布鲁氏菌新型疫苗的构建及其免疫原性研究
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摘要
布鲁氏菌病（Brucellosis）是由细胞内寄生的革兰氏阴性小杆菌布鲁氏菌引起的一种人畜共患病。它以发热，流产为主要症状，严重威胁着人畜健康。全世界每年出现500,000例人布鲁氏菌病，据美国农业部估计，仅在拉丁美洲布鲁氏菌病每年造成6亿美元的损失，而在九十年代的美国平均每年损失为1.5亿美元[5]。
控制布鲁氏菌病的传播是最终消灭它的关键，除了大规模地宰杀感染动物以外，群体疫苗接种是十分重要的手段，目前主要用于牛羊布鲁氏菌病预防的是牛型19号疫苗以及羊型Rev.弱毒苗，但是免疫保护率只有65％～75％。而利用基因工程制备的疫苗有很多优点:1.制备方便快捷，造价低；2. 安全稳定，靶向性明确，用量易于控制；3.可以用多种方式进行免疫。
本研究所选的L7/L12核蛋白为公认的T细胞保护性抗原，它具有高度的保守性，研究发现，重组L7/L12蛋白能特异性地刺激感染动物的单核细胞，并上调INF-γ的转录和表达，从而起到保护作用。以其为基础构建的重组蛋白苗和DNA疫苗都表现了较好的免疫保护效果；而OMP16外膜脂蛋白存在于所有6种布鲁氏菌中，也有一定的免疫保护作用；17.3kDa蛋白与布鲁氏菌18kDa的外膜蛋白（优秀的保护性抗原）的AA序列相似，因此，我们通过实验来验证17.3kDa蛋白是否具有保护作用。对翻译起始因子3蛋白进行抗原预测表明其具有免疫原性。本课题以4种蛋白分子为研究对象，分别构建了真核，原核表达质粒并表达了这4种蛋白，免疫动物后，研究其诱导产生的细胞免疫和体液免疫应答。
从布鲁氏菌中提取其全基因组DNA，利用PCR技术扩增出4种蛋白的编码基因，测序正确后分别克隆至原核表达载体PET32a(+)上，经诱导表达出相应的4种重组蛋白，WB鉴定后纯化待用；同时，4种编码基因隆至真核表达载体PCDNA3.1(+)上，将重组质粒转染COS-7细胞后经WB证实重组质粒构建成功。将重组蛋白混合弗氏佐剂免疫动物，同时DNA疫苗以PUMVC1-mGM-CSF重组质粒为佐剂（各100μg/只）肌肉免疫动物；每15d免疫一次，加强免疫三次后检测免疫应答指标，并进行组间比较。ELISA法检测到较高滴度的特异性抗体，WB也证实特异性抗体的产生；抗体亚型分类表明重组蛋白组的特异性IgG1/ IgG2a大于1，而DNA疫苗免疫组却小于1，说明重组蛋白疫苗诱导产生了Th2型为主免疫应答，DNA疫苗产生了Th1型为主的免疫应答。MTS法测小鼠T淋巴细胞增殖试验发现DNA组小鼠脾脏T淋巴细胞对ConA诱导的增殖反应增强，而重组蛋白组却无显著变化。进一步通过ELISPOT方法检测特异性活化的可
分泌IFN-γ的CD8+T细胞数，DNA免疫组的斑点数显著多于对照组（P＜0.05）；说明诱导了特异性CD8+T淋巴细胞应答。流式细胞仪所得到的淋巴细胞亚群分类也得到了同样的结果。
本课题利用真核表达载体PCDNA3.1成功构建了含４种布鲁氏菌编码蛋白的重组质粒，并利用原核表达载体表达了相应的重组蛋白。通过ELISA﹑WB﹑ELISPOT及淋巴细胞亚群分类检测疫苗在免疫小鼠体内诱导产生的体液及细胞免疫指标。 本研究成功构建了布鲁氏菌基因工程疫苗，在国内尚属首次，利用流式细胞仪的分型技术和ELISPOT法评价布鲁氏菌疫苗的细胞免疫指标将促进我国兽用疫苗及相关产品的实验室质控标准方法的建立和发展，为他们的临床应用奠定基础。 进一步的攻毒实验尚在进行中。
Brucellosis is a zoonotic disease caused by a small intracellular gram-negative bacterium which is pathogenic for humans as well as for many species of animals. This infection induces abortions, in livestock animals as well as undulant fever and osteomyelitis in humans leading to severe economic losses. For example, United States Department of Agriculture sources estimated that in the 1990s, on average about US$150 million were spent each year in the US.
