尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果
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摘要
链球菌病是威胁我国罗非鱼养殖产业健康发展的重要病害之一。为研制出免疫效果好、操作简便的罗非鱼链球菌病疫苗,本研究构建重组表达无乳链球菌Sip蛋白的穿梭质粒pNZ8124-Sip,通过酶切和测序验证后电转化乳酸乳球菌NZ9000,获得能够诱导重组表达无乳链球菌Sip蛋白的乳酸菌活菌载体疫苗。采用SPS-PAGE电泳摸索最佳诱导浓度和诱导时间以获得最大表达量,通过镍柱纯化目的蛋白并进行Western blot检测;利用不同浓度的重组乳酸菌活载体疫苗灌胃口服免疫尼罗罗非鱼,采用间接ELISA法测定免疫后血清抗体水平变化,通过人工腹腔注射感染无乳链球菌获得相对免疫保护率。研究结果显示,构建的重组乳酸乳球菌可通过nisin诱导表达大小为48 ku特异性蛋白,与目的蛋白大小一致;PAGE电泳显示,重组蛋白主要以可溶蛋白和包涵体2种形式存在,其中胞内可溶性蛋白浓度达7.65 mg/mL;诱导表达的最佳条件为100 ng/mL nisin诱导6 h;Western blot检测结果显示,诱导蛋白可与鼠抗His标签抗体特异性结合。口服免疫结果显示,中浓度组(2.24×10~(10) CFU/mL)和低浓度组(2.24×10~9 CFU/mL)免疫2次能够显著提高尼罗罗非鱼的血清抗体水平和抗无乳链球菌感染能力,中浓度免疫组的相对免疫保护率最高为41.0%。本研究可为罗非鱼链球菌病口服疫苗的研究奠定基础,具有广阔的应用前景。
Streptococcosis is a serious disease that threatens the development of Oreochromis niloticus industry in China. In order to make a vaccine of Streptococcus agalactiae with high immune efficiency and simple operation,we constructed a recombinant shuttle-plasmid pNZ8124-Sip which could express Sip protein of S. agalactiae. The recombinant plasmid was electro-transferred into Lactococcus lactis NZ9000 after being identified by enzyme digestion and sequencing analysis. The SDS-PAGE was used to obtain the optimumly induced concentration of nisin and induction time. The Sip protein was purified by Ni-chelating affinity chromatography and tested by Western blot. O. niloticus was vaccinated orally by gavage with different concentration of the recombinant L. lactis NZ9000-pNZ8124-Sip. The ELISA was used to test the change of serum antibody. The relative percent survival(RPS) was obtained by artificial abdominal injection with S. agalactiae.The SDS-PAGE showed that the molecular weight of expressed protein was 48 ku, which is equal to the expected protein size. The recombinant protein mainly existed as soluble protein and inclusion bodies. The concentration of purified protein could reach 7.65 mg/mL. The optimal condition was induction with 100 ng/mL nisin for 6 h. Western blot results showed that the recombinant Sip protein could be specifically combined with the mouse anti-His tag antibody. Oral immunization showed that the serum antibody and the resistance to S. agalactiae were significantly improved in the middle concentration group(2.24×1010 CFU/mL) and the low concentration group(2.24×109 CFU/mL). The RPS of the middle concentration was 41.0%, which was the highest. This study can lay a foundation for research on oral vaccine of O. niloticus against S. agalactiae and has a broad prospect of application.
