表达IBDV VP2蛋白的重组弱毒肠炎沙门菌的构建及其免疫原性的初步研究
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摘要
为研究肠炎沙门菌rfaH基因与致病性的关系及肠炎沙门菌rfaH基因缺失株作为疫苗载体的可行性,进一步探索传染性法氏囊病(IBD)的防控路径,通过λ-Red同源重组的方法构建了缺失rfaH基因的肠炎沙门菌(rSD),再以其为亲本菌株构建了稳定表达IBDV VP2蛋白的重组肠炎沙门菌(Se-VP2)。以1日龄SPF雏鸡为实验动物,分别检测了重组菌的致病力及免疫效果。结果显示,rSD株和Se-VP2株与野生株生长速度一致,体外连续传20代,基因缺失株和重组株均可稳定遗传;Western-blot表明,Se-VP2株可稳定表达VP2蛋白;构建的重组菌rSD株和Se-VP2株对SPF雏鸡无致病性,半数致死量(LD50)大于1.0×1010CFU,且重组菌Se-VP2株免疫动物后可同时诱导产生针对IBDV和沙门菌的抗体;重组菌Se-VP2株可以诱导T淋巴细胞产生Th1型细胞因子IFN-γ和Th2型细胞因子IL-4,并能刺激外周血淋巴细胞的增殖分化;攻毒保护性试验结果显示,Se-VP2株免疫动物后可对IBDV和沙门菌的感染提供100%的保护。结果表明,rfaH基因是沙门菌的毒力基因,重组弱毒肠炎沙门菌Se-VP2株作为疫苗候选株,对SPF雏鸡安全有效,为后续肠炎沙门菌作为表达载体的研究奠定了基础。
The pathogenic role of rfa H gene in Salmonella enteritidis,and feasibility of Salmonella as a vaccine carrier were studied for exploring new ways of prevention and control of infectious bursal disease(IBD).A mutant(rSD) with deficiency of gene rfaH of a Salmonella enteritidis strain was constructed using the λ-Red recombination system.A recombinant strain(Se-VP2) expressing VP2 protein of IBDV was then constructed using r SD as the parental strain.The pathogenicity and immune effects of the recombinant bacteria were tested respectively in one-day-old SPF chickens.In result,the growth rates of r SD and Se-VP2 were as the same as wild-type strain. After 20 successive passages in vitro,both r SD and Se-VP2 strains were stably heritable.Se-VP2 strain expressed VP2 protein stably as verified by Western-blot.The recombinant strain was safe for SPF chickens,lethal median dose(LD50) was greater than 1.0×1010 CFU,and Se-VP2 induced antibodies against IBDV and Salmonella simultaneously.Se-VP2 also induced the T lymphocyte to produce Th1 cytokine IFN-γ and Th2 cytokine IL-4,and significantly proliferated peripheral blood lymphocyte.Protective results showed that Se-VP2 strain provided 100% protection against IBDV and Salmonella infection.The results showed that rfa H gene is a virulence gene of Salmonella.As a vaccine candidate,the attenuated Salmonella enteritidis strain Se-VP2 is safe and effective for SPF chickens,which lay the foundation for further study on Salmonella enteritidis as an expression vector.
The pathogenic role of rfa H gene in Salmonella enteritidis,and feasibility of Salmonella as a vaccine carrier were studied for exploring new ways of prevention and control of infectious bursal disease(IBD).A mutant(rSD) with deficiency of gene rfaH of a Salmonella enteritidis strain was constructed using the λ-Red recombination system.A recombinant strain(Se-VP2) expressing VP2 protein of IBDV was then constructed using r SD as the parental strain.The pathogenicity and immune effects of the recombinant bacteria were tested respectively in one-day-old SPF chickens.In result,the growth rates of r SD and Se-VP2 were as the same as wild-type strain. After 20 successive passages in vitro,both r SD and Se-VP2 strains were stably heritable.Se-VP2 strain expressed VP2 protein stably as verified by Western-blot.The recombinant strain was safe for SPF chickens,lethal median dose(LD50) was greater than 1.0×1010 CFU,and Se-VP2 induced antibodies against IBDV and Salmonella simultaneously.Se-VP2 also induced the T lymphocyte to produce Th1 cytokine IFN-γ and Th2 cytokine IL-4,and significantly proliferated peripheral blood lymphocyte.Protective results showed that Se-VP2 strain provided 100% protection against IBDV and Salmonella infection.The results showed that rfa H gene is a virulence gene of Salmonella.As a vaccine candidate,the attenuated Salmonella enteritidis strain Se-VP2 is safe and effective for SPF chickens,which lay the foundation for further study on Salmonella enteritidis as an expression vector.
