牛分枝杆菌主要保护性抗原基因的克隆、表达及免疫研究
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摘要
牛结核病(Bovine tuberculosis)主要是由牛分枝杆菌(Mycobacterium bovis)引起的一种人兽共患传染病,该病的流行与传播不仅严重地影响着畜牧业的持续发展,而且也严重地威胁和危害着人类的生命与健康,被国际兽疫局(OIE)列为B类传染病。人结核病的10%以上是由牛分枝杆菌引起的。近年来,人结核病在世界范围内大幅回升,每年死亡人数高达300万,是其它传染病死亡人数之和,当属传染病之魁首。由于牛结核病的存在,致使人结核病最终无法根除。近年来,牛结核病在全球范围内也呈上升趋势,尤其是在广大的发展中国家流行严重,我国牛结核的阳性检出率为10%左右。在牛结核病的预防上,目前唯一可用的疫苗就是卡介苗(BCG),但是接种BCG后免疫效果不确定,同时还干扰牛PPD的变态反应检测,无法区别是自然感染还是人工免疫,给日常检疫工作带来了相当的困难。因此研发牛结核病的新型诊断和预防制剂已成为控制牛结核病的研究热点。
结核分枝杆菌是细胞内寄生菌,机体抵抗结核分枝杆菌的感染主要依靠细胞免疫。研究发现,结核分枝杆菌培养滤液中存在的大量分泌蛋白能有效地诱发对结核分枝杆菌的抵抗性,产生免疫保护作用,因此分泌蛋白是保护性抗原。DNA疫苗能有效地刺激机体产生持久的体液免疫和细胞免疫应答,而且DNA疫苗在细胞内表达的蛋白抗原通过MHC I类分子呈递抗原,激活CTL及杀灭靶细胞是DNA疫苗发挥保护作用的最主要机制之一,这正是抗结核感染所必需的。另有研究指出,DNA疫苗免疫后,不干扰结核病的变态反应检测。为了研制牛结核病敏感、特异的诊断试剂和新型、高效的预防制剂,尤其是DNA疫苗,本研究进行了以下几方面的工作:
1、牛分枝杆菌主要保护性抗原基因的克隆筛选了M. bovis Ag85A、Ag85B、MPB51、MPB63、MPB70、MPB83和ESAT-6七种分泌蛋白的保护性抗原基因。以M. bovis Vallee111染色体DNA为模板,以Ag85A、Ag85B、MPB51、MPB63、MPB70、MPB83和ESAT-6成熟蛋白基因特异性引物进行PCR扩增,分别扩增出888bp、858bp、801bp、390bp、492bp、603bp和288bp的DNA片段。通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,以α-互补法、质粒大小鉴定、酶切鉴定、PCR鉴定及序列分析鉴定重组克隆,成功地构建出克隆质粒pGEM-T-85A、pGEM-T-85B、pGEM-T-51、pGEM-T-63、pGEM-T-70、pGEM-T-83、pGEM-T-ESAT-6。序列测定及同源性分析表明,所获得的M. boris Vallee111成熟蛋白基因与M. bovis中其它菌株相应基因具有高度的同源性。
2、主要保护性抗原基因的原核表达 为了获得M. bovis七个保护性抗原基因的表达产
Mycobacterium bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings. The prevalence and circulation of this disease not only influence the development of stock raising severely, but also menace the health of human beings, so it was recognized as B infection by OIE. More than 10% of human tuberculosis is caused by M. bovis. In recent years, the incidence of human tuberculosis has raised in large extents, the death toll has reached 3 millions every year, which is the accumulation of death toll of other infections, so it is the ringleader among all the infections. Because of the prevalence of bovine tuberculosis, human tuberculosis can not be eradicated. Recently, the incidence of bovine tuberculosis has also raised largely, especially in the developing countries. In our country, the positive incidence is about 10%. As regards as the prevention of bovine tuberculosis, BCG is the only vaccine that can be used, but after the incubation of BCG, the immunized effects is not obvious, but it will disturb the allergy test of PPD, so we can not differentiate the natural infection and artificial immunization. Moreover, it made the routine quarantine more difficult. So the new vaccine for the diagnosis and prevention of the bovine tuberculosis has been the hot spot.M. bovis is a kind of parasitic bacterium in the cell. In the immunity of anti-mycobacterium, cell mediated immunity exerts leading effect. Some studies indicated that the secreted proteins of living bacterium exerted the mainly protective immunity, so the secreted proteins were protective antigens. DNA vaccine can induce permanent humoral and cell immunity, but also the expressed protein antigen in the cell is presented by MHC I molecule, the activation of CTL and the kill of target cell are the main mechanisms of DNA vaccine, this is necessary for the prevention of tuberculosis. Other studies indicated that the incubation of DNA vaccine do not disturb the allergy quarantine of tuberculosis. For the development of sensitive and special diagnosis and new and effective prevention, especially the DNA vaccine, in this study, the mainly study contents as follows: 1. the clone of the mainly protective antigen genes of M. bovisIn this study, we filtered seven protective antigen genes of secreted proteins:Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6. These mature protein genes were amplified from M. bovis Vallee111 chromosomal DNA by using PCR technique, the target gene is 888bp, 858bp, 801bp, 390bp, 492bp, 603bp and 288bp respectively. Through T-A clone, the PCR products were cloned into pGEM-T Vector system. The recombinant clone
