一种结核分枝杆菌蛋白组、其筛选方法及其应用与流程

1.本发明涉及一种结核分枝杆菌蛋白组、其筛选方法及其应用，属于生物学技术领域。


背景技术：

2.结核分枝杆菌(mycobacterium tuberculosis，mtb)感染引起的结核病(tu berculosis，tb)是一种慢性消耗性传染病，已成为威胁人类健康的全球性问题。who发布的《2021年全球结核病报告》显示，2020年，全球结核潜伏感染人群接近20亿，新发结核病患者987万，发病率为127/10万，估算发病数和发病率持续呈现下降趋势，但下降幅度较往年有所减缓。排除新冠肺炎疫情的影响，结核病曾是单一传染源的头号死亡原因，也是全球第13大死因。近年来，耐药结核病尤其是耐多药结核病(multidrug-resistant tuberculosis，mdr-tb)和广泛耐药结核病(extensively drug-resistant tuberculosis，xdr-tb)的出现，以及结核分枝杆菌耐药率的持续升高，给抗结核化学治疗提出了更高的挑战。此外，传统卡介苗(bcg)对儿童和成年人肺结核病的预防效果不稳定，且免疫学作用机制尚不完全清楚，这也导致结核病的预防陷入困境。然而，结核病的发生、发展和转归除了与机体载菌量及其毒力等因素有关，机体的免疫功能也起到了非常重要的作用。因此，充分了解结核分枝杆菌免疫应答与逃逸机制，对结核病的临床诊治、新型结核疫苗与免疫疗法的研究都具有重要的理论指导意义。
3.结核分枝杆菌是一种胞内致病菌，主要寄生于巨噬细胞中，其通过多种策略逃避宿主细胞的免疫杀伤作用，从而在在宿主体内缓慢生长并长期存活。这是通过特定病原体蛋白和宿主免疫细胞之间的分子相互作用发生的。由于分子间相互作用会影响分子功能发生改变，从而导致病原体改变其基因表达过程、调控病原体从复制(生长)状态到非复制(休眠)状态之间的转换并激发产生能量的替代机制。因此，这些分子间相互作用对结核分枝杆菌在宿主体内的存活至关重要，它们可以调节宿主对细菌感染的免疫应答并获取其生长所需的营养物质。这表明结核分枝杆菌感染时与宿主相互作用的蛋白，可能是设计新型抗结核药物和疫苗的潜在靶点。


技术实现要素：

4.为了克服现有技术的不足，本发明的第一个目的在于提供一种结核分枝杆菌蛋白组，对于结核病的诊断、治疗，特别是结核分枝杆菌的发病机制和开发结核疫苗的研究提供潜在的靶点。
5.本发明的第二个目的在于提供一种上述结核分枝杆菌蛋白组的筛选方法，该筛选方法结果客观，效率高且降低了成本，有一定的优越性。
6.本发明的第三个目的在于提供一种上述结核分枝杆菌蛋白组的应用，为将其用于制备检测试剂、治疗试剂或是用于研究结核分枝杆菌的作用机制，具有良好的辅助开发应用。
7.实现本发明的第一个目的可以通过采取如下技术方案达到：一种结核分枝杆菌蛋白组，包括rv3768、rv1848、rv0028、rv0301、rv1022、rv0095c、rv2523c、rv0546c、rv1778c、rv0100、rv1724c、rv1875、rv3735、rv3284、rv0577、rv3849、rv2697c、rv2360c、rv0489、rv3246c、rv3841、rv3285、rv0764c、rv2837c、rv2907c、rv3756c、rv3118、rv3291c、rv2534c、rv0865、rv1876、rv3688c、rv3295、rv3283、rv1404、rv0036、rv1070c、rv1719、rv3855、rv2731、rv2324、rv0815c、rv2754c、rv3257c、mt2455、rv0025、rv3013、rv0009、rv1628c、rv3420c、rv2779c、rv3089、rv0187、rv1356c、rv3503c、rv2528c、mt0946、rv0130、rv2098c、rv0730、rv0887c、rv2883c、rv2564、rv3042c、rv3788、rv2158c、rv2258c、rv2102、rv0398c、rv0861c、rv0459、rv1742、rv3009c、rv1284、rv0384c、rv1860、rv0390、rv2426c、rv2243、rv2233、rv2889c、rv1654、rv1094、rv1692、rv2043c、rv1828、rv3117、rv1389、rv2558、rv0678、rv3315c、rv2619c、rv3050c、rv0333、rv0793、rv1059、rv1405c、rv3836、rv1046c、rv1710、rv2438c、rv0046c、rv2460c、rv1604、rv0640、rv0571c、rv0551c、rv1657、rv1794、rv1142c、rv3198c、rv1896c、rv3279c、rv1531、rv2461c、rv3154、rv1015c、rv3007c、rv2511