Cloning,Bioinformatics Analysis and Prokaryotic Expression of IFN-γ Gene in Takin
The objective of this study was to explore the structure and function of the interferon y(IFN-γ)gene in takin,with the aim of providing a theoretical basis for enhancing the immune regula-tion capacity of this species.In this study,the IFN-γ gene of takin was cloned via RT-PCR,a compre-hensive analysis of its structure and function were conducted using variety of bioinformatics tools.The IFN-γ gene sequence was connected to the prokaryotic expression vector and transformed into E.coli to induce its expression.The results showed that the IFN-γ cDNA sequence of takin was 501 bp in length and encoded 166 amino acids.The prediction of the protein's higher order structure showed that the secondary structure was dominated by α helix,with 13 phosphorylation modification sites,a signal peptide,a low-grade complex region,a transmembrane domain and an IFN-γ domain.The IFN-γdomain is located in the extracellular region.Sequence alignment analysis showed that the nucleotide sequence homology with sheep,goat,cattle,buffalo,human,chimpanzee,mouse and chicken was 99.0％,98.8％,97.2％,96.2％,75.8％,75.4％,64.1％,and 54.1％,respectively.The amino acid se-quence homology was 99.4％,99.4％,95.8％,95.8％,62.7％,62.7％,43.9％,and 33.5％,respective-ly.The result of the phylogenetic tree analysis also showed that takin was most closely related to goats and sheep.SDS-PAGE and Western-blot showed that the recombinant IFN-γ protein was abundantly expressed in soluble form with a molecular weight of about 34 ku.Through optimization strategies for prokaryotic expression,such as codon optimization and IPTG induction expression at different concen-trations,it was found that codon optimization promoted the efficient expression of recombinant pro-teins,and the optimal induction concentration was determined to be 0.9 mmol·L-1.This study lays a foundational understanding for comprehensive functional exploration of IFN-γ gene in takin,as well as the potential application of interferon y in takin.These findings provide novel insights into takin con-servation efforts.
万方数据
维普