was identified by a -complementation, enzyme digestion, PCR identification and sequential analysis, then these recombinant plasmids pGEM-T-85A, pGEM-T-85B, pGEM-T-51, pGEM-T-63, pGEM-T-70, pGEM-T-83 and pGEM-T-ESAT-6 were constructed successfully. Sequential and homogeneous analysis indicated that between M. bovis Valleelll and other strains of M bovis, the related mature protein genes are homogeneous in large extents.2. the prokaryotic expression of the mainly protective antigen genesTo acquire the expression products of seven protective antigen genes of M. bovis, these seven protective antigen genes that were cloned into pGEM-T system were subcloned into prokaryotic expression vector pET28a and pGEX-4T-3, so prokaryotic expression plasmids pET28a-85A, pET28a-85B, pET28a-51, pET28a-63, pET28a-70, pET28a-83 and pGEX-4T-3-ESAT-6 were constructed successfully. These recombinant expression plasmids were transformed into E. coli BL21 (DE3), then through the induction of IPTG and SDS-PAGE analysis, these fused target proteins such as 32kDa, 30kDa, 30kDa, 18kDa, 25kDa, 26kDa and 34kDa were acquired respectively. Western-blot indicated that these target proteins have the antigenicity of M. bovis. Furthermore, except MPB63, which was expressed as the soluble proteins, others were expressed as inclusion body. These bacteria were broken by the cooling sonifier, then were washed by Triton X-100, so the inclusion body were abstracted. These target proteins were purified by SDS-PAGE, then were analyzed by SDS-PAGE, these purified proteins only presented a band, so they can be acted as the identified antigens for the eukaryotic expression plasmids.3. eukaryotic expression of the mainly protective antigen genesFor the construction of DNA vaccine, these seven protective antigen genes that were cloned into pGEM-T system were subcloned into eukaryotic expression vector pVAXl, then these seven eukaryotic expression plasmids pVAXl-85A, pVAXl-85B, pVAXl-51, pVAXl-63, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 were constructed successfully. These recombinant plasmids were transfected into BHK-21 cells with liposome, these expressions were detected with immunofluorescence and RT-PCR, which indicated that these target genes were expressed in BHK-21.4. Immunogenicity of eukaryotic expression plasmidsTo explore protective efficacy of eukaryotic expression plasmids, BALB/c mice was vaccinated with pVAXl-85A, PVAX1-85B, pVAXl-51, pVAXl-63, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 DNA vaccine respectively and combined vaccinated by pVAXl-85B, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 DNA vaccines, moreover, pVAXl, physiological saline and BCG immunization as control. The protective efficacy was detected by indirect
ELISA. The detection results indicated that the immunized mice harvested the special antibody against Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6, the antibody titer increased with the increase of immunity. Furthermore, the serum antibody titer of combined immunization was higher than the mono-immunization obviously (P<0.05), but the immunization efficacy was not as good as BCG, which is not the same with international reports that antibody titer of combined immunization was higher than BCG obviously. Lymphocyte transformation test indicated that except the pVAXl-85A, other recombinant plasmids induced cell meditated immunity that was as high as or higher than BCG. CD4+T and CD8+T cells determination results demonstrated that the recombinant plasmids immunization induced the mice to produce obvious cell immunity (combined immunity group), and the CDg+T cell was the chief aspect (PO.05). These results indicated that the expressed protein antigens by recombinant plasmids were mainly presented by MHC I molecule, CDg+T cells were activated. The protein antigen expressed by pVAXl-51 can be also presented by MHC II molecule and CD/T cells were activated, so the high level cell immunity can be induced. Furthermore, the cavy was vaccinated by pVAXl-70, pVAXl-83 respectively and combined immunized by pVAXl-70 and pVAXl-83, then pVAXl and BCG as control. Lymphocyte transformation test indicated that recombinant plasmids induced cell meditated immunity that was as high as or higher than BCG, the result was as same as that of little mice. The allergy quarantine of PPD indicated that it was different in large extents between the recombinant plasmids and BCG (PO.01, p=0.0001), this result is as same as the international reports.Those above results indicated that in this study, we acquired the prokaryotic expression products of seven protective antigen genes: Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6. So this laid a solid foundation for the study of biologic specialty and the new diagnosis reagents. The eukaryotic expression plasmids induced the experimental animals to produce special humoral immunity and cell immunity. So these results laid a solid foundation for the study of the protective efficacy of these genes, then we can filter the protective genes against M. bovis and develop the new and effective DNA vaccine.