、rv3677c、rv1463、rv0258c、rv2492、rv2614a、rv1034c、rv2514c、rv3322c、rv1112、rv0952、rv1919c、rv1636、rv0350、rv0119、rv0283、rv2996c、rv2862c、rv0984、rv2680、rv0351、rv0310c、mt3289、rv0396、rv3160c、rv1451、rv0056、mt1560.1、mt3268、rv3715c、rv0271c、rv0511、rv2478c、rv1413、rv2473、rv2540c、rv1957、rv0772、rv3309c、rv3301c、rv2841c、rv3799c、rv0684、rv2114、mt2330.1、rv2944、rv2026c、rv2505c、mt1547、rv2467、rv3395a、rv1292、rv2282c、rv1988、rv1716、rv3550、rv2914c、rv0582、rv2423、rv2525c、rv0078a、rv2471、rv0274、rv2291、rv3699、rv1852、rv1478、rv2096c、rv1717、rv2374c、rv2521、rv2851c、rv3099c、rv1677、rv3237c、rv1379、rv1377c、rv1347c、rv0491、rv2959c、rv2913c、rv2718c、rv1671、rv1019、rv1219c、rv2179c、rv2185c、rv1949c、rv2513、rv2149c、rv1683、rv3521、rv1003、rv1419、rv0726c、rv0970、rv0078、rv0821c、rv2689c、rv2825c、rv3442c、rv2436、rv3465、rv3748、rv0232、rv2477c、rv0045c、rv2376c、rv3311、rv0687、rv0937c、rv3559c、rv2847c、rv2703、rv3280、rv1827、rv3168、rv1013、rv2499c、rv3213c、rv0956、rv0429c、rv1718、rv0534c、rv3609c、rv0041、rv1056、rv1264、rv2293c、rv3899c、rv3676、rv3717、rv2711、rv3248c、mt3269、rv1080c、rv3002c、rv0191、mt2625、mt1196、rv2909c、mt3780、rv1667c、rv1202、rv0030、rv0113、rv0110、rv2765、rv1829、rv1414、rv1416、rv1685c、rv2310、rv0801、rv3678a、mt1364、rv0099、rv3709c、rv2202c、rv1045、rv1606、rv2333c、rv1331、rv3838c、rv3406、mt3103、rv2818c、rv3039c、rv3672c、rv1816、rv0518、rv1159a、rv2557、rv3791、rv2358、rv0831c、rv3567c、rv3705c、rv0737、rv1448c、rv0957、rv2546、rv1147、rv3592、rv0146、rv0158、rv0076c、rv1882c、rv1245c、rv2238c、rv3061c、rv2400c、rv3561、rv2626c、rv0164、rv0733、rv0195、mt0085.1、rv0138、rv2134c、rv3557c、rv0855、rv0583c、rv0361、rv1820、mt2068、rv3241c、rv1680、rv2927c、rv1725c、rv2712c、rv3573c、rv2234、rv3588c、rv2498c、rv0552、rv0422c、rv0385、rv2384、rv1894c、rv1428c、rv2133c、rv0364、rv2683、rv1025、rv3076、rv0566c、rv0178、rv1109c、mt0812、rv0302、rv1911c、rv2368c、rv0905、rv2503c、rv1337、rv3755c、rv2763c、rv3472、rv1837c-1、rv3427c、rv2912c、rv2299c、rv2226、rv1299、rv2696c、rv0273c、rv2407、rv0857、rv2115c、rv2592c、rv3121、rv1262c、rv1932、rv2535c、rv1395、rv0072、rv1766、rv1439c、rv1536、
rv2495c、rv0585c、rv1168c、rv3835、rv3341、mt1342、rv2937、rv1698、rv3437、rv1362c、rv3024c和rv0155中的至少一种。
