[目的]马拉色菌(Malassezia)是引起猫外耳炎的病因之一,具有脂质依赖性,脂肪酶SMG1基因能调控球形马拉色菌脂肪酶的合成.本研究旨在构建脂肪酶SMG1基因毕赤酵母表达载体,获得SMG1基因重组表达产物,并对脂肪酶酶活性进行分析,为后续马拉色菌脂肪酶SMG1基因功能研究提供依据.[方法]对1例疑似猫马拉色菌性外耳炎病例的致病菌进行真菌分离鉴定,并通过相似性比对确定菌种类型;利用DNAworks软件优化脂肪酶SMG1基因,构建克隆载体并鉴定;使用毕赤酵母表达系统构建SMG1基因真核表达载体,并对重组表达载体进行筛选及鉴定;对重组载体脂肪酶活性进行测定.[结果]试验成功从疑似患有马拉色菌性外耳炎的患猫耳道中分离得到1株马拉色菌菌株,鉴定为球形马拉色菌;通过对马拉色菌脂肪酶SMG1基因密码子的优化,将密码子适应指数(codon adaptation index,CAI)提高至0.95;成功构建脂肪酶SMG1基因克隆载体,成功构建重组质粒pGAPZaA-SMG1并转入毕赤酵母X33中,诱导表达后使用SDS-PAGE分析,蛋白产物大小为35 ku,脂肪酶酶活性测定为0.023 U/mL;重组脂肪酶SMG1在30 ℃酶活性最高(0.027 U/mL),与40、45 ℃组间差异均极显著(P＜0.01).[结论]球形马拉色菌在猫耳道皮肤温度附近有较高的脂肪酶活性,同时脂质促进了马拉色菌的生长;毕赤酵母表达系统可成功表达并分泌重组脂肪酶SMG1.试验结果为后续猫马拉色菌性外耳炎的致病机制研究、脂肪酶抑制剂研制及快速检测技术的开发奠定了基础.
[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.
王开开;赵懿侔;张含露;邱实;刘艺佳;张文鑫;杨丰利
长江大学动物科学技术学院,荆州 434025
畜牧业
猫;球形马拉色菌;脂肪酶;毕赤酵母;真核表达
cats;Malassezia globosa;lipase;Pichia pastoris;eukaryotic expression
《中国畜牧兽医》 2024 (005)
2037-2046 / 10
湖北省教育厅科学技术研究项目(D20191304)
10.16431/j.cnki.1671-7236.2024.05.025
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