引文
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[13]许信刚,赵红妮,陈光达,等.表达IBDV VP2蛋白重组减毒沙门菌活载体疫苗株的构建及免疫原性分析[J].中国兽医学报,2011,31(12):1686-1690.XU Xin-gang,ZHAO Hong-ni,CHEN Guang-da,et al.Construction of the recombinant attenuated Salmonella typhimurium live vector vaccine strain expressing IBDV VP2 protein and its immunogenicity analysis[J].Chinese Journal of Veterinary Science,2011,31(12):1686-1690.(in Chinese)
[14]单学强,李明义,高轩.传染性法氏囊病毒VP2基因原核表达及抗原性分析[J].动物医学进展,2012,33(5):18-21.SHAN Xue-qiang,LI Ming-yi,GAO Xuan.Prokaryotic expression of VP2 gene of infectious bursal disease virus and antigenicity of expressed products[J].Progress in Veterinary Medicine,2012,33(5):18-21.(in Chinese)
[15]孙化露,姜逸,李树纯,等.动物源性沙门菌的血清型、生物被膜形成能力和耐药性分析[J].畜牧兽医学报,2012,43(10):1630-1638.SUN Hua-lu,JIANG Yi,LI Shu-chun,et al.Analysis of serotype,biofilm-forming ability and antimicrobial resistance of Salmonella strains isolated from animals[J].Acta Veterinaria et Zootechnica Sinica,2012,43(10):1630-1638.(in Chinese)
[16]司微,于申业,陈利平,等.肠炎沙门菌tu-fA基因缺失株的构建及其生物学特性研究[J].中国预防兽医学报,2015,37(5):361-365.SI Wei,YU Shen-ye,CHEN Li-ping,et al.Construction and biological identification of a tu-fA gene deletion mutant from Salmonella enterica[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(5):361-365.(in Chinese)
[17]张春华,吕景生,赵斌.鼠疫菌毒力测定概念及计算公式的研讨[J].中国地方病防治杂志,2010,25(2):115-116.ZHANG Chun-hua,L譈Jing-sheng,ZHAO Bin,et al.The concept of the Yersinia pestis virulence test and the discussion of computation formula[J].Chinese Journal of Control of Endemic Diseases,2010,25(2):115-116.(in Chinese)
[18]康凯,陈小云,张敏,等.缺失rfaH基因的鸡白痢沙门菌株的构建及鉴定[J].中国预防兽医学报,2010,32(12):978-980.KANG Kai,CHEN Xiao-yun,ZHANG Min,et al.Construction of the rfaH gene delated Salmonella pullorum[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(12):978-980.(in Chinese)
[19] GONZALEZ-MACHADO C,CAPITA R,RIESCO-PELAEZ F,et al.Visualization and quantification of the cellular and extracellular components of Salmonella agona biofilms at different stages of development[J].PLoS One,2018,13(7):1-14.
[20] FOLEY S L,NAYAK R,HANNING I B,et al.Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production[J].Applied and Environmental Microbiology,2011,77(13):4273-4279.
[21] GEBREMICHAEL G,DANIEL A,YIMTUBEZINASH W/A,et al.Isolation and antimicrobial susceptibility profile of Shigella and Salmonella species from children with acute diarrhoea in Mekelle hospital and semen health center,Ethiopia[J].Ethiopian Journal of Health Sciences,2018,28(2):197-206.
[22] DOGGETT T A,CURTISS R 3RD.Delivery of antigens by recombinant avirulent Salmonella strains[J].Advances in Experimental Medicine and Biology,1992,327:165-173.
[23]鲜思美,李婷,冯将,等.携带DEV-gB/NP28重组减毒沙门菌诱导鸭的免疫应答研究[J].中国预防兽医学报,2018,40(4):330-335.XIAN Si-mei,LI Ting,FENG Jiang,et al.Study on the immune response of ducks induced by recombinant attenuated Salmonella carrying DEV-gB/NP28[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(4):330-335.(in Chinese)
[24] DING K,SHANG K,YU Z H,et al.Recombinant-attenuated Salmonella pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus(NDV)protects chickens against NDV and Salmonella pullorum challenge[J].Journal of Veterinary Science,2018,19(2):232-241.