结核分枝杆菌是细胞内寄生菌,机体抵抗结核分枝杆菌的感染主要依靠细胞免疫。研究发现,结核分枝杆菌培养滤液中存在的大量分泌蛋白能有效地诱发对结核分枝杆菌的抵抗性,产生免疫保护作用,因此分泌蛋白是保护性抗原。DNA疫苗能有效地刺激机体产生持久的体液免疫和细胞免疫应答,而且DNA疫苗在细胞内表达的蛋白抗原通过MHC I类分子呈递抗原,激活CTL及杀灭靶细胞是DNA疫苗发挥保护作用的最主要机制之一,这正是抗结核感染所必需的。另有研究指出,DNA疫苗免疫后,不干扰结核病的变态反应检测。为了研制牛结核病敏感、特异的诊断试剂和新型、高效的预防制剂,尤其是DNA疫苗,本研究进行了以下几方面的工作:
1、牛分枝杆菌主要保护性抗原基因的克隆筛选了M. bovis Ag85A、Ag85B、MPB51、MPB63、MPB70、MPB83和ESAT-6七种分泌蛋白的保护性抗原基因。以M. bovis Vallee111染色体DNA为模板,以Ag85A、Ag85B、MPB51、MPB63、MPB70、MPB83和ESAT-6成熟蛋白基因特异性引物进行PCR扩增,分别扩增出888bp、858bp、801bp、390bp、492bp、603bp和288bp的DNA片段。通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,以α-互补法、质粒大小鉴定、酶切鉴定、PCR鉴定及序列分析鉴定重组克隆,成功地构建出克隆质粒pGEM-T-85A、pGEM-T-85B、pGEM-T-51、pGEM-T-63、pGEM-T-70、pGEM-T-83、pGEM-T-ESAT-6。序列测定及同源性分析表明,所获得的M. boris Vallee111成熟蛋白基因与M. bovis中其它菌株相应基因具有高度的同源性。
2、主要保护性抗原基因的原核表达 为了获得M. bovis七个保护性抗原基因的表达产
Mycobacterium bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings. The prevalence and circulation of this disease not only influence the development of stock raising severely, but also menace the health of human beings, so it was recognized as B infection by OIE. More than 10% of human tuberculosis is caused by M. bovis. In recent years, the incidence of human tuberculosis has raised in large extents, the death toll has reached 3 millions every year, which is the accumulation of death toll of other infections, so it is the ringleader among all the infections. Because of the prevalence of bovine tuberculosis, human tuberculosis can not be eradicated. Recently, the incidence of bovine tuberculosis has also raised largely, especially in the developing countries. In our country, the positive incidence is about 10%. As regards as the prevention of bovine tuberculosis, BCG is the only vaccine that can be used, but after the incubation of BCG, the immunized effects is not obvious, but it will disturb the allergy test of PPD, so we can not differentiate the natural infection and artificial immunization. Moreover, it made the routine quarantine more difficult. So the new vaccine for the diagnosis and prevention of the bovine tuberculosis has been the hot spot.M. bovis is a kind of parasitic bacterium in the cell. In the immunity of anti-mycobacterium, cell mediated immunity exerts leading effect. Some studies indicated that the secreted proteins of living bacterium exerted the mainly protective immunity, so the secreted proteins were protective antigens. DNA vaccine can induce permanent humoral and cell immunity, but also the expressed protein antigen in the cell is presented by MHC I molecule, the activation of CTL and the kill of target cell are the main mechanisms of DNA vaccine, this is necessary for the prevention of tuberculosis. Other studies indicated that the incubation of DNA vaccine do not disturb the allergy quarantine of tuberculosis. For the development of sensitive and special diagnosis and new and effective prevention, especially the DNA vaccine, in this study, the mainly study contents as follows: 1. the clone of the mainly protective antigen genes of M. bovisIn this study, we filtered seven protective antigen genes of secreted proteins:Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6. These mature protein genes were amplified from M. bovis Vallee111 chromosomal DNA by using PCR technique, the target gene is 888bp, 858bp, 801bp, 390bp, 492bp, 603bp and 288bp respectively. Through T-A clone, the PCR products were cloned into pGEM-T Vector system. The recombinant clone
was identified by a -complementation, enzyme digestion, PCR identification and sequential analysis, then these recombinant plasmids pGEM-T-85A, pGEM-T-85B, pGEM-T-51, pGEM-T-63, pGEM-T-70, pGEM-T-83 and pGEM-T-ESAT-6 were constructed successfully. Sequential and homogeneous analysis indicated that between M. bovis Valleelll and other strains of M bovis, the related mature protein genes are homogeneous in large extents.2. the prokaryotic expression of the mainly protective antigen genesTo acquire the expression products of seven protective antigen genes of M. bovis, these seven protective antigen genes that were cloned into pGEM-T system were subcloned into prokaryotic expression vector pET28a and pGEX-4T-3, so prokaryotic expression plasmids pET28a-85A, pET28a-85B, pET28a-51, pET28a-63, pET28a-70, pET28a-83 and pGEX-4T-3-ESAT-6 were constructed successfully. These