表达猪δ冠状病毒S1蛋白植物乳杆菌的构建及验证  

Construction and verification of Lactobacillus plantarum expressing PDCoV S1 protein

作　　者：郝嘉翼 李乐天 张和平[3] 陈永福[3] 金宁一 王茂鹏 李昌 HAO Jiayi;LI Letian;ZHANG Heping;CHEN Yongfu;JIN Ningyi;WANG Maopeng;LI Chang(College of Animal Science and Technology/College of Animal Medicine,Jilin Agricultural University,Changchun 130118,China;Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses,Chinese Academy of Medical Sciences,Changchun Veterinary Research Insititute,Chinese Academy of Agricultural Sciences,Changchun 130122,China;Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Inner Mongolia Agricultural University,Huhhot 010018,China;Institute of Virology,Wenzhou University,Wenzhou,Zhejiang 325035,China)

机构地区：[1]吉林农业大学动物科技学院/动物医学院,吉林长春130118 [2]中国农业科学院长春兽医研究所,中国医学科学院人兽共患病毒病防控关键技术研究创新单元,吉林长春130122 [3]内蒙古农业大学乳品生物技术与工程教育部重点实验室,内蒙古呼和浩特010018 [4]温州大学病毒学研究所,浙江温州325035

出　　处：《中国兽医学报》2021年第12期2304-2310,2369,共8页Chinese Journal of Veterinary Science

基　　金：中国医学科学院医学与健康科技创新工程资助项目(2019RU059)。

摘　　要：本研究拟构建猪δ冠状病毒(PDCoV)S1蛋白的重组植物乳杆菌。通过PCR方法扩增含1320信号肽和DCpep优化的PDCoV S1基因,利用无缝克隆技术将扩增片段克隆至植物乳杆菌表达载体pSIP411,并将pSIP411电转至宿主乳酸乳球菌NZ3900,扩增后提取质粒并验证,获得重组质粒NZ3900:pSIP411-PDCoV S1。将重组质粒电转入植物乳杆菌LP18,获得重组植物乳杆菌LP18:pSIP411-PDCoV S1,对诱导时间、诱导剂质量浓度、诱导温度等表达条件进行优化,以获得最佳表达条件。结果显示,成功扩增出1939 bp经过修饰和优化的目的基因PDCoV S1,成功构建了重组植物乳杆菌LP18:pSIP411-PDCoV S1。Western blot、流式细胞术、免疫荧光等检测方法表明重组蛋白能够在重组植物乳杆菌中表达,最佳表达条件:诱导时间为6 h、诱导剂质量浓度为150μg/L、诱导温度为33℃。本试验成功构建了1株高表达PDCoV S1蛋白的植物乳杆菌,为PDCoV口服候选疫苗的研制奠定基础。Recombinant Lactobacillus plantarum with porcine deltacoronavirus(PDCoV)S1 protein was constructed and validated.The 1320 signal peptide and DCpep-optimized PDCoV S1 gene were amplified by PCR,ligated into the pSIP411 vector using the seamless cloning technique,and electroporated into the Lactocaccus NZ3900,the recombinant expressing plasmid NZ3900:pSIP411-PDCoV S1 was extracted and electroporated into Lactobacillus plantarum LP18,yielding recombinant Lactobacillus plantarum expressing PDCoV S1 protein.Meanwhile,the expression conditions including induction time,inducer concentration and induction temperature were optimized.The results showed that the modified and optimized PDCoV S1 gene was successfully amplified and the recombinant Lactobacillus plantarum LP18:pSIP411-PDCoV S1 was successfully constructed.The results of Western blot,flow cytometry,immunofluorescence and other detection methods showed that the target protein could be expressed in recombinant Lactobacillus plantarum.The optimal expression conditions were optimized and determined as follows:induction time was 6 h,optimal induction concentration was 150μg/L,and optimal induction temperature was 33°C.In conclusion,a strain of Lactobacillus plantarum with high expression of PDCoV S1 protein was successfully constructed,which laid a foundation for the development of PDCoV oral vaccine candidate.

关 键 词：PDCoV S1蛋白 植物乳杆菌 构建与表达 

分 类 号：S852.65[农业科学—基础兽医学]