外源抗原在蜡样芽胞杆菌细胞表面展示的可行性研究
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摘要
本研究室刘梅利用苏云金芽胞杆菌的S-层蛋白在苏云金芽胞杆菌细胞表面成功展示了禽流感病毒核衣壳蛋白(NP)、血凝素蛋白(HA1)和鸡毒霉形体粘附素蛋白(pMGA)等禽类病原体抗原,虽然实验证实苏云金芽胞杆菌BMB171对小鼠和鸡都安全无毒,但其是否具有益生作用,还未见相关报道;蜡样芽胞杆菌在畜牧业中常被用作微生态制剂来使用,本文在刘梅等研究的基础上利用细菌细胞表面展示技术,探索在蜡样芽胞杆菌细胞表面展示外源抗原的可行性,为研制能耐高温同时具有益生作用的禽用口服疫苗提供理论基础。
1.蜡样芽胞杆菌重组菌株的构建
以苏云金芽胞杆菌CH和BCCH菌株为出发菌株,进行质粒pCSHA1P、pCTC-HA1P和pMIL-CSA的抽提和酶切鉴定,将鉴定正确的重组质粒分别电转化至蜡样芽胞杆菌中,其中包含质粒pCSHA1P的重组菌株命名为BcA,同时含有两个质粒pCTC-HA1 P和pMIL-CSA的重组菌株命名为BcC。其中质粒pMIL-CSA携带有csaAB操纵元,在转化不含csaAB操纵元的重组质粒时要同时转化该质粒,以便给受体菌株提供csaAB操纵元。
2.检测外源抗原蛋白在重组菌株细胞表面的展示情况
用血凝和血凝抑制试验检测BcA和BcC这两个重组菌株的表面展示情况。结果,这两个重组菌株均表现出了预计的生物学活性,说明所构建的S-层融合蛋白基因在受体菌株中得到表达并展示到了细胞表面且具有活性。
3.检测重组菌株对雏鸡的免疫原性
将展示HA1蛋白的重组菌株BcA和BcC培养至芽胞形成期,用重组菌株的芽胞液通过口服途径免疫艾维因商品代肉雏鸡,用血凝抑制试验测定接种免疫鸡的血清抗体效价。结果,实验鸡未产生针对相应外源抗原的抗体,其机理有待于进一步研究。
S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avain influenza virus nucleoprotein (NP), hemagglutinin (HA1) or protein of Mycoplasma gallisepticum agglutinin (pMGA) on the cell surface of Bacillus thuringiensis. Although the experiment demonstrated Bacillus thuringiensis was safe and non-toxic for mice and chickens, but the role of probiotics, not related to reporting; Bacillus cereus, often used as probiotics, This study was used bacterial cell surface display technology to test the possibility of displaying heterologous antigens on the cell surface of bacillus cereus for the development of heat-stable, probiotic oral vaccine using B.cereus as a carrier.
1. Construction recombinant strains of Bacillus cereus
Bacillus thuringiensis recombinant strains CH and BCCH as the original beginning bacterial strains for plasmids extraction and endonuclease, being used for electroporation to Bacillus cereus. The resulting strains include plasmid PCSHAIP named BcA, include two plasmids: CTC- HA1P and pMIL-CSA named BcC. The plasmid pMIL-CSA harboring csaAB operon was co-transferred into B.cereus with the recombinant plasmids which did not harboring csaAB operon.
2. Assaying the display of heterologous antigens on the recombinant strains
Haemagglutination assay showed recombinant HAI proteins were displayed on the cell surface of respective recombinant strains. Hemagglutination inhibition assay showed recombinant HA1 proteins displayed on the cell surfaee of recombinants respectively were specific to standard positive serum of avian influenza virus, respectively.
3. Immunogenicity of recombinant strains to chiekens
The spores of recombinant strains BcA and BcC were prepared. The AV commercial broiler chickens were immunized by oral route with spores. The serum antibody titers of chiekens immunized with spores of BcA and BcC were assayed by haemagglutination inhibition test. The results showed that the recombinant strains did not elicite humoral response of chickens to respective heterologous antigens displayed on the cell surfaces and didn't exhibite immunogenieity. the mechanism need to be further studied.