recombinant expression plasmids were transformed into E. coli BL21 (DE3), then through the induction of IPTG and SDS-PAGE analysis, these fused target proteins such as 32kDa, 30kDa, 30kDa, 18kDa, 25kDa, 26kDa and 34kDa were acquired respectively. Western-blot indicated that these target proteins have the antigenicity of M. bovis. Furthermore, except MPB63, which was expressed as the soluble proteins, others were expressed as inclusion body. These bacteria were broken by the cooling sonifier, then were washed by Triton X-100, so the inclusion body were abstracted. These target proteins were purified by SDS-PAGE, then were analyzed by SDS-PAGE, these purified proteins only presented a band, so they can be acted as the identified antigens for the eukaryotic expression plasmids.3. eukaryotic expression of the mainly protective antigen genesFor the construction of DNA vaccine, these seven protective antigen genes that were cloned into pGEM-T system were subcloned into eukaryotic expression vector pVAXl, then these seven eukaryotic expression plasmids pVAXl-85A, pVAXl-85B, pVAXl-51, pVAXl-63, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 were constructed successfully. These recombinant plasmids were transfected into BHK-21 cells with liposome, these expressions were detected with immunofluorescence and RT-PCR, which indicated that these target genes were expressed in BHK-21.4. Immunogenicity of eukaryotic expression plasmidsTo explore protective efficacy of eukaryotic expression plasmids, BALB/c mice was vaccinated with pVAXl-85A, PVAX1-85B, pVAXl-51, pVAXl-63, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 DNA vaccine respectively and combined vaccinated by pVAXl-85B, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 DNA vaccines, moreover, pVAXl, physiological saline and BCG immunization as control. The protective efficacy was detected by indirect
ELISA. The detection results indicated that the immunized mice harvested the special antibody against Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6, the antibody titer increased with the increase of immunity. Furthermore, the serum antibody titer of combined immunization was higher than the mono-immunization obviously (P<0.05), but the immunization efficacy was not as good as BCG, which is not the same with international reports that antibody titer of combined immunization was higher than BCG obviously. Lymphocyte transformation test indicated that except the pVAXl-85A, other recombinant plasmids induced cell meditated immunity that was as high as or higher than BCG. CD4+T and CD8+T cells determination results demonstrated that the recombinant plasmids immunization induced the mice to produce obvious cell immunity (combined immunity group), and the CDg+T cell was the chief aspect (PO.05). These results indicated that the expressed protein antigens by recombinant plasmids were mainly presented by MHC I molecule, CDg+T cells were activated. The protein antigen expressed by pVAXl-51 can be also presented by MHC II molecule and CD/T cells were activated, so the high level cell immunity can be induced. Furthermore, the cavy was vaccinated by pVAXl-70, pVAXl-83 respectively and combined immunized by pVAXl-70 and pVAXl-83, then pVAXl and BCG as control. Lymphocyte transformation test indicated that recombinant plasmids induced cell meditated immunity that was as high as or higher than BCG, the result was as same as that of little mice. The allergy quarantine of PPD indicated that it was different in large extents between the recombinant plasmids and BCG (PO.01, p=0.0001), this result is as same as the international reports.Those above results indicated that in this study, we acquired the prokaryotic expression products of seven protective antigen genes: Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6. So this laid a solid foundation for the study of biologic specialty and the new diagnosis reagents. The eukaryotic expression plasmids induced the experimental animals to produce special humoral immunity and cell immunity. So these results laid a solid foundation for the study of the protective efficacy of these genes, then we can filter the protective genes against M. bovis and develop the new and effective DNA vaccine.
引文
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