中国畜牧兽医2024，Vol.51Issue(5)：2037-2046,10.DOI:10.16431/j.cnki.1671-7236.2024.05.025
猫球形马拉色菌脂肪酶SMG1基因的真核表达及重组载体脂肪酶活性分析
Eukaryotic Expression of Malassezia globosa Lipase SMG1 Gene in Cats and Lipase Activity Analysis of the Recombinant Vector
摘要
Abstract
[Objective]Malassezia is one of the causes of external otitis in cats,which is lipid-dependent.The lipase SMG1 gene can regulate the synthesis of Malassezia globosa lipase.The purpose of this study was to construct the expression vector of lipase SMG1 gene in Pichia pastoris,obtain the recombinant expression product of SMG1 gene,and analyze the activity of lipase enzyme,so as to provide basis for the subsequent research on the function of Malassezia lipase SMG1 gene.[Method]The pathogenic bacteria of a suspected case of Malassezia feline otitis externa were isolated and identified,and the fungi type was determined by similarity comparison.Lipase SMG1 gene was optimized by DNAworks software,the cloning vector of lipase SMG1 gene was constructed and identified.The eukaryotic expression vector of SMG1 gene was constructed using Pichia pastoris expression system,and the recombinant expression vector was screened and identified.The recombinant carrier lipase activity was determined.[Result]A strain of Malassezia was isolated from the ear canal in cats which was suspected to have Malassezia external otitis,and the isolate was identified as Malassezia globosa.By optimizing the codon of Malassezia lipase SMG1 gene,the codon adaptation index(CAI)was increased to 0.95.The cloning vector of lipase SMG1 gene was successfully constructed.The recombinant plasmid pGAPZaA-SMG1 was successfully constructed and transferred into Pichia pastoris X33.After the addition of methanol for induced expression,the expression product was analyzed by SDS-PAGE,the expression product of recombinant lipase SMG1 protein was 35 ku,and its enzyme activity was determined to be 0.023 U/mL.The enzyme activity of recombinant lipase SMG1 was the highest at 30 ℃(0.027 U/mL),which was extremely significantly different from that of 40 and 45 ℃ group(P＜0.01).[Conclusion]Malassezia globosa had higher lipase activity near the temperature of ear canal in cats,and the lipids promoted the growth of Malassezia.The expression system of Pichia pastoris could express and secrete recombinant lipase SMG1.The results laid a foundation for the pathogenesis of Malassezia externa otitis in cats,the development of lipase inhibitors,and the development of rapid detection technology.关键词
猫/球形马拉色菌/脂肪酶/毕赤酵母/真核表达Key words
cats/Malassezia globosa/lipase/Pichia pastoris/eukaryotic expression分类
农业科技引用本文复制引用
王开开,赵懿侔,张含露,邱实,刘艺佳,张文鑫,杨丰利..猫球形马拉色菌脂肪酶SMG1基因的真核表达及重组载体脂肪酶活性分析[J].中国畜牧兽医,2024,51(5):2037-2046,10.基金项目
湖北省教育厅科学技术研究项目(D20191304) （D20191304）
