非结核分枝杆菌的鉴定及胞内分枝杆菌HBHA表达和实验免疫研究
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摘要
非结核分枝杆菌(Nontuberculous mycobacterium,NTM)即除去结核杆菌复合群、麻风杆菌以外的分枝杆菌。其中一部分属致病菌或条件性致病菌。NTM病发生情况近年来呈明显上升趋势。该病与结核病临床表现上并无特异性,所以误诊机率极大。同时,致病菌对抗结核类药物呈现高耐药性。另外,大多数NTM对结核菌素变态反应(PPD)呈阴性,不易发现,给动物检验、检疫工作造成极大困难。
胞内分枝杆菌属致病性NTM的一种,分布广,可从气溶胶、饮用水、动物排泄物中分离。临床症状主要表现为慢性肺部感染,对获得性免疫缺陷综合征(acquired immune deficiency syndrome, AIDS)患者来说呈播散性感染。近年来,医学界认为胞内分枝杆菌是引起人结核病及肺结核样疾病的主要病原体(约占人NTM的90%以上),对于家畜而言,胞内分枝杆菌是猪抗酸菌病的主要病原体,人与猪之间存在交互感染的可能。
肝素结合血凝素(heparin-binding haeagglutinin, HBHA)为分枝杆菌分泌的一种细胞黏附素,是诱发肺外播散性结核的重要毒力因素之一。目前的研究主要集中于结核杆菌黏附素与宿主细胞之间相互作用与生物学特性等方面,国内对胞内分枝杆菌病诊断、防治等方面的研究则刚刚起步。
为了进一步了解吉林省家畜中NTM分布情况,为更有针对性的防治NTM感染提供数据支持和理论依据,本试验对21株(来自1050份结核菌素阴性猪、牛组织)分离于吉林省周边不同家畜不同分离部位的疑似NTM菌株进行了16S rRNA基因序列分析鉴定。
试验应用PCR方法成功扩增21株待检NTM菌株16S rDNA基因序列,Blast分析发现所测菌株16S rDNA基因序列均与标准序列呈现较高的同源性,构建系统发育树显示各菌株均得到良好区分,鉴定21株待检菌均为NTM。本试验中NTM总分离率为2.0%,牛及猪体内NTM分离率分别为1.82%和2.2%。除6株未最后定型以外,其他菌株均分型鉴定成功,在21株菌株中分得,浅黄分枝杆菌3株(14.3%)；母牛分枝杆菌1株(4.76%)；新金色分枝杆菌5株(23.8%)；偶发分枝杆菌3株(14.3%)；胞内分枝杆菌3株(14.3%)。本文从经过PPD变态反应筛选的屠宰猪、牛体内仍分离出NTM菌株,说明吉林省猪、牛群中NTM的流行情况不容小视。
试验选用自行鉴定的胞内分枝杆菌DFJ-1菌株为研究对象,应用巢式PCR成功扩增胞内分枝杆菌HBHA基因,测序结果与标准序列同源性高达100%,保证了表达蛋白的高度准确性。同时通过生物信息学软件对该蛋白分子特征进行预测,为HBHA可能做为诊断或免疫用抗原提供了理论依据。
为阐明胞内分枝杆菌HBHA在机体免疫应答中的作用,探索其在胞内分枝杆菌病诊断、预治等方面的应用,本试验成功构建原核表达载体pET32a-HBHA并获得良好表达,应用His-Binding-Resin进行分离纯化。利用纯化的可溶性HBHA蛋白免疫试验小鼠,结果发现,经过HBHA蛋白免疫的小鼠可以产生较高水平的特异性抗体；免疫组与对照组相比,淋巴细胞增殖水平有所增高但差异不显著(P>0.05);CD3+、CD4+、CD8+T细胞数量均有增加,其中CD3+T细胞数量增加最为明显,达到统计学显著水平(P<0.01),CD8+T细胞数量增加亦达到统计学上显著水平(P<0.05);免疫小鼠CD4+/CD8+T细胞比值较阴性对照组有所降低；小鼠IFN～γ检测结果显示,阳性组IFN-ν检测值有所增加,但尚未达到统计学显著水平(P>0.05);小鼠小鼠接种试验发现,被攻毒的阴性组小鼠肺组织病理切片可见弥散性病变,出现不同程度的间质增宽、肺气肿及肺脏局灶性坏死症状,但免疫阳性组病变不明显。结果表明,免疫HBHA蛋白的小鼠与阴性组小鼠相比呈现较好的免疫保护能力。
Nontuberculous mycobacterium is the Mycobacterium except the MTC and M. leprae. Most of them are pathogenic or opportunistic pathogen. The occurrence of NTM disease is obviously rising in recent years, Misdiagnosis is very likely to happen because of NTM's similar symptoms with tuberculosis. At the same time, the pathogens showed high resistance to anti-TB drugs. In addition, most of the NTM on the PPD were negative and were difficult to be found, which caused great difficulties for animal inspection and quarantine.