[25]张小会,张丹,梁恩涛,等.减毒沙门菌投递的TGEV S基因疫苗口服免疫猪的动态规律[J].中国人兽共患病学报,2016,32(1):33-38.ZHANG Xiao-hui,ZHANG Dan,LIANG En-tao,et al.Dynamic oral immuno-induction regulation of attenuated Salmonella typhimurium harboring DNA vaccine expressing S gene of transmissible gastroenteritisvirus in piglets[J].Chinese Journal of Zoonoses,2016,32(1):33-38.(in Chinese)
[26] PIKULA A,DOMA SKA-BLICHARZ K,CEPULIS R,et al.Identification of infectious bursal disease virus with atypical VP2 amino acid profile in Latvia[J].Journal of Veterinary Research,2017,61(2):145-149.
[27] NAGY G,DANINO V,DOBRINDT U,et al.Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhymurium rfaH mutant a promising live-attenuated vaccine candidate[J].Infection and Immunity,2006,74(10):5914-5925.
[28] ZHANG J,DE MASI L,JOHN B,et al.Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella[J].Microbial Cell Factories,2014,13(80):1-12.
[2] LI Z H,WANG Y Q,LI X,et al.Critical roles of glucocorticoid-induced leucine zipper in infectious bursal disease virus(IBDV)-induced suppression of typeⅠinterferon expression and enhancement of IBDV growth in host cells via interaction with VP4[J].Journal of Virology,2013,87(2):1221-1231.
[3] VUKEA P R,WILLOWS-MUNRO S,HORNER R F,et al.Phylogenetic analysis of the polyprotein coding region of an infectious South African bursal disease virus(IBDV)strain[J].Infection,Genetics and Evolution,2014,21(1):279-286.
[4] IKUTA N,EL-ATTRACHE J,VILLEGAS P,et al.Molecular characterization of Brazilian infectious bursal disease viruses[J].Avian Diseases,2001,45(2):297-306.
[5] BERG T P.Acute infectious bursal disease in poultry:a review[J].Avian Pathology,2000,29(3):175-194.
[6] MULLER H,MUNDT E,ETERRADOSSI N,et al.Current status of vaccines against infectious bursal disease[J].Avian Pathology,2012,41(2):133-139.
[7] TORO H,PENNINGTON D,GALLARDO R A,et al.Infectious bronchitis virus subpopulations in vaccinated chicken after challenge[J].Avian Diseases,2012,56(3):501-508.
[8] JACKWOOD D J.Advances in vaccine research against economically important viral diseases of food animals:infectious bursal disease virus[J].Veterinary Microbiology,2017,206:121-125.
[9] PRADHAN S N,PRINCE P R,MADHUMATHI J,et al.Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens[J].Veterinary Immunology and Immunopathology,2012,148(3-4):293-301.
[10] DU A F,WANG S H.Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens[J]. International Journal for Parasitology,2005,35(7):777-785.
[11] CONG W,JIN H,JIANG C D,et al.Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine[J].Veterinary Research,2010,41(3):1-11.
[12] DEY S,CHELLAPPA M M,PATHAK D C,et al.Newcastle disease virus vectored bivalent vaccine against virulent infectious bursal disease and Newcastle disease of chickens[J].Vaccines(Basel),2017,5(4):3-14.