8.进一步地，rv3768、rv1848、rv0028、rv0301、rv1022、rv0095c、rv2523c、rv0546c、rv1778c、rv0100、rv1724c、rv1875、rv3735、rv3284、rv0577、rv3849、rv2697c、rv2360c、rv0489、rv3246c、rv3841、rv3285、rv0764c、rv2837c、rv2907c、rv3756c、rv3118、rv3291c、rv2534c、rv0865、rv1876、rv3688c、rv3295、rv3283、rv1404、rv0036、rv1070c、rv1719、rv3855、rv2731、rv2324、rv0815c、rv2754c、rv3257c、mt2455、rv0025、rv3013、rv0009、rv1628c、rv3420c、rv2779c、rv3089、rv0187、rv1356c、rv3503c、rv2528c、mt0946、rv0130、rv2098c、rv0730、rv0887c、rv2883c、rv2564、rv3042c、rv3788、rv2158c、rv2258c、rv2102、rv0398c、rv0861c、rv0459、rv1742、rv3009c、rv1284、rv0384c、rv1860、rv0390、rv2426c、rv2243、rv2233、rv2889c、rv1654、rv1094、rv1692、rv2043c、rv1828、rv3117、rv1389、rv2558、rv0678、rv3315c、rv2619c、rv3050c、rv0333、rv0793、rv1059、rv1405c、rv3836、rv1046c、rv1710、rv2438c、rv0046c、rv2460c、rv1604、rv0640、rv0571c、rv0551c、rv1657、rv1794、rv1142c、rv3198c、rv1896c、rv3279c、rv1531、rv2461c、rv3154、rv1015c、rv3007c、rv2511、rv3677c、rv1463、rv0258c、rv2492、rv2614a、rv1034c、rv2514c、rv3322c、rv1112、rv0952、rv1919c、rv1636、rv0350、rv0119、rv0283、rv2996c、rv2862c、rv0984、rv2680、rv0351、rv0310c、mt3289、rv0396、rv3160c、rv1451、rv0056、mt1560.1、mt3268、rv3715c、rv0271c、rv0511、rv2478c、rv1413、rv2473、rv2540c、rv1957、rv0772、rv3309c、rv3301c、rv2841c、rv3799c、rv0684、rv2114、mt2330.1、rv2944、rv2026c、rv2505c、mt1547、rv2467、rv3395a、rv1292、rv2282c、rv1988、rv1716、rv3550、rv2914c、rv0582、rv2423、rv2525c、rv0078a、rv2471、rv0274、rv2291、rv3699、rv1852、rv1478、rv2096c、rv1717、rv2374c、rv2521、rv2851c、rv3099c、rv1677、rv3237c、rv1379、rv1377c、rv1347c、rv0491、rv2959c、rv2913c、rv2718c、rv1671、rv1019、rv1219c、rv2179c、rv2185c、rv1949c、rv2513、rv2149c、rv1683、rv3521、rv1003、rv1419、rv0726c、rv0970、rv0078、rv0821c、rv2689c、rv2825c、rv3442c、rv2436、rv3465、rv3748、rv0232、rv2477c、rv0045c、rv2376c、rv3311、rv0687、rv0937c、rv3559c、rv2847c、rv2703、rv3280、rv1827、rv3168、rv1013、rv2499c、rv3213c、rv0956、rv0429c、rv1718、rv0534c、rv3609c、rv0041、rv1056、rv1264、rv2293c、rv3899c、rv3676、rv3717、rv2711、rv3248c和mt3269中的至少一种为非结核病患者和结核病患者的共有蛋白；
