热带医学》2014年第9期1035-1038,1049,共5页China Tropical Medicine

基　　金：山东省高等学校科技计划项目(No.J12LK56);山东省自然基金(No.ZR2012HM037);教育部留学回国人员科研启动基金(No.20101174);山东省高等学校青年骨干教师国内访问学者项目(2012年度)

摘　　要：目的将人粒细胞-巨噬细胞集落刺激因子基因(GM-CSF)与EB病毒即刻早期基因(BZLF1)重组为融合基因GCBF,并转化入卡介苗(BCG)中进行表达。方法用随机引物Oligo(d T)15进行RT-PCR,分别获得人GM-CSF和BZLF1编码序列的c DNA。将纯化GM-CSF和BZLF1 PCR扩增产物插入分泌表达载体p MV261,并转化入感受态细胞Escherichia coli DH5α(E.coli DH5α),在LB培养基上进行卡那霉素抗性选择,测序正确的序列进行剪接式重叠延伸,将目的基因GM-CSF和BZLF1编码经多肽接头(Gly4Ser)3序列连接,构建融合基因GCBF,将GCBF克隆至p MV261,转化感受态细胞E.coli DH5α,在LB培养基平板上进行卡那霉素抗性选择,阳性克隆提取质粒后转化感受态BCG,western-blot检测GCBF在r BCG中的表达。结果目的基因GM-CSF和BZLF1 RT-PCR产物大小分别为461 bp和788bp,与预期值一致。构建的重组质粒经双酶切、扩增及测序鉴定证实,融合基因GCBF(1209bp)正确插入载体,成功转化入BCG感受态细胞,且被正确表达。结论融合基因GCBF修饰的r BCG构建及表达成功,为进一步探讨r BCG杀伤EB病毒阳性肿瘤的免疫活性研究奠定了实验基础。Objective To investigate the construction and expression of rBCG combining human granulocyte-macrophage colony stimulating factor（GM-CSF） and EB virus （EBV） encoding immediate early gene（BZLF1）. Methods Oligo（dT）15 was used to obtain the cDNA sequence of GM-CSF and BZLF1, the purified GM-CSF and BZLF1 were amplified by PCR and inserted into plasmid pMV261, then transformed them into Escbericbia coli DH5a（E.coli. DH5α）. In LB culture medium containing kanamycin, the positive colony was selected, the correct sequence of the GM-CSF and BZLF1 were connected by the polypeptide （Gly4Ser）3 series with the splicing overlap extension technology. The fusion gene GCBF was constructed and cloned into pMV261, transformed competent bacteria and selected on LB culture medium flat containing kanamycin. Plasmid pMVGCBF extracted from positive clones were transformed into competent BCG. Western-blot was employed for determination of expression of GCBF. Results The RT-PCR product sizes of objective gene GM-CSF and BZLF1 were 461bp and 788bp, consistented with expected values. The recombinant plasmid was confirmed by restriction double enzymes, amplification and sequencing ,then the fusion gene （1209bp） was correctly inserted into the vector.pMVGCBF were correctly transformed into competent BCG.The expression of fusion protein GCBF was detected in rBCG.Conclusion rBCG encoding GCBF fusion gene was successfully constructed to have provided basis for further study of the function of rBCG.

关 键 词：融合基因GCBF 基因重组 重组卡介苗 

分 类 号：R784[医药卫生—口腔医学]