Streptococcosis is a serious disease that threatens the development of Oreochromis niloticus industry in China. In order to make a vaccine of Streptococcus agalactiae with high immune efficiency and simple operation,we constructed a recombinant shuttle-plasmid pNZ8124-Sip which could express Sip protein of S. agalactiae. The recombinant plasmid was electro-transferred into Lactococcus lactis NZ9000 after being identified by enzyme digestion and sequencing analysis. The SDS-PAGE was used to obtain the optimumly induced concentration of nisin and induction time. The Sip protein was purified by Ni-chelating affinity chromatography and tested by Western blot. O. niloticus was vaccinated orally by gavage with different concentration of the recombinant L. lactis NZ9000-pNZ8124-Sip. The ELISA was used to test the change of serum antibody. The relative percent survival(RPS) was obtained by artificial abdominal injection with S. agalactiae.The SDS-PAGE showed that the molecular weight of expressed protein was 48 ku, which is equal to the expected protein size. The recombinant protein mainly existed as soluble protein and inclusion bodies. The concentration of purified protein could reach 7.65 mg/mL. The optimal condition was induction with 100 ng/mL nisin for 6 h. Western blot results showed that the recombinant Sip protein could be specifically combined with the mouse anti-His tag antibody. Oral immunization showed that the serum antibody and the resistance to S. agalactiae were significantly improved in the middle concentration group(2.24×1010 CFU/mL) and the low concentration group(2.24×109 CFU/mL). The RPS of the middle concentration was 41.0%, which was the highest. This study can lay a foundation for research on oral vaccine of O. niloticus against S. agalactiae and has a broad prospect of application.
引文
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[13]Chen M,Wang R,Luo F G,et al.Streptococcus agalactiae isolates of serotypes Ia,III and V from human and cow are able to infect tilapia[J].Veterinary Microbiology,2015,180(1-2):129-135.
[14]Li W,Wang H Q,He R Z,et al.Major surfome and secretome profile of Streptococcus agalactiae from Nile tilapia(Oreochromis niloticus):insight into vaccine development[J].Fish&Shellfish Immunology,2016,55:737-746.
[15]Maione D,Margarit I,Rinaudo C D,et al.Identification of a universal Group B Streptococcus vaccine by multiple genome screen[J].Science,2005,309(5731):148-150.
[16]Brodeur B R,Boyer M,Charlebois I,et al.Identification of group B streptococcal Sip protein,which elicits crossprotective immunity[J].Infection and Immunity,2000,68(10):5610-5618.
[17]Xue G H,Yu L H,Li S T,et al.Intranasal immunization with GBS surface protein Sip and ScpB induces specific mucosal and systemic immune responses in mice[J].FEMS Immunology&Medical Microbiology,2010,58(2):202-210.
[18]Manning S D,Wood S,Kasha K,et al.Naturally occurring antibodies for the group B streptococcal surface immunogenic protein(Sip)in pregnant women and newborn babies[J].Vaccine,2006,24(47-48):6905-6912.
[19]Li L P,Wang R,Liang W W,et al.Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro[J].Fish&Shellfish Immunology,2015,45(2):955-963.
[20]Zhu L,Yang Q,Huang L Y,et al.Effectivity of oral recombinant DNA vaccine against Streptococcus agalactiae in Nile tilapia[J].Developmental&Comparative Immunology,2017,77:77-87.
[21]余丽芸,王桂华,唐彦君.乳酸菌作为口服疫苗载体的研究进展[J].生物技术通报,2008(5):48-50.Yu L Y,Wang G H,Tang Y J,et al.Advancement of lactic acid bacteria as oral vaccine[J].Biotechnology Bulletin,2008(5):48-50(in Chinese).
[22]Li Y J,Ma G P,Li G W,et al.Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production[J].Journal of Biomedicine and Biotechnology,2010,2010:708460.
[23]Anuradha K,Foo H L,Mariana N S,et al.Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia(Oreochromis niloticus)[J].Journal of Applied Microbiology,2010,109(5):1632-1642.
[24]De Vos W M.Gene expression systems for lactic acid bacteria[J].Current Opinion in Microbiology,1999,2(3):289-295.
[25]Shigemori S,Yonekura S,Sato T,et al.Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis[J].Applied Microbiology and Biotechnology,2013,97(8):3603-3611.
[26]Arya R,Sabir J S M,Bora R S,et al.Optimization of culture parameters and novel strategies to improve protein solubility.In García-Fruitós E.Insoluble Proteins:Methods and Protocols[M].New York,NY:Humana Press,2015:45-63.
[27]朱向东,徐波.乳酸菌蛋白质分泌表达研究进展[J].中国微生态学杂志,2008,20(1):91-92.Zhu X D,Xu B.Research progress of secretion expression of lactic acid bacteria proteins[J].Chinese Journal of Microecology,2008,20(1):91-92(in Chinese).
