猪肺炎支原体p97R1区基因与LTB基因的重组表达及重组蛋白的免疫原性研究
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摘要
猪支原体肺炎(Swine mycoplasmal pneumomia SWP)又称猪喘气病或猪地方性流行性肺炎,是由猪肺炎支原体(Mycoplasma hyopneumoniae Mhp)引起的一种接触性慢性呼吸道疾病,普遍存在于世界各地,是造成养猪业经济损失最重要的疾病之一。控制该病主要依靠弱毒疫苗和灭活疫苗,但是存在成本高效果低的缺点。本课题重点针对猪肺炎支原体P97蛋白的主要抗原决定区R1区的表达和猪肺炎支原体黏膜免疫特性开展研究,将具有黏膜免疫佐剂作用的不耐热肠毒素B亚单位(The B subunit of the heat-labile enterotoxin of Escherichia coli LTB)与具有气管纤毛结合表位的p97粘附因子的重复序列R1区进行重组,扩增得到重组序列LTBR1,构建原核表达质粒pET28a(+)-rLTBRl,为了对比免疫效果同时构建了原核表达质粒pET28a(+)-rR1。分别表达获得了两个与六个组氨酸融合表达的重组蛋白rLTBR1和rR1。采用亲和层析的方法对目的蛋白进行纯化。LTB的黏膜免疫作用需要检测该蛋白与受体神经节苷酯(ganglioside GM1)结合的亲和性。通过ELISA方法检测结果证明所表达的重组蛋白rLTBR1具有与受体GM1结合的高亲和性。通过SDS-PAGE和Western Blot试验对目的蛋白进行检测,鉴定,结果显示:表达的目的蛋白为特异性目的蛋白。
为了研究rLTBR1蛋白在体内的免疫原性,将rLTBR1蛋白和对照蛋白rR1蛋白分别通过皮下和鼻内接种两个免疫途径免疫小鼠。通过ELISA试验检测免疫鼠血清中猪肺炎支原体p97粘附因子的抗体水平,结果证明:无论皮下接种还是鼻内接种rLTBR1蛋白,免疫鼠均可产生特异性抗猪肺炎支原体p97蛋白的抗体;皮下单独接种rR1蛋白同样可以产生特异性的抗rR1蛋白的抗体,然而鼻内单独接种rR1没有检测到特异性的抗体。通过ELISA方法检测黏膜抗体水平和脾细胞上清液中IFN-γ水平,鼻内接种rLTBR1蛋白产生了比较高的黏膜免疫抗体IgA,并且在鼻内免疫rLTBR1蛋白的小鼠脾细胞中诱导产生了IFN-γ,而皮下免疫rLTBR1蛋白组和鼻内,皮下免疫rR1蛋白组没有诱导机体产生有效的黏膜免疫抗体和细胞免疫。说明LTB利R1融合表达后可以更好的刺激小鼠的黏膜免疫和细胞免疫反应。本研究结果显示:LTB增强了猪肺炎支原体p97R1基因蛋白的黏膜免疫和细胞免疫作用。为猪肺炎支原体基因重组亚单位疫苗的研究奠定了重要的基础。
Swine mycoplasmal pneumonia(SMP),caused by Mycoplasma hyopneumoniae(Mhp),is one of the most important respiratory diseases in swine breeding.The infection rate is high,but the mortality is low.The disease would lead to significant economical losses in pig industry.The commonly used vaccines to control this disease are inactivated whole cells and attenuated live vaccine, whose bio-products costs are high but the efficiency are limited.The objective of this study was to develop a recombinant subunit vaccine rLTBR1 containing R1 region of P97 adhesin of M. hyopneumoniae and B subunit of the heat-labile enterotoxin of Escherichia coli(LTB).Inserted the sequence into the express vector pET-28a(+) and induced two recombinant proteins rLTBR1 and rR1.The protein rR1 was expressed in the form of solubility and the recombinant protein rLTBR1 was inclusion body.The recombinant protein was purified through affinity chromatography method.The purified rLTBR1 was also tested for its biological activity by studies on its ability to bind to ganglioside acceptor in ELISA experiments.The result indicates that rLTBR1 revealed GM1 ganglioside-binding capacity.The antigenicity was evaluated by the methods of SDS-PAGE and Western Blot.Both of the results were positive.