There are six species Brucella. Abortus、 B. suis、 B. melitensis 、B. canis、 B.ovis and B.neotomae. Control of the spread of the disease in domesticated animals is the use of vaccines for whole-herd vaccination programs. But the killed vaccines and live vaccines have been limited because of their unacceptable production costs, poor protection and serological problem which interfere with the diagnosis of field infection. And the recombinant subunit vaccine has many advantages: first, it’s easy to make and does not cost much money; second, it’s safe and easy to master the quantity; finally, it can be used through all kinds of delivery system. DNA vaccine also attracted the eyes of many scientists, after injection into the muscles, it can express the protein for a long time, and the protein degrades into peptides and associates with major histocompatibility complex (MHC) class I molecules. These peptide/MHC complexes stimulate cytolytic T cells, induce cell mediated immune response(CMI) to control the infection.
L7/L12 ribosome protein is the best known T-cell antigen .Studies have confirmed that recombinant L7/L12 protein could specifically stimulate mononulclear cells from B.abortus reactive animals and upregulate the transcription and expression of IFN-γand L7/L12 DNA vaccine also engendered significant protection.The OMP16 outer membrane lipoprotein exists in all the six species of Brucella. It has been proved to be a protective antigen too; The sequence of 17.3kDa protein shows homology with that of lumazine synthase, indicates that the two protein may share the same characters, and the lumazine synthase is an enzyme with lumazine synthase activity which can induce T-cell
activity. Because of this reason, the 17.3kDa protein should be a protective protein. Using B epitope soft assay suggests that the initialfactor 3 has immungenicity, although we can not find anti-IF3 antibody in the sera of Brucella infected animal, probably the protein can elicit cytokines and co-react with other antibody.
We selected these 4 proteins, constructed recombinant eukaryotic protein and expressed the corresponding recombinant protein at the same time, immunized animals and evaluated their capability in inducing humoral immunity and celluar immunity. The whole genome was isolated from the B.melitensis, and amplicated the corresponding gene using PCR techniques. The genes were inserted into pET32a(+) prokaryotic expression vector and then expressed the proteins;genes were cloned into pCDNA3.1 too, transfected the recombinant plasmids and identified proteins expression using Western blot analyses and immunochemistry. Injection of these