鸡趋化因子受体2和趋化因子受体5真核表达载体的构建及鉴定

Construction and Identification of Eukaryotic Expression Vector of Chemokine Receptor 2 and Chemokine Receptor 5 of Chicken

试验旨在构建鸡趋化因子受体2(CCR2)和趋化因子受体5(CCR5)真核表达系统,并瞬时转染中国仓鼠卵巢细胞(CHO-K1)观察其表达情况。以鸡基因组DNA为模板,PCR克隆出鸡的CCR2和CCR5基因的编码序列(CDS),将克隆出的片段插入真核表达载体pc DNA3.1(+)中,构建重组真核表达质粒pc DNA3.1(+)-CCR2、pc DNA3.1(+)-CCR5,酶切鉴定后测序。加Kozak序列和myc分子标签后,瞬时转染CHO-K1细胞,用反转录PCR(RT-PCR)、Western blot分别从转录、翻译水平鉴定重组质粒是否能在真核细胞中表达。结果显示,成功克隆出鸡CCR2、CCR5的CDS序列,酶切和测序结果表明pc DNA3.1(+)-CCR2-myc、pc DNA3.1(+)-CCR5-myc构建成功,RT-PCR、Western blot证实转染的重组质粒能在CHO-K1细胞中转录和翻译。本试验成功构建出鸡CCR2和CCR5真核表达载体,并能在真核细胞中表达,为进一步研究CCR2、CCR5的功能奠定了基础。

This emperiment was to construct eukaryotic expression vector of chemokine receptor 2（CCR2）and chemokine receptor 5（CCR5）of chicken,and observe the expression in transiently transfected CHO-K1 cells. The coding sequence（CDS）of CCR2 gene and CCR5 gene was amplified by PCR from genomic DNA of chicken and cloned into pc DNA3.1（＋）vector,then inserted Kozak sequence and myc-tag. The recombinant vectors were confirmed by enzyme digestion and sequencing.The expression vectors were transiently transfected into CHO-K1 cells.The transcription and expression of CCR2 gene and CCR5 gene of chicken in eukaryotic cells were verified by RT-PCR and Western blot.The results showed that the CDS of c CCR2 and c CCR5 were cloned successfully the recombinant vectors（pc DNA3.1（＋）-CCR2-myc and pc DNA3.1（＋）-CCR5-myc）were correct. And the results were confirmed by RT-PCR and Western blot.Two functional eukaryotic expression plasmids had been established successfully. The study laid a foundation for further study on c CCR2 gene and c CCR5 gene function in vitro.