山羊痘病毒毒力因子KLP1互作蛋白的筛选与初步鉴定

Screening and preliminary identification of the interaction protein of goat pox virus virulence factor KLP1

为进一步研究山羊痘病毒毒力因子KLP1,了解其生物学功能,本试验构建了羊皮肤组织酵母cDNA文库及GTPV KLP1蛋白基因的诱饵质粒,并做了质粒的自激活检测。采用酵母双杂交方法,从羊皮肤组织酵母cDNA文库中筛选出与KLP1蛋白相互作用的蛋白,结果成功构建了羊皮肤组织cDNA文库,容量为1.05×10^(7) CFU,成功构建pGBKT7-KLP1诱饵质粒,且在酵母双杂交系统中没有毒性和自激活作用;酵母双杂交筛选出与KLP1蛋白互作的蛋白克隆25个,经过测序比对合并得9种蛋白,对其进行LacZ回转验证和共定位试验验证,证实NOTCH1蛋白与KLP1蛋白具有特异的相互作用。本试验为阐明GTPV毒力因子KLP1的生物学特征及其作用机制提供参考。

In order to study the goat pox virus virulence factor KLP1 and understand its biological function, a yeast cDNA library of sheep skin tissue and the GTPV KLP1 gene decoy plasmid were constructed, and the plasmid self-activation was detected.The proteins interacting with KLP1 protein were selected from the yeast cDNA library using yeast two-hybrid method.The result showed that the sheep skin tissue cDNA library was successfully constructed with a capacity of 1.05×10^(7) CFU,and the pGBKT7-KLP1 bait plasmid was successfully constructed, and there was no toxicity and self-activation in the yeast two-hybrid.Twenty-five protein clones interacting with the KLP1 protein were screened by the yeast two-hybrid system.After sequencing and comparison, 9 proteins were obtained, they were verified by LacZ retransformation assay and co-localization experiment.It was confirmed that Notch1 protein and KLP1 protein have a specific interaction.This study provides a reference for clarifying the biological characteristics and mechanism of action of GTPV virulence factor KLP1.