Prokaryotic expression and egg yolk antibody of canine ELF4 gene

【Objective】 To clone canine transcription factor ELF4 gene （cELF4），recombinant cELF4 protein （rcELF4） expressed in vitro and prepare egg yolk antibody（IgY）against this protein，so as to provide fundamental material for exploring canine ELF4 protein function.【Method】The cELF4 gene was amplified by RT-PCR，and the target gene was cloned into pMD19-T vector to construct recombinant plasmid and sequenced.Then nucleotide sequence and amino acid sequence characteristics were analyzed by bioinformatics software.The cELF4 gene was cloned into the prokaryotic expression vector pET-28a，and the recombinant expression plasmid pET28a-cELF4 was constructed and transformed into Escherichia coli BL21（DE3）and Rosetta（DE3）competent cells，respectively.After expression induction，expression product was identified by SDS-PAGE and western blotting.The rcELF4 protein was emulsified with 3 adjuvants（ISA 15AVG，ISA 71VG and ISA 201VG），immunized with egg chickens，antibody titers and IgY content were detected.The eukaryotic recombinant expression vector pEGFP-cELF4 was constructed，cells were transfected，and the immune activity of cELF4-specific IgY was analyzed by the immunoperoxidase cell monolayer assay（IPMA）.【Result】The cELF4 gene （GenBank accession number MZ198105）had an open reading frame of 1992 bp and encoded 663 amino acid residues.The cELF4 gene had the highest similarity with the predicted fox ELF4 gene（99.3%），while it had the lowest similarity with small mouse（81.5%）.The phylogenetic analysis showed that canine cELF4 gene were most closely related to the gene in animals such as foxes，pandas，European ferrets，Lontra canadensis and were relatively distantly related to the gene in primates such as humans and monkeys and rodent rats.Molecular weight of rcELF4 protein was about 100 kD and the protein can stimulate chicken to produce antibodies.Titers of serum antibodies in ISA 15AVG，ISA 71VG and ISA 201VG groups peaked at 38 d after the first immunization，and the titer of ISA 201VG group was the highest（>128000 times）.The rcELF4-specific IgY antibodies in all three groups turned positive at 14 d after the first immunization，and peaked at 35 d after the first immunization.The titer of ISA 201VG group was 256000 times.The highest IgY content in egg yolk of the 3 groups was 4.09 mg/10 mL egg yolk liquid，which had a specific reaction with rcELF4 protein，and it also cross-reacted with human ELF4 protein.rcELF4 protein located in the nucleus.【Conclusion】 The rcELF4 protein is successfully expressed in E.coli and it is significantly modified post-translation.Different types of adjuvants have different effects on the immunogenicity of rcELF4 protein，and the prepared rcELF4 antibody have good immune activity and was cross-reacted with human ELF4 protein.