recombinant plasmid DNA into BALB/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene and recombinant proteins mixed with Freund’s adjuvant via an intraperitoneal injection elicited both humoral and cellular immune responses. Experimental animals exhibited specific antibodies that were detectable 15 days after the first injection and increased steadily, the IgG2a-specific antibody titers of DNA groups were higher than IgG1-specific antibody titers, and the recombinant protein groups were contrary. The DNA vaccines elicited a T-cell-proliferative responses as well as IFN-γ production upon restimulation with the specific antigens using ELISPOT assay; the ratio of CD4+/CD8+ were lower than that of the control mice. Furthermore, immunoglobulin G isotype analysis indicated that the ratio of IgG1 and IgG2a in protein vaccinated group is higher than 1 and that of DNA vaccine groups lower than 1,suggesting the i
控制布鲁氏菌病的传播是最终消灭它的关键，除了大规模地宰杀感染动物以外，群体疫苗接种是十分重要的手段，目前主要用于牛羊布鲁氏菌病预防的是牛型19号疫苗以及羊型Rev.弱毒苗，但是免疫保护率只有65％～75％。而利用基因工程制备的疫苗有很多优点:1.制备方便快捷，造价低；2. 安全稳定，靶向性明确，用量易于控制；3.可以用多种方式进行免疫。
本研究所选的L7/L12核蛋白为公认的T细胞保护性抗原，它具有高度的保守性，研究发现，重组L7/L12蛋白能特异性地刺激感染动物的单核细胞，并上调INF-γ的转录和表达，从而起到保护作用。以其为基础构建的重组蛋白苗和DNA疫苗都表现了较好的免疫保护效果；而OMP16外膜脂蛋白存在于所有6种布鲁氏菌中，也有一定的免疫保护作用；17.3kDa蛋白与布鲁氏菌18kDa的外膜蛋白（优秀的保护性抗原）的AA序列相似，因此，我们通过实验来验证17.3kDa蛋白是否具有保护作用。对翻译起始因子3蛋白进行抗原预测表明其具有免疫原性。本课题以4种蛋白分子为研究对象，分别构建了真核，原核表达质粒并表达了这4种蛋白，免疫动物后，研究其诱导产生的细胞免疫和体液免疫应答。
从布鲁氏菌中提取其全基因组DNA，利用PCR技术扩增出4种蛋白的编码基因，测序正确后分别克隆至原核表达载体PET32a(+)上，经诱导表达出相应的4种重组蛋白，WB鉴定后纯化待用；同时，4种编码基因隆至真核表达载体PCDNA3.1(+)上，将重组质粒转染COS-7细胞后经WB证实重组质粒构建成功。将重组蛋白混合弗氏佐剂免疫动物，同时DNA疫苗以PUMVC1-mGM-CSF重组质粒为佐剂（各100μg/只）肌肉免疫动物；每15d免疫一次，加强免疫三次后检测免疫应答指标，并进行组间比较。ELISA法检测到较高滴度的特异性抗体，WB也证实特异性抗体的产生；抗体亚型分类表明重组蛋白组的特异性IgG1/ IgG2a大于1，而DNA疫苗免疫组却小于1，说明重组蛋白疫苗诱导产生了Th2型为主免疫应答，DNA疫苗产生了Th1型为主的免疫应答。MTS法测小鼠T淋巴细胞增殖试验发现DNA组小鼠脾脏T淋巴细胞对ConA诱导的增殖反应增强，而重组蛋白组却无显著变化。进一步通过ELISPOT方法检测特异性活化的可
分泌IFN-γ的CD8+T细胞数，DNA免疫组的斑点数显著多于对照组（P＜0.05）；说明诱导了特异性CD8+T淋巴细胞应答。流式细胞仪所得到的淋巴细胞亚群分类也得到了同样的结果。
本课题利用真核表达载体PCDNA3.1成功构建了含４种布鲁氏菌编码蛋白的重组质粒，并利用原核表达载体表达了相应的重组蛋白。通过ELISA﹑WB﹑ELISPOT及淋巴细胞亚群分类检测疫苗在免疫小鼠体内诱导产生的体液及细胞免疫指标。 本研究成功构建了布鲁氏菌基因工程疫苗，在国内尚属首次，利用流式细胞仪的分型技术和ELISPOT法评价布鲁氏菌疫苗的细胞免疫指标将促进我国兽用疫苗及相关产品的实验室质控标准方法的建立和发展，为他们的临床应用奠定基础。 进一步的攻毒实验尚在进行中。
Brucellosis is a zoonotic disease caused by a small intracellular gram-negative bacterium which is pathogenic for humans as well as for many species of animals. This infection induces abortions, in livestock animals as well as undulant fever and osteomyelitis in humans leading to severe economic losses. For example, United States Department of Agriculture sources estimated that in the 1990s, on average about US$150 million were spent each year in the US.
