Construction of Eukaryotic Expression Vector of African Swine Fever Virus O174L Protein and Analysis of Molecular Characteristics
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 type Ⅱ isolates 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 type Ⅰisolates SD/DY-I/2021 and HeN/ZZ-P1/2021 were different from other Chinese type Ⅱ isolates 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 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.
国家科技期刊平台
NSTL
万方数据
维普