[13]许信刚,赵红妮,陈光达,等.表达IBDV VP2蛋白重组减毒沙门菌活载体疫苗株的构建及免疫原性分析[J].中国兽医学报,2011,31(12):1686-1690.XU Xin-gang,ZHAO Hong-ni,CHEN Guang-da,et al.Construction of the recombinant attenuated Salmonella typhimurium live vector vaccine strain expressing IBDV VP2 protein and its immunogenicity analysis[J].Chinese Journal of Veterinary Science,2011,31(12):1686-1690.(in Chinese)
[14]单学强,李明义,高轩.传染性法氏囊病毒VP2基因原核表达及抗原性分析[J].动物医学进展,2012,33(5):18-21.SHAN Xue-qiang,LI Ming-yi,GAO Xuan.Prokaryotic expression of VP2 gene of infectious bursal disease virus and antigenicity of expressed products[J].Progress in Veterinary Medicine,2012,33(5):18-21.(in Chinese)
[15]孙化露,姜逸,李树纯,等.动物源性沙门菌的血清型、生物被膜形成能力和耐药性分析[J].畜牧兽医学报,2012,43(10):1630-1638.SUN Hua-lu,JIANG Yi,LI Shu-chun,et al.Analysis of serotype,biofilm-forming ability and antimicrobial resistance of Salmonella strains isolated from animals[J].Acta Veterinaria et Zootechnica Sinica,2012,43(10):1630-1638.(in Chinese)
[16]司微,于申业,陈利平,等.肠炎沙门菌tu-fA基因缺失株的构建及其生物学特性研究[J].中国预防兽医学报,2015,37(5):361-365.SI Wei,YU Shen-ye,CHEN Li-ping,et al.Construction and biological identification of a tu-fA gene deletion mutant from Salmonella enterica[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(5):361-365.(in Chinese)
[17]张春华,吕景生,赵斌.鼠疫菌毒力测定概念及计算公式的研讨[J].中国地方病防治杂志,2010,25(2):115-116.ZHANG Chun-hua,L譈Jing-sheng,ZHAO Bin,et al.The concept of the Yersinia pestis virulence test and the discussion of computation formula[J].Chinese Journal of Control of Endemic Diseases,2010,25(2):115-116.(in Chinese)
[18]康凯,陈小云,张敏,等.缺失rfaH基因的鸡白痢沙门菌株的构建及鉴定[J].中国预防兽医学报,2010,32(12):978-980.KANG Kai,CHEN Xiao-yun,ZHANG Min,et al.Construction of the rfaH gene delated Salmonella pullorum[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(12):978-980.(in Chinese)
[19] GONZALEZ-MACHADO C,CAPITA R,RIESCO-PELAEZ F,et al.Visualization and quantification of the cellular and extracellular components of Salmonella agona biofilms at different stages of development[J].PLoS One,2018,13(7):1-14.
[20] FOLEY S L,NAYAK R,HANNING I B,et al.Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production[J].Applied and Environmental Microbiology,2011,77(13):4273-4279.
[21] GEBREMICHAEL G,DANIEL A,YIMTUBEZINASH W/A,et al.Isolation and antimicrobial susceptibility profile of Shigella and Salmonella species from children with acute diarrhoea in Mekelle hospital and semen health center,Ethiopia[J].Ethiopian Journal of Health Sciences,2018,28(2):197-206.
[22] DOGGETT T A,CURTISS R 3RD.Delivery of antigens by recombinant avirulent Salmonella strains[J].Advances in Experimental Medicine and Biology,1992,327:165-173.
[23]鲜思美,李婷,冯将,等.携带DEV-gB/NP28重组减毒沙门菌诱导鸭的免疫应答研究[J].中国预防兽医学报,2018,40(4):330-335.XIAN Si-mei,LI Ting,FENG Jiang,et al.Study on the immune response of ducks induced by recombinant attenuated Salmonella carrying DEV-gB/NP28[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(4):330-335.(in Chinese)
[24] DING K,SHANG K,YU Z H,et al.Recombinant-attenuated Salmonella pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus(NDV)protects chickens against NDV and Salmonella pullorum challenge[J].Journal of Veterinary Science,2018,19(2):232-241.
[25]张小会,张丹,梁恩涛,等.减毒沙门菌投递的TGEV S基因疫苗口服免疫猪的动态规律[J].中国人兽共患病学报,2016,32(1):33-38.ZHANG Xiao-hui,ZHANG Dan,LIANG En-tao,et al.Dynamic oral immuno-induction regulation of attenuated Salmonella typhimurium harboring DNA vaccine expressing S gene of transmissible gastroenteritisvirus in piglets[J].Chinese Journal of Zoonoses,2016,32(1):33-38.(in Chinese)
[26] PIKULA A,DOMA SKA-BLICHARZ K,CEPULIS R,et al.Identification of infectious bursal disease virus with atypical VP2 amino acid profile in Latvia[J].Journal of Veterinary Research,2017,61(2):145-149.
[27] NAGY G,DANINO V,DOBRINDT U,et al.Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhymurium rfaH mutant a promising live-attenuated vaccine candidate[J].Infection and Immunity,2006,74(10):5914-5925.
[28] ZHANG J,DE MASI L,JOHN B,et al.Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella[J].Microbial Cell Factories,2014,13(80):1-12.