东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达

doi:10.16431/j.cnki.1671-7236.2021.01.008

Abstract

Abstract: The purpose of this experiment was to clone the Siberian tiger γ-interferon (IFN-γ) gene,studied its molecular characteristics and predicted the biological function of the protein,making preliminary preparations for the subsequent study of the antiviral activity of interferon.The Siberian tiger IFN-γ gene was amplified from ConA-induced Siberian tiger blood lymphocytes by RT-PCR and sequenced to obtain the sequence.Bioinformatics was used for sequence analysis.The results showed that IFN-γ gene coding region consisted of 504 nucleotides,encoding a total of 167 amino acids.The relative molecular weight of the Siberian tiger IFN-γ amino acid was 19.59 ku,the isoelectric point was 9.03.The encoded protein was a basic hydrophilic protein,of which the first 23 amino acids might be signal peptides.The conserved domain of the IFN-γ encoded protein was the IFN-γ superfamily,and there was a transmembrane structure,in which amino acids 1－6 were intracellular regions and amino acids 7－28 were transmembrane regions,amino acids 29－167 were extracellular regions.The secondary structure of IFN-γ encoded protein was mainly α-helix (58.08%) and random coil (33.53%).There were 5 potential B cell epitopes and 3 potential N-glycosylation sites.Sequence analysis showed that the Siberian tiger IFN-γ had 80.4%－99.8% nucleotide similarity to the Siberian tiger,African lion,leopard,puma,cheetah,domestic cat,Canadian lynx and wild boar published on GenBank,and the amino acid similarity was 70.5%－100%,nucleotide genetic evolution analysis showed that Siberian tiger IFN-γ had the closest relationship with African lions and leopards,followed by puma,cheetah,domestic cat and lynx,and wild boars were farthest.By synthesizing the modified Siberian tiger IFN-γ gene,a recombinant plasmid pPIC9K-IFN-γ capable of secreting and expressing IFN-γ protein was constructed,which was introduced into a highly efficient expression system-Pichia pastoris to induce expression,and analyzed by SDS-PAGE,the molecular weight of the expressed protein was about 17.8 ku,which was consistent with the expected size,indicating that the Siberian tiger IFN-γ was successfully expressed.

Key words: Siberian tiger; γ-interferon (IFN-γ); gene cloning; bioinformatics; Pichia pastoris

CLC Number:

S865.31

ZHAI Junqiong, WU Yajiang, DAI Junwei, CHEN Anni, ZHOU Niu, WANG Chen, DU Xueqing, LUO Manlin, CHEN Wu. Cloning and Bioinformatics Analysis of the Siberian Tiger γ-interferon Gene and Its Expression in Pichia pastoris[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 72-82.

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Cloning and Bioinformatics Analysis of the Siberian Tiger γ-interferon Gene and Its Expression in Pichia pastoris

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