[28]Mercenier A,Müller-Alouf H,Grangette C.Lactic acid bacteria as live vaccines[J].Current Issues in Molecular Biology,2000,2(1):17-25.
[29]Strobel S,Mowat A M.Immune responses to dietary antigens:oral tolerance[J].Immunology Today,1998,19(4):173-181.
[30]江强,周必英.寄生虫疫苗免疫剂量的研究现状[J].国际医学寄生虫病杂志,2014,41(2):90-93.Jiang Q,Zhou B Y.The research status of immune dose of the parasite vaccine[J].International Journal of Medical Parasitic Diseases,2014,41(2):90-93(in Chinese).
[31]Rioux S,Martin D,Ackermann H W,et al.Localization of surface immunogenic protein on group Bstreptococcus[J].Infection and Immunity,2001,69(8):5162-5165.
[32]黎炯,叶星,可小丽,等.罗非鱼无乳链球菌Sip基因的克隆、表达及免疫原性分析[J].水生生物学报,2012,36(4):626-633.Li J,Ye X,Ke X L,et al.Cloning,expression and immunogenicity analysis of surface immunogenic protein(Sip)of tilapia Streptococcus agalactiae[J].Acta Hydrobiologica Sinica,2012,36(4):626-633(in Chinese).
[2]Liu G J,Zhu J L,Chen K M,et al.Development of Streptococcus agalactiae vaccines for tilapia[J].Diseases of Aquatic Organisms,2016,122(2):163-170.
[3]卢迈新,黎炯,叶星,等.广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析[J].微生物学通报,2010,37(5):766-774.Lu M X,Li J,Ye X,et al.Identification and characterizations of Streptococcus agalactiae isolated from tilapia cultured in Guangdong and Hainan provinces[J].Microbiology China,2010,37(5):766-774 (in Chinese).
[4]Pretto-Giordano L G,Müller E E,De Freitas J C,et al.Evaluation on the pathogenesis of Streptococcus agalactiae in Nile tilapia(Oreochromis niloticus)[J].Brazilian Archives of Biology and Technology,2010,53(1):87-92.
[5]Robinson J A,Meyer F P.Streptococcal fish pathogen[J].Journal of Bacteriology,1966,92(2):512.
[6]Plumb J A,Schachte J H,Gaines J L,et al.Streptococcus sp.from marine fishes along the Alabama and Northwest Florida coast of the gulf of Mexico[J].Transactions of the American Fisheries Society,1974,103(2):358-361.
[7]Garcia J C,Klesius P H,Evans J J,et al.Non-infectivity of cattle Streptococcus agalactiae in Nile tilapia,Oreochromis niloticus and channel catfish,Ictalurus punctatus[J].Aquaculture,2008,281(1-4):151-154.
[8]Mian G F,Godoy D T,Leal C A G,et al.Aspects of the natural history and virulence of S.agalactiae infection in Nile tilapia[J].Veterinary Microbiology,2009,136(1-2):180-183.
[9]Slotved H C,Kong F R,Lambertsen L,et al.Serotype IX,a proposed new Streptococcus agalactiae serotype[J].Journal of Clinical Microbiology,2007,45(9):2929-2936.
[10]Paoletti L C,Kasper D L.Conjugate vaccines against group B Streptococcus types IV and VII[J].The Journal of Infectious Diseases,2002,186(1):123-126.
[11]Yi T,Li Y W,Liu L,et al.Protection of Nile tilapia(Oreochromis niloticus L.)against Streptococcus agalactiae following immunization with recombinant FbsA andα-enolase[J].Aquaculture,2014,428-429:35-40.
[12]Kayansamruaj P,Pirarat N,Kondo H,et al.Genomic comparison between pathogenic Streptococcus agalactiae isolated from Nile tilapia in Thailand and fish-derived ST7 strains[J].Infection,Genetics and Evolution,2015,36:307-314.
[13]Chen M,Wang R,Luo F G,et al.Streptococcus agalactiae isolates of serotypes Ia,III and V from human and cow are able to infect tilapia[J].Veterinary Microbiology,2015,180(1-2):129-135.
