TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定

Construction and bio-characteristic of the recombinant goatpox virus with thymidine kinase gene (TK) deletion

【目的】利用同源重组技术构建TK基因缺陷的山羊痘病毒(GTPV)毒株,为研制出更安全、高效的GTPV弱毒疫苗和病毒载体提供备选毒株。【方法】采用PCR克隆GTPV AV41株TK基因(ORF56)及其侧翼基因组片段,在TK基因内部插入报告基因EGFP和抗性基因gpt表达盒,构建GTPV重组转移载体pTK-Eg。将重组转移载体pTK-Eg与GTPV AV41株共转染Vero细胞,通过空斑纯化筛选阳性重组病毒,鉴定其生长特性和遗传稳定性,并接种山羊评价其安全性和免疫原性。【结果】成功构建获得一株TK基因缺陷的重组病毒vTK-Eg。与亲本毒株GTPV AV41相比,vTK-Eg的生长特性及其形成的细胞病变均未发生显著改变,只是毒价略微下降100.5个数量级;在原代牛睾丸(BT)细胞上连续传代,至少在10代内保持遗传性状和病毒滴度稳定。接种vTK-Eg的山羊精神和食欲均正常,接种后体温升高和局部反应的严重程度均较接种亲本毒株GTPV AV41的山羊有所降低;vTK-Eg与GTPV AV41株诱导山羊产生的GTPV中和抗体水平无明显差异(P>0.05)。【结论】构建的TK基因重组缺陷病毒vTK-Eg具有良好的遗传稳定性和免疫原性,较亲本毒株其安全性也有所提高,可作为研制GTPV基因工程弱毒疫苗和活载体疫苗的备选毒株。

[Objective ]This experiment was conducted to construct a recombinant goatpox virus （GTPV） with inactivat- ing thymidine kinase gene （TK） in order to develop a safe and high effective live attenuated GTPV vaccine and viral vector. [Method]Genome fragments of GTPV AV41 strain containing TK gene （ORF56） were cloned by PCR as flanking sequences for homologous recombinant. The transfer shuttle plasmid pTK-Eg was constructed by inserting the expression cassettes of EGFP gene and gpt gene. The pTK-Eg and GTPV AV41 were co-transfected into Vero cell, and recombinant viruses were selected and screened by fluorescence. Then, recombinant viruses were examined by fluorescence and tittering during seri- al passaging. Finally, the goats were injected with vTK-Eg in order to evaluate its safety and immunogenicity. [Result]A recombinant GTPV with TK gene deleted was obtained and named as vTK-Eg, vTK-Eg remained almost the same growth pattern and specific CPE of its parental virus （AV41） in cell culture. The differentee between the titers of vTK-Eg and that of AV41 was only 10-0.5 fold. It kept genetic and titer stable in at least 10 generation in BT cell. Furthermore, it had lower virulence in goats, causing less skin lesions and fever than those of its parental virus. However, there was no significant different （P〉0.05） between the neutralizing antibody levels of goats injected by vTK-Eg and those of goats injected by AV41. [Conclusion]vTK-Eg had good genetic stability and immunogenicity, as well as beuer bio-safety. Therefore, it has the potential to be further developed as a genetic engineering attenuate vaccine strain.