There are six species Brucella. Abortus、 B. suis、 B. melitensis 、B. canis、 B.ovis and B.neotomae. Control of the spread of the disease in domesticated animals is the use of vaccines for whole-herd vaccination programs. But the killed vaccines and live vaccines have been limited because of their unacceptable production costs, poor protection and serological problem which interfere with the diagnosis of field infection. And the recombinant subunit vaccine has many advantages: first, it’s easy to make and does not cost much money; second, it’s safe and easy to master the quantity; finally, it can be used through all kinds of delivery system. DNA vaccine also attracted the eyes of many scientists, after injection into the muscles, it can express the protein for a long time, and the protein degrades into peptides and associates with major histocompatibility complex (MHC) class I molecules. These peptide/MHC complexes stimulate cytolytic T cells, induce cell mediated immune response(CMI) to control the infection.
L7/L12 ribosome protein is the best known T-cell antigen .Studies have confirmed that recombinant L7/L12 protein could specifically stimulate mononulclear cells from B.abortus reactive animals and upregulate the transcription and expression of IFN-γand L7/L12 DNA vaccine also engendered significant protection.The OMP16 outer membrane lipoprotein exists in all the six species of Brucella. It has been proved to be a protective antigen too; The sequence of 17.3kDa protein shows homology with that of lumazine synthase, indicates that the two protein may share the same characters, and the lumazine synthase is an enzyme with lumazine synthase activity which can induce T-cell
activity. Because of this reason, the 17.3kDa protein should be a protective protein. Using B epitope soft assay suggests that the initialfactor 3 has immungenicity, although we can not find anti-IF3 antibody in the sera of Brucella infected animal, probably the protein can elicit cytokines and co-react with other antibody.
We selected these 4 proteins, constructed recombinant eukaryotic protein and expressed the corresponding recombinant protein at the same time, immunized animals and evaluated their capability in inducing humoral immunity and celluar immunity. The whole genome was isolated from the B.melitensis, and amplicated the corresponding gene using PCR techniques. The genes were inserted into pET32a(+) prokaryotic expression vector and then expressed the proteins;genes were cloned into pCDNA3.1 too, transfected the recombinant plasmids and identified proteins expression using Western blot analyses and immunochemistry. Injection of these recombinant plasmid DNA into BALB/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene and recombinant proteins mixed with Freund’s adjuvant via an intraperitoneal injection elicited both humoral and cellular immune responses. Experimental animals exhibited specific antibodies that were detectable 15 days after the first injection and increased steadily, the IgG2a-specific antibody titers of DNA groups were higher than IgG1-specific antibody titers, and the recombinant protein groups were contrary. The DNA vaccines elicited a T-cell-proliferative responses as well as IFN-γ production upon restimulation with the specific antigens using ELISPOT assay; the ratio of CD4+/CD8+ were lower than that of the control mice. Furthermore, immunoglobulin G isotype analysis indicated that the ratio of IgG1 and IgG2a in protein vaccinated group is higher than 1 and that of DNA vaccine groups lower than 1,suggesting the i
引文
[103] 金冬雁，黎孟枫译（1992）:分子克隆实验指南[M]，北京，科学出版社，第二版。
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Michaux-Charachon S , Bourg G,.Jumas-Bilak Eet al. Genome structure and phylogeny in the genus Brucella[J]. J. Bacteriol. 1997. 179:3244-3249.
Montaraz J A , Winter A J, Hunter D M, et al. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies[J]. Infect. Immun. 1986.51:961-963.
Schurig G G, Roop R M, Bagchi J T, et al. Biological properties of RB51; a stable rough strain of Brucella abortus[J]. Vet. Microbiol. 1991.28:171-188.
Hoffmann E M., and Houle J J. Contradictory roles for antibody and complement in the interaction of Brucella abortus with its host[J]. Crit. Rev. Microbiol. 1995.21:153-163.
Ko J , Gendron-Fitzpatrick A., and Splitter G A. Susceptibility of interferon regulatory factor (IRF-1) and interferon consensus sequence binding protein (ICSBP) deficient mice to brucellosis[J]. J. Immunol. 2002.168:2433-2440.
Zhan Y , Kelso A, and Cheers C. Cytokine production in the murine response to Brucella infection or immunization with antigenic extracts[J]. Immunology 1993. 80:458-464.