Mycobacterium intracellular is a kind of pathogenic NTM,which is spread widely and can be isolated from the aerosol, drinking water, animal excreta. It mainly causes chronic lung infection of patients, and mycobacterium intracellular is usually easy to lead to disseminated infection with acquired immune deficiency syndrome(AIDS). In recent years, it is thought that Mycobacteria intracellular are main pathogens for human tuberculosis (about90%of NTM above). For livestock, Mycobacterium intracellular is the main pathogen of Mycobacterial Infectiou on pig.There are possibilities of cross infection between man and pig.
The heparin-binding hemagglutinin (HBHA) is one kind of cell adhesion secreted by mycobacterium, and it is an important factor inducing disseminated tuberculosis. At present, The research of HBHA was focused primarily on host cells and bacteria adhesion and the biological characteristics of HBHA the research of the diagnosis, prevention and control of mycobacterium intracellular disease has just started in China.
In order to further study on NTM distribution in livestocks of Jilin province and provide basis on NTM prevention, the16S rRNA gene sequences of21suspected NTM strains (from1050PPD negative tissues of livestocks) were identified from different parts of livestocks. In the test, the16S rDNA gene sequences of21strains NTM were successfully amplified by PCR. It was found by Blast Analysis that the sequences of16S rDNA have highter homology with the standard sequence of NTM strains. Results showed all strains were distinguished in good condition by Phylogenetic Tree. In this test, the total isolation rate of NTM was2.0%, cattle and pigs of NTM isolation rates were1.82%and2.2%respectively. Other strains were all identified successfully except6ones. In which:3strains as mycobacterium gilvum(14.3%),1strains as mycobacterium vaccae(4.76%),5strains as mycobacterium neoaurum(23.8%),3strains as mycobacteria fortuitum(14.3%) and3strains as mycobacteria intracellular respectively(14.3%). The NTM strains which were isolated from the pigs and cattle were identified as negative by PPD. It is revealed that The prevalence of NTM in pigs and cattle can not be overlooked in Jilin Province.
DFJ-1strains of Mycobacterium intracellular was identified in our laboratory, and it was selected as an object for study. The HBHA gene of Mycobacterium intracellulare has been amplified successfully by nested PCR. Results showed the homology between the HBHA sequence and the standard sequence is up to100%, and it guaranteed the accuracy of HBHA protein expressing. At the same time, the molecular characteristics of HBHA protein were predicted by bioinformatics software.It offered theoretical basis for HBHA as possible diagnostic or immune antigen.
To illustrate the role of Mycobacterium intracellular HBHA in the immune response and to explore the application on the disease diagnosis, pre-treatment and other aspects. In this experiment, the pET32a-HBHA as a Prokaryotic expression vector was successfully constructed, and it was got a good expression.
The soluble protein of HBHA were separated and purified by His-Binding-Resin. At the same time, BALB/c mice was vaccinated with the soluble protein of HBHA, moreover, the sodium chloride acts as control. Detection showed that there was a specific antibody against HBHA.Lymphocyte transformation test showed that cell proliferation activity in HBHA group was higher than sodium chloride control group, but there is no significant difference(P>0.05). CD3+, CD4+, CD8+T cells have increased in quantity. For example, the numbers of CD3+T cells increased with especially significant difference (P<0.01); the numbers of CD8+T cells increased with significant difference (P<0.05), compared to the sodium chloride control group, the ratio of CD4+/CD8+T cells have reduced. The IFN-y test results showed that the IFN-y level of HBHA group was increase with no significant difference(P>0.05). Both the HBHA group and the sodium chloride control group were both injected with Mycobacterium intracellular. The results showed that the diffuse lesions can be seen in lung tissue of sodium chloride control mice. Pulmonary symptoms:widened interstitial, emphysema and lung focal necrosis, but immunoreactive lesions of the HBHA group were not obvious. The result reveals that the mice immuned to HBHA protein have better immune protection than those of the sodium chloride control group.
