【摘要】 目的构建人粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因与EB病毒(EBV)潜伏膜蛋白基因LMP2A融合基因的原核表达载体。方法用随机引物Oligo(dT)15进行RT-PCR,分别获得人GM-CSF和LMP2A编码序列的cDNA。将纯化GM-CSF和EB病毒LMP2APCR扩增产物插入高效克隆载体pMD18-T,并转化入感受态细胞Esche-richia coli DH10B,在含有x-gal、IPTG和氨苄青霉素的LB培养基平板上进行蓝白斑选择,测序正确的序列进行剪接式重叠延伸,将目的基因GM-CSF和LMP2A编码经多肽接头(Gly4Ser)3序列连接,构建融合基因GC2A,将GC2A克隆至pMD18-T,转化感受态细胞E.coliDH10B,在含有x-gal、IPTG和氨苄青霉素的LB培养基平板上进行蓝白斑选择。结果目的基因GM-CSF和LMP2ART-PCR产物大小分别为470bp和1 545bp,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(1 961bp)正确插入载体。结论融合基因正确插入pMD18-T,并成功转化入感受态细胞E.coli DH10B,为进一步研究奠定了基础。更多还原

【Abstract】 Objective To construct a prokaryotic expression vector of a fusion gene combining human granulocyte-macrophage colony stimulating factor(GM-CSF) and latent membrane protein gene LMP2A of the EB virus(EBV).Methods Oligo(dT)15 was used to obtain the cDNA sequence of GM-CSF and LMP2A.Purified GM-CSF and LMP2A were amplified with PCR,inserted into the plasmid pMD18-T,and then transformed into Escherichia coli DH10B.In an ampicillin LB culture medium containing x-gal and IPTG,colonies were selected by blue-white screening.Splicing by overlap extension was used to connect the correct sequence of GM-CSF and LMP2A using the polypeptide(Gly4Ser)3.The fusion gene GC2A was constructed and cloned into pMD18-T and transformed into competent bacteria.Blue-white screening was then done in an ampicillin LB culture plate containing x-gal and IPTG.Results According to RT-PCR,the target genes GM-CSF and LMP2A were 470 bp and 1 545 bp in length,which was consistent with expected values.The recombinant plasmid was confirmed by double restriction enzymes,amplification,and sequencing,and then the fusion gene(1,961 bp) was correctly inserted into the vector.Conclusion A fusion gene was correctly inserted into pMD18-T and successfully transformed into E.coli DH10B,providing a basis for further study.更多还原