猪附红细胞体ENO基因重组腺病毒穿梭质粒的构建及真核表达

为构建猪附红细胞体ENO基因重组腺病毒穿梭质粒，试验根据GenBank中登录的猪附红细胞体ENO基因序列（登录号：CP002525.1）设计特异性引物，对ENO基因进行PCR扩增，并将纯化后的PCR产物克隆到pMD19-T载体中。用Kpn Ⅰ和Xho Ⅰ对pMD19T-ENO进行双酶切后，将其亚克隆至腺病毒穿梭载体PCR259中，构建PCR259-ENO重组质粒，提取重组质粒进行鉴定。应用脂质体介导转染法将鉴定正确的PCR259-ENO重组穿梭质粒转染293细胞，应用间接免疫荧光法（IFTA）检测ENO基因在293细胞中的表达。结果显示，试验克隆的ENO基因长为1 182 bp，编码393个氨基酸，与GenBank中ENO基因序列（登录号：CP002525.1）同源性为99%。构建的重组腺病毒穿梭质粒PCR259-ENO经PCR和酶切鉴定正确，并且能在293细胞中表达，表明ENO基因成功插入腺病毒穿梭质粒PCR259中，重组腺病毒穿梭质粒PCR259-ENO构建成功。

Construction and Eukaryotic Expression of Recombinant Adenovirus Shuttle Plasmid of Mycoplasma suis ENO Gene

To construct recombinant adenovirus shuttle plasmid of ENO gene of Mycoplasma suis and express it in eukaryotic cells,a pair of primers was designed and synthesized according to the ENO gene sequence of Mycoplasma suis in GenBank (accession No.:CP002525.1) in this study.ENO gene was amplified by PCR method,and the products were cloned into the pMD19-T vector.The pMD19T-ENO plasmid was digested with Kpn Ⅰ and Xho Ⅰ, and then subcloned into the adenovirus shuttle vector PCR259.The PCR259-ENO plasmid was constructed and then identified by PCR and enzyme digestion.The PCR259-ENO recombinant shuttle plasmid was transfected into 293 cells by liposome-mediated transfection,and the expression of ENO gene in 293 cells was detected by IFTA technology.The results showed that the cloned ENO gene was 1 182 bp in length and encoded 393 amino acids with a homology of 99% with the ENO gene sequence in GenBank (accession No.:CP002525.1).The constructed recombinant adenovirus shuttle plasmid PCR259-ENO was identified by PCR and restriction enzyme digestion correctly, and was able to be expressed in 293 cells,indicating that ENO gene was successfully inserted into adenovirus shuttle plasmid PCR259 and the recombinant shuttle plasmid was successfully constructed.