文献类型：期刊文献

中文题名：犬PD-1、PD-L1胞外区蛋白的原核表达及鉴定

英文题名：Prokaryotic expression and identification of extracellular domain proteins of canine PD-1 and PD-L1

作者：何海汛[1,2];孙国鹏[1];李丹[1];陈威[1];蒋力维[1];刘金宵[1];王选年[1]

第一作者：何海汛

机构：[1]新乡学院生物技术研究中心,河南新乡453000;[2]郑州大学生命科学学院,郑州450000

第一机构：新乡学院生命科学技术学院

年份：2021

期号：7

起止页码：131-135

中文期刊名：黑龙江畜牧兽医

外文期刊名：Heilongjiang Animal Science And veterinary Medicine

收录：北大核心:【北大核心2020】；

基金：国家自然科学基金面上项目(31672540);新乡学院青年骨干教师项目(1244160006)。

语种：中文

中文关键词：犬;PD-1;PD-L1;胞外区;原核表达

外文关键词：canine;PD-1;PD-L1;extracellular domain;prokaryotic expression

摘要：为研究免疫抑制信号通路程序性细胞死亡因子-1(programed cell death 1, PD-1)及其配体-1(ligands of programmed death-1,PD-L1)在犬肿瘤等免疫抑制类疾病中发挥的作用,进一步寻求治疗肿瘤的方法和手段,试验根据在GenBank中查询的犬PD-1及PD-L1基因序列信息,结合生物信息学技术,以及在其他种源中二者蛋白质结构研究的相关结果,克隆编码胞外区主要结构域的基因片段,构建相应原核表达载体并诱导表达,并应用Western-blot对目标蛋白质产物进行鉴定。结果表明:成功构建了表达犬PD-1(25~168 aa)及PD-L1(19~238 aa)胞外区主要结构域的原核重组表达载体pET-30a-PD-1和pET-30a-PD-L1,诱导表达的目标蛋白质产物分子质量分别约为27 ku和33 ku,二者均以包涵体形式表达,纯化后蛋白质产物纯度可达到80%,且均能够被商品化抗体特异性识别,具有良好的免疫原性。
The aim of this study was to investigate the role of programmed cell death 1( PD-1) and its ligands of programmed death-1( PD-L1)in immunosuppressive diseases such as canine tumors,and further explore methods and means in the treatment of tumors. Taking the sequence information of canine PD-1 and PD-L1 genes in Gen Bank as basis,combining with bioinformatics technology,as well as the relevant results of their protein structure study in other provenances,the gene fragments encoding the main domains of extracellular regions were cloned,the corresponding prokaryotic expression vectors were constructed and induced for expression,and the target protein products were identified by Western-blot assay.The results showed that the prokaryotic expression vectors p ET-30 a-PD-1 and p ET-30 a-PD-L1 expressing the main extracellular domain of canine PD-1( 25-168 aa) and PD-L1( 19-238 aa) were successfully constructed. The molecular weights of the induced target protein products were about 27 ku and 33 ku,respectively,and both of them were expressed in the form of inclusion bodies. After purification,the purity of the purified protein products could reach 80%,and they could be specifically recognized by commercial antibodies with good immunogenicity.