猪圆环病毒2型ORF2基因序列分析及真核表达载体的构建

根据ＧｅｎＢａｎｋ中猪圆环病毒２型（ＰＣＶ－２）ＯＲＦ２基因序列，设计一　对引物，应用ＰＣＲ从疑患断奶仔猪多系统消耗综合症（ＰＭＷＳ）的死亡仔猪组织病料中扩增出ＯＲＦ　２全基因（７０２ｂｐ）。将此片段克隆入ｐＧＥＭ－Ｔ　ｅａｓｙ载体，筛选获得重组质粒ｐＴＯＲＦ２，并对此质　粒中的插入序列进行了测序分析，结果表明本试验克隆的ＯＲＦ２与美国ＰＣＶ－２分离株ＡＦ２６４０３９　的核苷酸及氨基酸序列同源性均达到１００％，与其他ＰＣＶ－２毒株同源性分别为９２．３％～９８．　６％和　９２．３％～９６．６％。重组质粒ｐＴＯＲＦ２经　Ｂａｍ　Ｈ　Ｉ、Ｅｃｏ　Ｒ　Ｖ双酶切，回收ＯＲＦ２基因，转　移入真　核表达载体ｐＳｅｃＴａｇ２／ＨｙｇｒｏＢ的相应酶切位点之间，构建成重组质粒ｐＳｅｃＴａｇＯＲＦ２。此重组表　达载体的构建成功为进一步研究ＯＲＦ２编码蛋白的生物学活性及建立ＰＣＶ诊断试剂盒打下基础。

Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2

According to the published ORF2 gene sequence of PCV-2, two primers were designed, and ORF2 gene was amplified from suspected PMWS sampl e using PCR. Then the PCR product was cloned into pGEM-T easy vector, and recom binant plasmid named pTORF2 was obtained. The cloned ORF2 gene in pTORF2 was seq uenced and compared with other PCV isolates in GenBank, the results demonstrated that the ORF2 gene is closely related with a USA isolate AF264039, the homology of their nucleotide and amino acid sequence both were 100%. In addition, there existed 92.3% to 98.6% nucleotide sequence identity and 92.3% to 96.6% amino aci d sequence identity with other PCV-2 isolates. Recombinant plasmid pTORF2 was d igested with Bam H I Eco R V, ORF2 gene was purified, and introduced into eu karyotic expressing vector pSecTag2/HygroB between BamH I and EcoR V enz yme site, then pSecTagORF2 recombinant plasmid was constructed. Further research is underw ay to study the biological activity of ORF2 protein and establish the diagnostic kit of PCV.