1.蜡样芽胞杆菌重组菌株的构建
以苏云金芽胞杆菌CH和BCCH菌株为出发菌株,进行质粒pCSHA1P、pCTC-HA1P和pMIL-CSA的抽提和酶切鉴定,将鉴定正确的重组质粒分别电转化至蜡样芽胞杆菌中,其中包含质粒pCSHA1P的重组菌株命名为BcA,同时含有两个质粒pCTC-HA1 P和pMIL-CSA的重组菌株命名为BcC。其中质粒pMIL-CSA携带有csaAB操纵元,在转化不含csaAB操纵元的重组质粒时要同时转化该质粒,以便给受体菌株提供csaAB操纵元。
2.检测外源抗原蛋白在重组菌株细胞表面的展示情况
用血凝和血凝抑制试验检测BcA和BcC这两个重组菌株的表面展示情况。结果,这两个重组菌株均表现出了预计的生物学活性,说明所构建的S-层融合蛋白基因在受体菌株中得到表达并展示到了细胞表面且具有活性。
3.检测重组菌株对雏鸡的免疫原性
将展示HA1蛋白的重组菌株BcA和BcC培养至芽胞形成期,用重组菌株的芽胞液通过口服途径免疫艾维因商品代肉雏鸡,用血凝抑制试验测定接种免疫鸡的血清抗体效价。结果,实验鸡未产生针对相应外源抗原的抗体,其机理有待于进一步研究。
S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avain influenza virus nucleoprotein (NP), hemagglutinin (HA1) or protein of Mycoplasma gallisepticum agglutinin (pMGA) on the cell surface of Bacillus thuringiensis. Although the experiment demonstrated Bacillus thuringiensis was safe and non-toxic for mice and chickens, but the role of probiotics, not related to reporting; Bacillus cereus, often used as probiotics, This study was used bacterial cell surface display technology to test the possibility of displaying heterologous antigens on the cell surface of bacillus cereus for the development of heat-stable, probiotic oral vaccine using B.cereus as a carrier.
1. Construction recombinant strains of Bacillus cereus
Bacillus thuringiensis recombinant strains CH and BCCH as the original beginning bacterial strains for plasmids extraction and endonuclease, being used for electroporation to Bacillus cereus. The resulting strains include plasmid PCSHAIP named BcA, include two plasmids: CTC- HA1P and pMIL-CSA named BcC. The plasmid pMIL-CSA harboring csaAB operon was co-transferred into B.cereus with the recombinant plasmids which did not harboring csaAB operon.
2. Assaying the display of heterologous antigens on the recombinant strains
Haemagglutination assay showed recombinant HAI proteins were displayed on the cell surface of respective recombinant strains. Hemagglutination inhibition assay showed recombinant HA1 proteins displayed on the cell surfaee of recombinants respectively were specific to standard positive serum of avian influenza virus, respectively.
3. Immunogenicity of recombinant strains to chiekens
The spores of recombinant strains BcA and BcC were prepared. The AV commercial broiler chickens were immunized by oral route with spores. The serum antibody titers of chiekens immunized with spores of BcA and BcC were assayed by haemagglutination inhibition test. The results showed that the recombinant strains did not elicite humoral response of chickens to respective heterologous antigens displayed on the cell surfaces and didn't exhibite immunogenieity. the mechanism need to be further studied.
引文
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3.郭军,李立民.一株肠出血性大肠杆菌O157H7免疫奶牛实验即抗ETEC O157H7免疫乳的试制【J】.食品科学,2002,23(8):254-25
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8.雷迎峰,杨敬.抗HBV与HCV免疫乳的实验免疫研究[J].生物技术通讯,2005,16(5):632-634
9.李国强,庞广昌.抗哮喘免疫乳研究初报【J】.食品科学,2003,24(1):75—77
10.刘克琳,何明清.益生菌对鲤鱼免疫功能影响的研究[J].饲料工业,2000,21(6):24-25
11.刘梅.禽类病原体抗原在苏云金芽胞杆菌细胞表面的展示.【博士学位论文】.武汉:华中农业大学图书馆,2007
12.聂志武,王采先,胡志高.益生菌及其在猪鸡饲粮中的应用.山西农业科学,1998,26(1):85-89
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15.石英,周育森,郭彦,肖文君,于虹,吴吴,陈月,Phalguni Gup ta.应用梭状芽孢杆菌口服疫苗载体表达HⅣ p24蛋白的研究.科学技术与工程,2007,7(17):4265-4270
16.孙敬方.动物实验方法学.北京:人民卫生出版社,2001,356-367
17.唐丽杰,魏颖,葛俊伟,任晓峰,汪淼,徐毅刚,李一经.表达猪传染性胃肠炎病毒S蛋白重组乳酸乳球菌在模拟动物胃肠道内的稳定性检测.中国微生态学杂志,2007,5(19):401-403
18.王长青.疫苗食品悄然兴起【J].四川畜牧兽医,1999,10(26):48-49
19.王士长,何若刚.益生素(活菌和死菌)防治哺乳仔猪的下痢,黑龙江畜牧兽医,1998,(1):23-24
20.吴怀光,刘欣,孙明,喻子牛.利用S-层蛋白在细胞表面展示α-淀粉酶和金属硫蛋白.微生物学报,2004,44:655-662
21.吴占福.生物统计学.北京:科学出版社,2005,49-57
22.向贵友,谢后清,刘克琳.鸡微生物饲料添加剂对肉仔鸡促生长的研究.四川农业大学学报,1994,12(增刊):602-605
23.姚火春.兽医微生物学实验指导.北京:中国农业出版社,2002,46-47:105-107
24.张广民.芽孢杆菌益生素在肉仔鸡饲料中的应用研究.[博士学位论文】.武汉:华中农业大学图书馆,2003
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27.Anja Kotiranta,Kari Lounatmaa and Markus Haapasalo.Epidemiology and pathogenesis of Bacillus cereus infections.Microbes and Infection.2000,189-198
28.Baumeister W,Lembcke G.Structural features of archaebaecterial cell envelopes.J Bioenerg Biomembr,1992,24:567-575
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32.Fuller R M.Probiotics in man and animals.J APPL Bacteriol.1989,66(5):365-378
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2.陈思维,钟瑾,还连栋.微生物学报,2007,47(6):987—991.
