山羊痘病毒非必需基因区gp024基因的鉴定

Identification of non-essential gp024 gene in Goatpox virus

为筛选山羊痘病毒(GTPV)的外源基因插入区,本研究以GTPV Pellor株为模板,设计2对引物,PCR扩增AV41株GTPV_gp024基因相邻上下游长度约1.1kb的同源重组片段,分别插入质粒pGPT-EGFP中。插入部位在痘病毒双向启动子p11～p7.5启动的报告基因EGFP-GPT上下游,构建转移载体pEG024,并转染已感染GTPV AV41病毒的LT细胞,经GPT加压筛选7代后得到重组病毒。结果显示,重组病毒稳定表达报告基因EGFP,从而表明GTPV_gp024基因能够作为外源基因的插入区。

In this study, two circumferential fragments covering 1.1 kb approximately of the locus of ＂GTPV_gp024＂ were amplified by PCR with specific primers based on GTPV strain Pellor. The PCR fragments were cloned into upstream and downstream of the enhanced green fluorescent protein （EGFP） of pGPT-EGFP vector modulated by bi-directional promoter p11-p7.5 originated from fowlpox virus. The recombinant transfer vector was co-transinfected into GTPV strain AV 41 infected LT cells. A recombinant GTPV strain was obtained after 7 rounds screening under GPT pressure. The results showed that EGFP was expressed stably by the recombinant GTPV, and the GTPV_gp024 locus could be used as a novel region for expression of foreign genes.