Zhan Y , and Cheers C. Endogenous gamma interferon mediates resistance to Brucella abortus infection[J]. Infect. Immun. 1993. 61:4899-4901.
Zhan Y , Z Liu, and Cheers C. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms[J]. Infect. Immun. 1996. 64:2782-2786.
Zhan Y F, Stanley E R, and Cheers C. Prophylaxis or treatment of experimental brucellosis with
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Doyle A G , Halliday W J , Barnett C J ,et al. Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice. Infect. Immun. 1992. 60:1465-1472.
Nicoletti P. Vaccination against Brucella[J]. Advances in Biotechnology Processes,1990 ,13:147-168.
Buck J M , Studies of vaccination during calfhood to prevent bovine infectious abortion[J].J.Agric.Res. 1930.41,667.
Mingle C K ,Manthei C A ,Jasmin A M , The stability of reduced virulence exhibited by Brucella abortus[J]. J.Am. Vet.MED.Assoc. 1941.99,203.
Horwell F D, Van Drimmelen G G, Brucella menlitensis strain Rev.1 as a vaccine in cattle[C].S.Afr.Vet.Med.Assoc. 1971.42,233-235.
Garcia-Carrillo C , Comparison of Brucella menlitensis Rev.1 and B abortus strain 19 as a vaccine against bucellosis in cattle[J]. Zentralbl. Verteraermedicine 27,131-138.
Blasco J M , Marin C ,Jimenez de Bagues et al. Efficacy of Brucella suis strains.Vaccine 1980.11(3),1291-1294.
Shang D Q , An advance about vaccne products of brucellosis[C].Endem. Dis .Bull. China1,2000 .5(1)70-73 .
Verger J M , Grayon M ,Zundel E ,et al. Compariso of the efficacy of Brucella suis strain 2 and Brucella menlitensis Rev.1 live vaccines against a Brucella menlitensis experimental infection in pregnant ewes [J]. Vaccine 1995.13(2) ,191-196.
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McEwen a d , Experiments on contagious abortion. The immunity of cattle inoculated with vaccines of graded virulence[J]. Vet.Rec. 1940.52.815.
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Lord V R , Schurig G G , Cherwonogrodzky J W , et al. Field study of vaccination of cattle with Brucella abortus strain RB51 and 19 under low disease prevalence[J]. Am.J.Vet.Res. 1998a.59,1016-1020.
Jimenz de Bagues, Barberan M P, Marin C M , et al. The Brucella abortus RB51 vaccine does not confer protection against Brucella ovis in rams[J]. Vaccine 1995.13,301-304.
Vemulaplli R., He Y, Buccolo L S , et al. Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attention[J]. Infect. Immun. 2000a.68,3927-3932.
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Schurig G G, Roop R M, Bagchi J T, et al. Biological properties of RB51; a stable rough strain of Brucella abortus[J]. Vet. Microbiol. 1991.28:171-188.
Hoffmann E M., and Houle J J. Contradictory roles for antibody and complement in the interaction of Brucella abortus with its host[J]. Crit. Rev. Microbiol. 1995.21:153-163.
Ko J , Gendron-Fitzpatrick A., and Splitter G A. Susceptibility of interferon regulatory factor (IRF-1) and interferon consensus sequence binding protein (ICSBP) deficient mice to brucellosis[J]. J. Immunol. 2002.168:2433-2440.
Zhan Y , Kelso A, and Cheers C. Cytokine production in the murine response to Brucella infection or immunization with antigenic extracts[J]. Immunology 1993. 80:458-464.
Zhan Y , and Cheers C. Endogenous gamma interferon mediates resistance to Brucella abortus infection[J]. Infect. Immun. 1993. 61:4899-4901.
Zhan Y , Z Liu, and Cheers C. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms[J]. Infect. Immun. 1996. 64:2782-2786.
Zhan Y F, Stanley E R, and Cheers C. Prophylaxis or treatment of experimental brucellosis with
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Doyle A G , Halliday W J , Barnett C J ,et al. Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice. Infect. Immun. 1992. 60:1465-1472.
