非洲猪瘟病毒O174L蛋白的真核表达载体构建及分子特征分析

Construction of Eukaryotic Expression Vector of African Swine Fever Virus O174L Protein and Analysis of Molecular Characteristics

为分析非洲猪瘟病毒O174L基因,通过同源重组方式将O174L基因连接至p RK5M-C-2×Strep载体,构建重组质粒,经PCR扩增和测序鉴定正确后,将重组质粒转染至猪小肠上皮细胞系(porcine intestinal columnar epithelial cells,IPEC-J2)中,通过免疫荧光和Western blot检测O174L蛋白的表达情况。PCR及测序结果显示,p RK5M-C-2×Strep-O174L重组质粒构建成功。免疫荧光和Western blot检测结果显示,O174L蛋白能够在IPEC-J2细胞中稳定表达。生物信息学分析结果显示,基于O174L基因和B646L(p72)基因序列构建各分离毒株的2个系统发育树间排列高度相似。来自中国的16株分离株中,O174L基因序列的相似性高达96.76%~100.00%。其中,与中国爆发的其他Ⅱ型分离株相比,China/2018/Anhui XCGQ在O174L蛋白的第67、75及110位氨基酸存在差异,GZ201801在第110位氨基酸存在差异。Ⅰ型分离株SD/DY-I/2021和HeN/ZZ-P1/2021的O174L蛋白的氨基酸序列分别在第13、73、93、95、113和114位上与其他中国Ⅱ型分离株存在差异。O174L蛋白为稳定的亲水蛋白,没有信号肽和跨膜区;其二级结构由α螺旋、β折叠和无规则卷曲组成,三级结构预测结果与二级结构预测相符。以上结果为深入研究ASFV O174L蛋白与宿主间的相互作用和遗传进化提供了基础。

To study the O174L gene of African swine fever virus(ASFV),the O174L gene was connected to the vector pRK5M-C-2×Strep through homologous recombination,and the eukaryotic expression vector of ASFV was constructed.After PCR amplification and sequencing identification,the recombinant plasmid was transfected into porcine intestinal epithelial cells(IPEC-J2).The expression of O174L protein was detected by immunofluorescence and Western blot.The PCR and sequencing results showed that recombinant plasmid pRK5M-C-2×Strep-O174L was successfully constructed.The results of immunofluorescence and Western blot showed that O174L protein could be stably expressed in IPEC-J2 cells.Bioinformatics analysis of O174L protein showed that the arrangement of the branches and isolates of the phylogenetic tree based on the O174L gene sequence was highly similar to that based on the B646L(p72)gene sequence.Among the 16 isolates from China,the similarity of the O174L gene sequence between the isolates was as high as 96.76%~100.00%.Compared with other typeⅡisolates in China,China/2018/AnhuiXCGQ had differences in the 67th,75th and 110th amino acids of O174L,and GZ201801 had difference in the 110th amino acids.The O174L amino acid sequence of typeⅠisolates SD/DY-I/2021 and HeN/ZZ-P1/2021 were different from other Chinese typeⅡisolates at 13th,73th,93th,95th,113th and 114th amino acids,respectively.O174L protein was a stable hydrophilic protein without signal peptide and transmembrane region.The secondary structure of O174L protein was composed ofαhelix,βstrand and random coil,and the prediction result of tertiary structure was consistent with the secondary structure.Above results provided the basis and experimental materials for studying the protein interaction and genetic evolution between ASFV and host.