结核分枝杆菌和牛分枝杆菌耐药及非耐药菌株的黏附素筛选

Tuberculosis(TB) is a facultative global epidemic. China is strikingly listed on the 2nd place with a heavy burden of TB disease（just better than Indian）. Facing today's difficult situations of TB control in the world especially in China, both drug-sensitive and multidrug-resistant strains of Mycobacterium. tuberculosis and Mycobacterium. bovis (4 strains) are chosen in present study. First, all cell-wall related proteins from each Mycobacterium strain are separated and purified based on successive four steps: removal of capsule and then mycolic acid in cell wall, followed by preparative pH successive gradient electrophoresis and liquid chromatography. Then sample the proteins on glass to produce low-density protein-chip. Next, cell adehesion assay on protein-chip is performed using macrophages originated from human against M. tuberculosis and from newborn cows against M. bovis respectively. Finally, the first purpose of present study will be achieved- - - - the complete array of adhesins from each of this 4 Mycobacterium strains are respectively screened out by differential display using fluorescent antibodies. The second purpose of present study is to find different /same adhesins by comparison, between this two Mycobacterium species, drug-sensitive and resistant strains of each species and two macrophages originated from human and cow, and common ashesins among all strains. And the third part of this research is, based on cell invasion and inhibition assay to further select phagocytosis-inducing adhesins. The importance and urgency of present study are mainly demonstrated as three aspects: First, display of the whole array of adhesins from M. tuberculosis and M. bovis including knowledge about those common/different/same adhesins between two species/strains and targeting cells, are big advances in the pathogenic theory of Mycobacterium. 2nd, selection and analysis on adhesins, is the first but decisive step for a safe and high efficacy "multi-valent" adhesin vaccine. This creative and unique vaccine could actually perform "multi-dimensional" control against TB by blocking antigenic variation strain, drug-resistant strain, HIV combined infection and targeting both human and animal. And 3rd, the methodology of high throughput and high sensitivity in purification of cell-wall-related proteins and selection of adhesins based on cell adhesion assay on protein-chip could serve as a universal platform for other bacterial pathogen's research.

结核病是重大人兽共患病，中国负担沉重排全球第二。本研究选结核和牛分枝杆菌的多重耐药和非耐药四菌株，经除荚膜…除分枝菌酸…pH连续梯度制备电泳…制备液相色谱四步，制备其胞壁蛋白；点样低密度芯片，经芯片-细胞黏附（结核/牛分枝杆菌：人/牛巨噬细胞）和荧光抗体差异显示，筛出4株菌的全部黏附素。比对分析两菌种间、两细胞间、耐药/非耐药菌株间的共同/差异/保守黏附素。用细胞吞噬抑制试验筛出吞噬诱导性黏附素。其意义：1澄清结核和牛分枝杆菌耐药/非耐药菌株的完整黏附素阵容及共同/差异/保守/吞噬诱导性黏附素，是其致病机理的突破性进展；是全面揭示黏附及吞噬分子机理的新起点；2为多价黏附素分子疫苗奠定基础。此疫苗的独创性在于：可针对抗原变异、耐药菌株、HIV感染和人兽通用，实现"多途径控制"！3 高通量高效率高灵敏的的蛋白纯化方法和蛋白质-细胞芯片筛选黏附素技术，为其它病原微生物的研究提供通用的技术平台。

结核病是人兽共患病，结核分枝和牛分枝杆菌均可引起，耐药结核正威胁人类生命安全。黏附是病原菌建立感染的第一步。本项目完成了全部计划内容—非耐药和耐药分枝杆菌黏附素筛选。研究结果：.（1）建立了分枝杆菌两级多重PCR诊断方法。一级两重PCR靶标16S rRNA鉴定属和群；二级七重PCR靶标结核分枝杆菌复合群染色体，依扩增谱型鉴定种。（2）建立了牛结核病PPD-ESAT-6/CFP-10—IFN-γ诊断方法。PPD和ESAT-6/CFP-10串联使用，比PPD特异性提高4.3%，并联使用比PPD敏感性提高3.6%。（3）2013-2016年间，共扑杀阳性牛215头，剖检后60.87%可见明显干酪样结节，其中85.71%为肺外结核。共分离到179株菌。（4）其中28株为多重耐药，对4种抗结核病一线药物利福平、异烟肼、乙胺丁醇，链霉素全部耐药，PCR扩增到全部耐药基因（RIF/ rpoB/543 bp、INH/ katG/inhA/455 bp、EMB/ embB /490 bp和rrs/SM/516 bp）。（5）结核/牛分枝杆菌黏附素相同，人/牛巨噬细胞黏附素相同。非耐药菌株和耐药菌株分别筛选到5种（HBHA、 Cpn60.2、FBP、Hlp、Apa）和7种黏附素（HBHA、 Cpn60.2、Hlp、Apa、FBP、Mces、19-kDa蛋白），其中肝素结合血凝素/HBHA为诱导巨噬细胞吞噬性粘附素。（6）分枝杆菌hbha、fbpA、fbpB、fbpC、apa基因的原核表达并验证了其粘附素功能。（7）HBHA基因在耻垢分枝杆菌中的表达及其黏附特性的鉴定， hbha- pMV361- mc2155 重组菌特异性粘附A549 细胞，提高菌体胞内存活数量(P<0.05)，可被HBHA重组蛋白或其抗体阻断。（8）HBHA-HSP65融合黏附素对小鼠的免疫保护力。HBHA-HSP65亚单位疫苗免疫BALB/c小鼠，血清抗体效价达1:16000，显著高于HBHA、HSP65和BCG组(P<0.05)；显著降低在小鼠脾脏的荷菌量(P<0.05)，几乎完全切断分枝杆菌对肺脏的感染(P<0.01)。（9）延伸进行了布鲁氏菌病和牦牛的病毒病（tetraparvovirus 1 (bovine hokovirus1), Hepatitis E virus）流行病学调查。