The immunogenicity of rLTBR1 was detected also by BALB/c mice.The antibody level was evaluated through ELISA test.The results suggested that inoculated rLTBR1 through the intranasal(IN) or intramuscular(IM) route both produced specified anti-Mhp systemetic and mucosal antibody (sIgA).The group inoculated R1 by IM route also produced specified anti-Mhp systemetic antibody, but through the IN route didn't induce specified and mucosal antibody.Group administrated rLTBR1 through the IN route also induced IFN-γsecretion by lymphocytes.Howerver,the other groups didn't induced IFN-γ,compared to the negative group.In conclusion,LTB can enhance the mucosal and cellular immunity level through the IN route.Certainly,the immune responses of mice should not be extrapolated,so the potential of the rLTBR1 for the control of SMP requires further studies in pigs.This study suggested that rLTBR1 vaccine may establish a new strategy for preventing infection by Mycoplasma hyopneumoniae.
为了研究rLTBR1蛋白在体内的免疫原性,将rLTBR1蛋白和对照蛋白rR1蛋白分别通过皮下和鼻内接种两个免疫途径免疫小鼠。通过ELISA试验检测免疫鼠血清中猪肺炎支原体p97粘附因子的抗体水平,结果证明:无论皮下接种还是鼻内接种rLTBR1蛋白,免疫鼠均可产生特异性抗猪肺炎支原体p97蛋白的抗体;皮下单独接种rR1蛋白同样可以产生特异性的抗rR1蛋白的抗体,然而鼻内单独接种rR1没有检测到特异性的抗体。通过ELISA方法检测黏膜抗体水平和脾细胞上清液中IFN-γ水平,鼻内接种rLTBR1蛋白产生了比较高的黏膜免疫抗体IgA,并且在鼻内免疫rLTBR1蛋白的小鼠脾细胞中诱导产生了IFN-γ,而皮下免疫rLTBR1蛋白组和鼻内,皮下免疫rR1蛋白组没有诱导机体产生有效的黏膜免疫抗体和细胞免疫。说明LTB利R1融合表达后可以更好的刺激小鼠的黏膜免疫和细胞免疫反应。本研究结果显示:LTB增强了猪肺炎支原体p97R1基因蛋白的黏膜免疫和细胞免疫作用。为猪肺炎支原体基因重组亚单位疫苗的研究奠定了重要的基础。
Swine mycoplasmal pneumonia(SMP),caused by Mycoplasma hyopneumoniae(Mhp),is one of the most important respiratory diseases in swine breeding.The infection rate is high,but the mortality is low.The disease would lead to significant economical losses in pig industry.The commonly used vaccines to control this disease are inactivated whole cells and attenuated live vaccine, whose bio-products costs are high but the efficiency are limited.The objective of this study was to develop a recombinant subunit vaccine rLTBR1 containing R1 region of P97 adhesin of M. hyopneumoniae and B subunit of the heat-labile enterotoxin of Escherichia coli(LTB).Inserted the sequence into the express vector pET-28a(+) and induced two recombinant proteins rLTBR1 and rR1.The protein rR1 was expressed in the form of solubility and the recombinant protein rLTBR1 was inclusion body.The recombinant protein was purified through affinity chromatography method.The purified rLTBR1 was also tested for its biological activity by studies on its ability to bind to ganglioside acceptor in ELISA experiments.The result indicates that rLTBR1 revealed GM1 ganglioside-binding capacity.The antigenicity was evaluated by the methods of SDS-PAGE and Western Blot.Both of the results were positive.
The immunogenicity of rLTBR1 was detected also by BALB/c mice.The antibody level was evaluated through ELISA test.The results suggested that inoculated rLTBR1 through the intranasal(IN) or intramuscular(IM) route both produced specified anti-Mhp systemetic and mucosal antibody (sIgA).The group inoculated R1 by IM route also produced specified anti-Mhp systemetic antibody, but through the IN route didn't induce specified and mucosal antibody.Group administrated rLTBR1 through the IN route also induced IFN-γsecretion by lymphocytes.Howerver,the other groups didn't induced IFN-γ,compared to the negative group.In conclusion,LTB can enhance the mucosal and cellular immunity level through the IN route.Certainly,the immune responses of mice should not be extrapolated,so the potential of the rLTBR1 for the control of SMP requires further studies in pigs.This study suggested that rLTBR1 vaccine may establish a new strategy for preventing infection by Mycoplasma hyopneumoniae.
引文
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