3.郭军,李立民.一株肠出血性大肠杆菌O157H7免疫奶牛实验即抗ETEC O157H7免疫乳的试制【J】.食品科学,2002,23(8):254-25
4.郭兴华.益生菌基础与应用.北京:北京科学技术出版社,2002,260-261
5.何明清.8901、8902活菌制剂对猪、禽和鱼的抗病及增产试验初报.中国微生态学杂志.1989,1(1)封4
6.何明清.动物微生态学.北京:中国农业出版社,1994,79-87
7.李林,喻子牛.苏云金芽胞杆菌无质粒突变株BMB171的转化和表达性能.应用与环境生物学报,1999,5:395-399
8.雷迎峰,杨敬.抗HBV与HCV免疫乳的实验免疫研究[J].生物技术通讯,2005,16(5):632-634
9.李国强,庞广昌.抗哮喘免疫乳研究初报【J】.食品科学,2003,24(1):75—77
10.刘克琳,何明清.益生菌对鲤鱼免疫功能影响的研究[J].饲料工业,2000,21(6):24-25
11.刘梅.禽类病原体抗原在苏云金芽胞杆菌细胞表面的展示.【博士学位论文】.武汉:华中农业大学图书馆,2007
12.聂志武,王采先,胡志高.益生菌及其在猪鸡饲粮中的应用.山西农业科学,1998,26(1):85-89
13.潘国忠.现代胃肠病学.科学出版社,1994,877
14.潘康成,吕道俊,何明清.正常菌群对动物免疫功能的影响研究进展.饲料工业,1999,20(9):24-26
15.石英,周育森,郭彦,肖文君,于虹,吴吴,陈月,Phalguni Gup ta.应用梭状芽孢杆菌口服疫苗载体表达HⅣ p24蛋白的研究.科学技术与工程,2007,7(17):4265-4270
16.孙敬方.动物实验方法学.北京:人民卫生出版社,2001,356-367
17.唐丽杰,魏颖,葛俊伟,任晓峰,汪淼,徐毅刚,李一经.表达猪传染性胃肠炎病毒S蛋白重组乳酸乳球菌在模拟动物胃肠道内的稳定性检测.中国微生态学杂志,2007,5(19):401-403
18.王长青.疫苗食品悄然兴起【J].四川畜牧兽医,1999,10(26):48-49
19.王士长,何若刚.益生素(活菌和死菌)防治哺乳仔猪的下痢,黑龙江畜牧兽医,1998,(1):23-24
20.吴怀光,刘欣,孙明,喻子牛.利用S-层蛋白在细胞表面展示α-淀粉酶和金属硫蛋白.微生物学报,2004,44:655-662
21.吴占福.生物统计学.北京:科学出版社,2005,49-57
22.向贵友,谢后清,刘克琳.鸡微生物饲料添加剂对肉仔鸡促生长的研究.四川农业大学学报,1994,12(增刊):602-605
23.姚火春.兽医微生物学实验指导.北京:中国农业出版社,2002,46-47:105-107
24.张广民.芽孢杆菌益生素在肉仔鸡饲料中的应用研究.[博士学位论文】.武汉:华中农业大学图书馆,2003
25.张和平,吴红,郭军等.免疫乳抗炎作用研究[J].营养学报,2006,28(5):386-390
26.Alexopoulos C,I E Georgoulakis,A Tzivara,C S Kyriakis,A Govads,S C Kyriakis.Field evaluation of the effect of a probiotic-containing Bacillus licheniformis and Bacillus subtilis spores on the health status,performance and carcass quality of grower and finisher pigs.Vet.Med.A Physiol.Pathol.Clin.Med,2004,51:306-312
27.Anja Kotiranta,Kari Lounatmaa and Markus Haapasalo.Epidemiology and pathogenesis of Bacillus cereus infections.Microbes and Infection.2000,189-198
28.Baumeister W,Lembcke G.Structural features of archaebaecterial cell envelopes.J Bioenerg Biomembr,1992,24:567-575
29.Bouche F B,Marquet-Blouin E,Yanagi Y,et al.Neutralising immunogenicity of a polyepitope antigen expressed in a transgenic food plant:a novel antigen to protect against measles[J].Vaccine,2003,21(17-18):2065-2072
30.Daniell H,Lee S B,Panchal T,et al.Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts[J].J Mol Biol,2001,311(5):1001-1009
31.Egelseer E M,Scliocher I,Sara M,Sleytr U B.The S-layer from Bacillus stearothermopliilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.J Bacteriol,1995,177:1444-1451
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