Nicoletti P. Vaccination against Brucella[J]. Advances in Biotechnology Processes,1990 ,13:147-168.
Buck J M , Studies of vaccination during calfhood to prevent bovine infectious abortion[J].J.Agric.Res. 1930.41,667.
Mingle C K ,Manthei C A ,Jasmin A M , The stability of reduced virulence exhibited by Brucella abortus[J]. J.Am. Vet.MED.Assoc. 1941.99,203.
Horwell F D, Van Drimmelen G G, Brucella menlitensis strain Rev.1 as a vaccine in cattle[C].S.Afr.Vet.Med.Assoc. 1971.42,233-235.
Garcia-Carrillo C , Comparison of Brucella menlitensis Rev.1 and B abortus strain 19 as a vaccine against bucellosis in cattle[J]. Zentralbl. Verteraermedicine 27,131-138.
Blasco J M , Marin C ,Jimenez de Bagues et al. Efficacy of Brucella suis strains.Vaccine 1980.11(3),1291-1294.
Shang D Q , An advance about vaccne products of brucellosis[C].Endem. Dis .Bull. China1,2000 .5(1)70-73 .
Verger J M , Grayon M ,Zundel E ,et al. Compariso of the efficacy of Brucella suis strain 2 and Brucella menlitensis Rev.1 live vaccines against a Brucella menlitensis experimental infection in pregnant ewes [J]. Vaccine 1995.13(2) ,191-196.
WHO Report of WHO Working Group Meeting on Oral/Conjunctiva Brucella suis Strain 2 Vaccine[C]. Rome,2-7 October 1991,pp.2-4 .
Shumilov K V , Kasyanov A N , Romakhov V A ,et al. Properities of vaccine strain 104-M of Brucella abortus[C]. Trudy Vses. Inst. Eksp. 1984.Vet.61,10; Abstract 5815 in Vet. Bull.
Buck J M, Studies of vaccination during calfhood to prevent bovine infectious abortion [J].J.Agric.Res. 1930.41,667.
McEwen a d , Experiments on contagious abortion. The immunity of cattle inoculated with vaccines of graded virulence[J]. Vet.Rec. 1940.52.815.
Meyer M E , Gibbons R W , Results of trial use of H-38 vaccine for immunizing beef heifers against experimental exposure to Brucella abortus, strain 2308[C]. In:Proceedings of the 82nd Annual Meting of US Animal Health Association, 1978.vol. 82,pp106-119.
Plommet M , Renoux G, Philppon A ,et al. Brucellose experimental. I.Comparision del’dfficacite des vaccines B19 and H38 vaccine[C].Ann.Rech.Vet. 1970.1,189-201.
Renoux G, Nicholas J A, Imbert R, et al. Calf vaccination against Brucella abortus:comparison of
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Kolar J , Brucellosis in eastern European countries. In:Young, E.J.,Corbel,M.J.,(Eds), Brucellosis:Clinical and Laboratory Aspects.CRC Press[C], Boca Raton, 1989.Chapter 12,pp.163-172.
Schurig G G ,Roop R M ,Bagchi T,et al. Biological properities of RB51; a stable rough strain of Brucella abortus[J]. Vet. Microbiol. 1991.28,171-188.
Cheville N F ,Jensen A E ,Halling S M , et al. Immunology: bacterial survival, lymph node changes, and immunologic response of cattle vaccinated with standard and mutant strains of Brucella abortus[J] .Am. J.Vet. Res1992.53,1881-1888.
Lord V R , Schurig G G , Cherwonogrodzky J W , et al. Field study of vaccination of cattle with Brucella abortus strain RB51 and 19 under low disease prevalence[J]. Am.J.Vet.Res. 1998a.59,1016-1020.
Jimenz de Bagues, Barberan M P, Marin C M , et al. The Brucella abortus RB51 vaccine does not confer protection against Brucella ovis in rams[J]. Vaccine 1995.13,301-304.
Vemulaplli R., He Y, Buccolo L S , et al. Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attention[J]. Infect. Immun. 2000a.68,3927-3932.
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