伪狂犬病病毒Min-A株gD基因的克隆及其真核表达质粒的构建

Cloning and sequencing of the gD gene of pseudorabies virus Min-A strain

参考GeneBank收录的伪狂犬病病毒gD基因的序列设计了一对引物,对PRVMin＿A株进行了PCR扩增,扩增产物克隆于pGEM＿TEasy载体。对重组质粒PGTE＿gD进行限制性内切酶分析和基因测序,证实了克隆片段的可靠性。测序结果表明目的片段包含一个1203bp的开放性阅读框(ORF),编码400个氨基酸组成的多肽。将gD基因亚克隆至真核表达载体pcDVA3.1＿的CMV启动子下游,构建了真核表达质粒pcDNA＿gD,为下一步基因免疫奠定了基础。

A pair of primers were designed according to the sequences pulished by the GenBank in order to amplifiy gD gene of the pseudorabies virus Min-A strain.The gD gene were obtained by polymerase chain reaction(PCR), and then cloned into the pGEM-T Easy vector. The recombinant plasmid of pGTE-gD was identified by restriction enzyme analysis and sequencing ,Which proved completely its validity. Nucleotide sequencing revealed that this fragment contained an open reading frame of 1203 bp encoding a 400 aa protein. The gD geng is subcloned into pcDNA3.1-,an eukaryotic expressing vector.The constructed expressing plasmid pcDNA-gD will be used for future gene vaccination.