植物乳杆菌表达重组新冠病毒M抗原表位及免疫原性分析  

Expression and immunogenicity analysis of recombinant SARS-CoV-2 M peptide epitope by Lactiplantibacillus plantarum

作　　者：邓安琦 叶丹妮 艾雪言 唐秀兰 陈文聪 陈嘉豪 郝嘉翼 邓玲聪 李昌 陈永福[3] 金俊杰[4] 王茂鹏 DENG Anqi;YE Danni;AI Xueyan;TANG Xiulan;CHEN Wencong;CHEN Jiahao;HAO Jiayi;DENG Lingcong;LI Chang;CHEN Yongfu;JIN Junjie;WANG Maopeng(Wenzhou Key Laboratory of Virology and Immunology,Institute of Virology,Wenzhou University,Wenzhou,Zhejiang 325035,China;Changchun Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences,Changchun 130122,China;Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Inner Mongolia Agricultural University,Hohhot Ol0018,China;Wenzhou Vocational College of Science and Technology(Wenzhou Institute of Agricultural Science),Wenzhou,Zhejiang325006,China)

机构地区：[1]温州大学病毒学研究所温州市病毒学与免疫学重点实验室,浙江温州325035 [2]中国农业科学院长春兽医研究所,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]温州科技职业学院(温州市农业科学研究院),浙江温州325006

出　　处：《中国兽医学报》2024年第8期1719-1727,共9页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划(2022YFC2604204);国家生猪技术创新中心先导科技项目(NTCIP-XD/B11);温州市特派员专项(X2023085,N2023076);温州市农业丰收计划(FSJH2022003)。

摘　　要：新型冠状病毒(SARS-CoV-2)是引起新冠肺炎的病原体,其变异快、传播强,导致疫情广泛流行,以疫苗接种为主要防控措施。尽管已有大量新冠肽表位疫苗和黏膜型疫苗的研究,但靶向黏膜的肽表位疫苗和其功能评价的研究鲜有报道。本研究以IEDB数据库预测SARS-CoV-2结构蛋白M肽表位作为抗原靶点,设计含3050及1229信号肽和DCpep优化的MS-3S基因,插入MS2噬菌体衣壳蛋白中,通过PCR和无缝克隆技术构建表达质粒pSIP:MS-3S,转化到植物乳杆菌LP18中获得重组菌LP18:MS-3S。通过对诱导时间、诱导剂质量浓度、转速和初始pH值等表达条件进行优化及鼻内免疫实验,验证重组菌免疫效果。结果显示,获得出916 bp修饰和优化的目的基因MS-3S,并成功构建重组菌LP18:MS-3S,经Western blot、流式细胞术、免疫荧光等检测方法验证,获得重组蛋白表达的最佳条件:诱导时间为4 h,诱导肽质量浓度为100μg/L,培养基初始pH值为7.0,转速为100 r/min。通过ELISA试验验证,在小鼠的血清、肺泡灌洗液和粪便稀释液中,检测出抗肽表位的特异性抗体。本研究成功构建了1株表达新冠M蛋白肽表位抗原的重组菌,并且可诱导机体产生体液和黏膜免疫,为新冠黏膜免疫候选疫苗的研制奠定了基础。Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the main pathogen that causes COVID-19,which is fast-mutating and highly transmissible.The infection has led to a global epidemic.As the main preventive and control measure,vaccination plays a critical role in fighting against COVID-19.Although a large number of epitope-based and mucosal vaccines have been studied,few peptide epitope vaccines targeting the mucosa and their functional evaluation have been reported.In this study,we used SARS-CoV-2 structural protein M peptide epitope predicted by the IEDB database as an antigenic target to design the MS-3Sgene containing 3050and 1229signal peptides and DCpep optimized for insertion into MS2phage coat proteins.The expression plasmid pSIP:MS-3Swas constructed by cloning the PCR fragments seamlessly and was transformed into Lactiplantibacillus plantarum 18to obtain the recombinant bacterium LP18:MS-3S.Expression conditions such as induction time,inducer concentration,rotational speed and initial pH were optimized.The intranasal immunization experiments were performed to examine the vaccine efficacy.The results showed that the 916bp-long target gene MS-3Smodified and optimized was amplified and used to successfully construct the recombinant bacterial strain LP18:MS-3S.The optimal conditions for recombinant protein expression were obtained and verified by Western blot,flow cytometry,immunofluorescence and other detection methods.The optimal expression conditions were determined as follows:induction time was 4hwith 100μg/L of SppIP as the optimal induction concentration.Antibody-specific for the epitope was verified by ELISA experiments in serum,alveolar lavage fluid and fecal dilutions of mice.In summary,a recombinant bacterial strain expressing the epitope antigen of the SARS-CoV-2 M protein peptide was constructed.The obtained protein can induce the body to produce humoral and mucosal immunity,which lays the foundation for the development of a vaccine candidate for the mucosal immunity of COVID-19.

关 键 词：SARS-CoV-2 M蛋白 肽表位 植物乳杆菌 鼻内免疫 

分 类 号：S852.65[农业科学—基础兽医学]