牛坏死杆菌白细胞毒素PL2蛋白的生物信息学分析及其原核表达鉴定  

Bioinformatics Analysis and Prokaryotic Expression Identification of Leukotoxin PL2 Protein of Fusobacterium necrophorum

作　　者：于思雯 肖佳薇 毕栏 王天硕 贺显晶[1] 郭东华[1] Yu Siwen;Xiao Jiawei;Bi Lan;Wang Tianshuo;He Xianjing;Guo Donghua(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《黑龙江八一农垦大学学报》2024年第3期38-43,98,共7页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省自然科学基金项目(No.LH2021C070)。

摘　　要：为确定牛坏死杆菌(Fusobacterium necrophorum)白细胞毒素PL2蛋白的组成结构及生物信息学特性。根据NCBI数据库中的牛坏死杆菌白细胞毒素PL2基因序列及其编码蛋白的氨基酸序列,利用生物信息学软件对PL2蛋白的理化性质、B细胞及T细胞抗原表位等进行了预测研究,并利用原核表达系统构建重组质粒pGEX-6p-1-PL2-1、pGEX-6p-1-PL2-2、pGEX-6p-1-PL2-3,采用SDS-PAGE检测PL2蛋白的表达,将获得的重组蛋白经Western blot验证。PL2蛋白是由332个氨基酸构成的富含丝氨酸的稳定亲水蛋白,二级结构以无规卷曲和α螺旋居多。预测该蛋白含有10个B细胞优势抗原表位和16个T细胞优势抗原表位,利用PCR扩增得到的pGEX-6p-1-PL2-1为228 bp、pGEX-6p-1-PL2-2为300 bp、pGEX-6p-1-PL2-3为588 bp。SDS-PAGE证明3个蛋白成功表达,蛋白大小为34 kDa,Western blot结果表明,该重组蛋白PL2-1具有良好的反应原性。牛坏死杆菌白细胞毒素PL2蛋白为膜外蛋白,不存在跨膜区,含有多个磷酸化位点及多个B细胞、T细胞抗原表位,根据抗原表位预测结果获得的重组蛋白PL2-1具有良好的反应原性,可作为潜在抗原用于牛坏死杆菌病亚单位疫苗研发。To determine the composition,structure and bioinformatics properties of Fusobacterium necrophorum leukotoxin PL2 protein,according to gene sequence and the amino acid sequence of the leukotoxin PL2 of Fusobacterium necrophorum in the NCBI database,the physicochemical properties,B cell and T cell epitopes of leukotoxin PL2 protein were predicted by bioinformatics software,and recombinant plasmids pGEX-6p-1-PL2-1,pGEX-6p-1-PL2-2,pGEX-6p-1-PL2-3 were constructed by prokaryotic expression system,and the expression of leukotoxin PL2 protein was detected by SDS-PAGE.The obtained recombinant protein was validated by Western blot.PL2 protein was a stable hydrophilic protein rich in serine composed of 332 amino acids,and the secondary structure was mostly random curl and α helix.It was predicted that the protein contained 10 B cell dominant epitopes and 16 T cell dominant epitopes,and the pGEX-6p-1-PL2-1 obtained by PCR amplification was 228 bp,pGEX-6p-1-PL2-2 was 300 bp,and pGEX-6p-1-PL2-3 was 588 bp.The SDS-PAGE results demonstrated that 3 proteins were successfully expressed with a protein size of 34 kDa,and the western blot results showed that the recombinant protein PL2-1 had immunogenicity was proved.Leukotoxin PL2 protein of Fusobacterium necrophorum was an extramembrane protein,there was no transmembrane region,containing multiple phosphorylation sites and multiple B cell and T cell epitopes,and the recombinant PL2-1 protein obtained according to the prediction results of epitopes has immunogenicity was proved,which could be used as a potential antigen for the development of subunit vaccines for Fusobacterium necrophorum.

关 键 词：牛坏死杆菌 白细胞毒素PL2蛋白 生物信息学分析 原核表达 

分 类 号：S858.2[农业科学—临床兽医学]