胞内分枝杆菌属致病性NTM的一种,分布广,可从气溶胶、饮用水、动物排泄物中分离。临床症状主要表现为慢性肺部感染,对获得性免疫缺陷综合征(acquired immune deficiency syndrome, AIDS)患者来说呈播散性感染。近年来,医学界认为胞内分枝杆菌是引起人结核病及肺结核样疾病的主要病原体(约占人NTM的90%以上),对于家畜而言,胞内分枝杆菌是猪抗酸菌病的主要病原体,人与猪之间存在交互感染的可能。
肝素结合血凝素(heparin-binding haeagglutinin, HBHA)为分枝杆菌分泌的一种细胞黏附素,是诱发肺外播散性结核的重要毒力因素之一。目前的研究主要集中于结核杆菌黏附素与宿主细胞之间相互作用与生物学特性等方面,国内对胞内分枝杆菌病诊断、防治等方面的研究则刚刚起步。
为了进一步了解吉林省家畜中NTM分布情况,为更有针对性的防治NTM感染提供数据支持和理论依据,本试验对21株(来自1050份结核菌素阴性猪、牛组织)分离于吉林省周边不同家畜不同分离部位的疑似NTM菌株进行了16S rRNA基因序列分析鉴定。
试验应用PCR方法成功扩增21株待检NTM菌株16S rDNA基因序列,Blast分析发现所测菌株16S rDNA基因序列均与标准序列呈现较高的同源性,构建系统发育树显示各菌株均得到良好区分,鉴定21株待检菌均为NTM。本试验中NTM总分离率为2.0%,牛及猪体内NTM分离率分别为1.82%和2.2%。除6株未最后定型以外,其他菌株均分型鉴定成功,在21株菌株中分得,浅黄分枝杆菌3株(14.3%)；母牛分枝杆菌1株(4.76%)；新金色分枝杆菌5株(23.8%)；偶发分枝杆菌3株(14.3%)；胞内分枝杆菌3株(14.3%)。本文从经过PPD变态反应筛选的屠宰猪、牛体内仍分离出NTM菌株,说明吉林省猪、牛群中NTM的流行情况不容小视。
试验选用自行鉴定的胞内分枝杆菌DFJ-1菌株为研究对象,应用巢式PCR成功扩增胞内分枝杆菌HBHA基因,测序结果与标准序列同源性高达100%,保证了表达蛋白的高度准确性。同时通过生物信息学软件对该蛋白分子特征进行预测,为HBHA可能做为诊断或免疫用抗原提供了理论依据。
为阐明胞内分枝杆菌HBHA在机体免疫应答中的作用,探索其在胞内分枝杆菌病诊断、预治等方面的应用,本试验成功构建原核表达载体pET32a-HBHA并获得良好表达,应用His-Binding-Resin进行分离纯化。利用纯化的可溶性HBHA蛋白免疫试验小鼠,结果发现,经过HBHA蛋白免疫的小鼠可以产生较高水平的特异性抗体；免疫组与对照组相比,淋巴细胞增殖水平有所增高但差异不显著(P>0.05);CD3+、CD4+、CD8+T细胞数量均有增加,其中CD3+T细胞数量增加最为明显,达到统计学显著水平(P<0.01),CD8+T细胞数量增加亦达到统计学上显著水平(P<0.05);免疫小鼠CD4+/CD8+T细胞比值较阴性对照组有所降低；小鼠IFN～γ检测结果显示,阳性组IFN-ν检测值有所增加,但尚未达到统计学显著水平(P>0.05);小鼠小鼠接种试验发现,被攻毒的阴性组小鼠肺组织病理切片可见弥散性病变,出现不同程度的间质增宽、肺气肿及肺脏局灶性坏死症状,但免疫阳性组病变不明显。结果表明,免疫HBHA蛋白的小鼠与阴性组小鼠相比呈现较好的免疫保护能力。
Nontuberculous mycobacterium is the Mycobacterium except the MTC and M. leprae. Most of them are pathogenic or opportunistic pathogen. The occurrence of NTM disease is obviously rising in recent years, Misdiagnosis is very likely to happen because of NTM's similar symptoms with tuberculosis. At the same time, the pathogens showed high resistance to anti-TB drugs. In addition, most of the NTM on the PPD were negative and were difficult to be found, which caused great difficulties for animal inspection and quarantine.