[14]Li W,Wang H Q,He R Z,et al.Major surfome and secretome profile of Streptococcus agalactiae from Nile tilapia(Oreochromis niloticus):insight into vaccine development[J].Fish&Shellfish Immunology,2016,55:737-746.
[15]Maione D,Margarit I,Rinaudo C D,et al.Identification of a universal Group B Streptococcus vaccine by multiple genome screen[J].Science,2005,309(5731):148-150.
[16]Brodeur B R,Boyer M,Charlebois I,et al.Identification of group B streptococcal Sip protein,which elicits crossprotective immunity[J].Infection and Immunity,2000,68(10):5610-5618.
[17]Xue G H,Yu L H,Li S T,et al.Intranasal immunization with GBS surface protein Sip and ScpB induces specific mucosal and systemic immune responses in mice[J].FEMS Immunology&Medical Microbiology,2010,58(2):202-210.
[18]Manning S D,Wood S,Kasha K,et al.Naturally occurring antibodies for the group B streptococcal surface immunogenic protein(Sip)in pregnant women and newborn babies[J].Vaccine,2006,24(47-48):6905-6912.
[19]Li L P,Wang R,Liang W W,et al.Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro[J].Fish&Shellfish Immunology,2015,45(2):955-963.
[20]Zhu L,Yang Q,Huang L Y,et al.Effectivity of oral recombinant DNA vaccine against Streptococcus agalactiae in Nile tilapia[J].Developmental&Comparative Immunology,2017,77:77-87.
[21]余丽芸,王桂华,唐彦君.乳酸菌作为口服疫苗载体的研究进展[J].生物技术通报,2008(5):48-50.Yu L Y,Wang G H,Tang Y J,et al.Advancement of lactic acid bacteria as oral vaccine[J].Biotechnology Bulletin,2008(5):48-50(in Chinese).
[22]Li Y J,Ma G P,Li G W,et al.Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production[J].Journal of Biomedicine and Biotechnology,2010,2010:708460.
[23]Anuradha K,Foo H L,Mariana N S,et al.Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia(Oreochromis niloticus)[J].Journal of Applied Microbiology,2010,109(5):1632-1642.
[24]De Vos W M.Gene expression systems for lactic acid bacteria[J].Current Opinion in Microbiology,1999,2(3):289-295.
[25]Shigemori S,Yonekura S,Sato T,et al.Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis[J].Applied Microbiology and Biotechnology,2013,97(8):3603-3611.
[26]Arya R,Sabir J S M,Bora R S,et al.Optimization of culture parameters and novel strategies to improve protein solubility.In García-Fruitós E.Insoluble Proteins:Methods and Protocols[M].New York,NY:Humana Press,2015:45-63.
[27]朱向东,徐波.乳酸菌蛋白质分泌表达研究进展[J].中国微生态学杂志,2008,20(1):91-92.Zhu X D,Xu B.Research progress of secretion expression of lactic acid bacteria proteins[J].Chinese Journal of Microecology,2008,20(1):91-92(in Chinese).
[28]Mercenier A,Müller-Alouf H,Grangette C.Lactic acid bacteria as live vaccines[J].Current Issues in Molecular Biology,2000,2(1):17-25.
[29]Strobel S,Mowat A M.Immune responses to dietary antigens:oral tolerance[J].Immunology Today,1998,19(4):173-181.
[30]江强,周必英.寄生虫疫苗免疫剂量的研究现状[J].国际医学寄生虫病杂志,2014,41(2):90-93.Jiang Q,Zhou B Y.The research status of immune dose of the parasite vaccine[J].International Journal of Medical Parasitic Diseases,2014,41(2):90-93(in Chinese).
[31]Rioux S,Martin D,Ackermann H W,et al.Localization of surface immunogenic protein on group Bstreptococcus[J].Infection and Immunity,2001,69(8):5162-5165.
[32]黎炯,叶星,可小丽,等.罗非鱼无乳链球菌Sip基因的克隆、表达及免疫原性分析[J].水生生物学报,2012,36(4):626-633.Li J,Ye X,Ke X L,et al.Cloning,expression and immunogenicity analysis of surface immunogenic protein(Sip)of tilapia Streptococcus agalactiae[J].Acta Hydrobiologica Sinica,2012,36(4):626-633(in Chinese).