关键词: 非洲猪瘟病毒, O174L 基因, 真核表达, 分子特征

Abstract:

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 O174Lprotein 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 type Ⅱ isolates in China， China/2018/AnhuiXCGQ had differences in the 67^th， 75^th and 110^th amino acids of O174L， and GZ201801 had difference in the 110^th amino acids. The O174Lamino acid sequence of type Ⅰ isolates SD/DY-I/2021 and HeN/ZZ-P1/2021 were different from other Chinese type Ⅱ isolates at 13^th， 73^th， 93^th， 95^th， 113^th and 114^th amino acids， respectively. O174Lprotein was a stable hydrophilic protein without signal peptide and transmembrane region. The secondary structure of O174Lprotein was composed of α helix， β strand 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.

Key words: African swine fever virus, O174L gene, eukaryotic expression, molecular characteristics