Mycobacterium intracellular is a kind of pathogenic NTM,which is spread widely and can be isolated from the aerosol, drinking water, animal excreta. It mainly causes chronic lung infection of patients, and mycobacterium intracellular is usually easy to lead to disseminated infection with acquired immune deficiency syndrome(AIDS). In recent years, it is thought that Mycobacteria intracellular are main pathogens for human tuberculosis (about90%of NTM above). For livestock, Mycobacterium intracellular is the main pathogen of Mycobacterial Infectiou on pig.There are possibilities of cross infection between man and pig.
The heparin-binding hemagglutinin (HBHA) is one kind of cell adhesion secreted by mycobacterium, and it is an important factor inducing disseminated tuberculosis. At present, The research of HBHA was focused primarily on host cells and bacteria adhesion and the biological characteristics of HBHA the research of the diagnosis, prevention and control of mycobacterium intracellular disease has just started in China.
In order to further study on NTM distribution in livestocks of Jilin province and provide basis on NTM prevention, the16S rRNA gene sequences of21suspected NTM strains (from1050PPD negative tissues of livestocks) were identified from different parts of livestocks. In the test, the16S rDNA gene sequences of21strains NTM were successfully amplified by PCR. It was found by Blast Analysis that the sequences of16S rDNA have highter homology with the standard sequence of NTM strains. Results showed all strains were distinguished in good condition by Phylogenetic Tree. In this test, the total isolation rate of NTM was2.0%, cattle and pigs of NTM isolation rates were1.82%and2.2%respectively. Other strains were all identified successfully except6ones. In which:3strains as mycobacterium gilvum(14.3%),1strains as mycobacterium vaccae(4.76%),5strains as mycobacterium neoaurum(23.8%),3strains as mycobacteria fortuitum(14.3%) and3strains as mycobacteria intracellular respectively(14.3%). The NTM strains which were isolated from the pigs and cattle were identified as negative by PPD. It is revealed that The prevalence of NTM in pigs and cattle can not be overlooked in Jilin Province.
DFJ-1strains of Mycobacterium intracellular was identified in our laboratory, and it was selected as an object for study. The HBHA gene of Mycobacterium intracellulare has been amplified successfully by nested PCR. Results showed the homology between the HBHA sequence and the standard sequence is up to100%, and it guaranteed the accuracy of HBHA protein expressing. At the same time, the molecular characteristics of HBHA protein were predicted by bioinformatics software.It offered theoretical basis for HBHA as possible diagnostic or immune antigen.
To illustrate the role of Mycobacterium intracellular HBHA in the immune response and to explore the application on the disease diagnosis, pre-treatment and other aspects. In this experiment, the pET32a-HBHA as a Prokaryotic expression vector was successfully constructed, and it was got a good expression.
The soluble protein of HBHA were separated and purified by His-Binding-Resin. At the same time, BALB/c mice was vaccinated with the soluble protein of HBHA, moreover, the sodium chloride acts as control. Detection showed that there was a specific antibody against HBHA.Lymphocyte transformation test showed that cell proliferation activity in HBHA group was higher than sodium chloride control group, but there is no significant difference(P>0.05). CD3+, CD4+, CD8+T cells have increased in quantity. For example, the numbers of CD3+T cells increased with especially significant difference (P<0.01); the numbers of CD8+T cells increased with significant difference (P<0.05), compared to the sodium chloride control group, the ratio of CD4+/CD8+T cells have reduced. The IFN-y test results showed that the IFN-y level of HBHA group was increase with no significant difference(P>0.05). Both the HBHA group and the sodium chloride control group were both injected with Mycobacterium intracellular. The results showed that the diffuse lesions can be seen in lung tissue of sodium chloride control mice. Pulmonary symptoms:widened interstitial, emphysema and lung focal necrosis, but immunoreactive lesions of the HBHA group were not obvious. The result reveals that the mice immuned to HBHA protein have better immune protection than those of the sodium chloride control group.
引文
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