9.rv1080c、rv3002c、rv0191、mt2625、mt1196、rv2909c、mt3780、rv1667c、rv1202、rv0030、rv0113、rv0110、rv2765、rv1829、rv1414、rv1416、rv1685c、rv2310、rv0801、rv3678a、mt1364、rv0099、rv3709c、rv2202c、rv1045、rv1606、rv2333c、rv1331、rv3838c、rv3406、mt3103、rv2818c、rv3039c、rv3672c、rv1816、rv0518、rv1159a、rv2557、rv3791、rv2358、rv0831c、rv3567c、rv3705c、rv0737、rv1448c、rv0957、rv2546、rv1147、rv3592、rv0146、rv0158、rv0076c、rv1882c、rv1245c、rv2238c、rv3061c、rv2400c、rv3561、rv2626c、rv0164、rv0733、rv0195、mt0085.1、rv0138、rv2134c、rv3557c、rv0855、rv0583c、rv0361、rv1820、mt2068、rv3241c、rv1680、rv2927c、rv1725c、rv2712c、rv3573c、rv2234、rv3588c、rv2498c、rv0552、rv0422c、rv0385、rv2384、rv1894c、rv1428c、rv2133c、rv0364、rv2683、rv1025、rv3076、rv0566c、rv0178、rv1109c、mt0812、rv0302、rv1911c、rv2368c、rv0905、
rv2503c、rv1337、rv3755c、rv2763c、rv3472、rv1837c-1、rv3427c、rv2912c、rv2299c、rv2226、rv1299、rv2696c、rv0273c、rv2407、rv0857、rv2115c、rv2592c、rv3121、rv1262c、rv1932、rv2535c、rv1395、rv0072、rv1766、rv1439c、rv1536、rv2495c、rv0585c、rv1168c、rv3835、rv3341、mt1342、rv2937、rv1698、rv3437、rv1362c、rv3024c和rv0155为非结核病患者的特异性蛋白。
10.进一步地，结核分枝杆菌蛋白组与t细胞蛋白杂交呈阳性。
11.实现本发明的第二个目的可以通过采取如下技术方案达到：一种结核分枝杆菌蛋白组的筛选方法，包括将人t细胞蛋白和结核分枝杆菌全蛋白质组芯片进行杂交，选择呈阳性位点的结核分枝杆菌蛋白，得到结核分枝杆菌蛋白组。
12.进一步地，人t细胞蛋白包括结核病患者的t细胞蛋白和非结核病患者的t细胞蛋白。
13.进一步地，人t细胞蛋白通过流式细胞术获得；流式细胞术检测细胞免疫型为cd3
+
t淋巴细胞百分比并进行分选，得到人t细胞蛋白。
14.实现本发明的第三个目的可以通过采取如下技术方案达到：一种结核分枝杆菌蛋白组的应用，结核分枝杆菌蛋白组用于制备与结核分枝杆菌相关的检测试剂；试剂包括结核分枝杆菌蛋白组；或编码结核分枝杆菌蛋白组的dna分子；或由含有dna分子的重组菌产生的重组蛋白。
15.进一步地，试剂为结核分枝杆菌的检测试剂或结核免疫功能状态的检测试剂。
16.或，一种结核分枝杆菌蛋白组的应用，结核分枝杆菌蛋白组用于制备结核治疗试剂。
17.进一步地，结核治疗试剂包括以结核分枝杆菌蛋白组作为免疫原获得的多克隆抗体和/或识别结核分枝杆菌蛋白组抗原蛋白的单克隆抗体。
18.或，一种结核分枝杆菌蛋白组的应用，结核分枝杆菌蛋白组用于制备疫苗。
19.或，一种结核分枝杆菌蛋白组的应用，结核分枝杆菌蛋白组用于研究结核分枝杆菌的作用机制。
20.相比现有技术，本发明的有益效果在于：
21.1、本发明结核分枝杆菌蛋白组，对于结核病的诊断、治疗，特别是结核分枝杆菌的发病机制和开发结核疫苗的研究提供潜在的靶点；
22.2、本发明结核分枝杆菌蛋白组的筛选方法，为通过蛋白质-蛋白质功能的相互作用获得，筛选方法结果客观，效率高且降低了成本，有一定的优越性；
23.3、本发明结核分枝杆菌蛋白组的应用广泛，可以将mtb作为一个系统分析其功能，并识别该系统的模式和属性；用于制备检测试剂、治疗试剂或是用于研究结核分枝杆菌的作用机制，具有良好的辅助开发应用。
附图说明
24.图1为hv组的sds-page检测结果；
25.图2为tb组的sds-page检测结果；
26.图3为tb组芯片扫描视图；
27.图4为hv组芯片扫描视图；
28.图5为jurkat组的sds-page检测结果；
29.图6为western blot检测结果；
30.图7-8为dot blot检测结果；
31.图9为jurkat组芯片扫描视图；
32.图10为venn图。
具体实施方式
33.下面，结合附图以及具体实施方式，对本发明做进一步描述：
34.结核分枝杆菌蛋白组的筛选方法，包括
35.t细胞蛋白获得步骤：人t细胞蛋白通过流式细胞术获得；流式细胞术检测细胞免疫型为cd3
+
t淋巴细胞百分比并进行分选，得到人t细胞蛋白；
36.杂交步骤：将结核病患者的t细胞蛋白和非结核病患者的t细胞蛋白分别和结核分枝杆菌全蛋白质组芯片进行杂交，选择呈阳性位点的结核分枝杆菌蛋白，得到结核分枝杆菌蛋白组。
37.结核分枝杆菌蛋白组，包括非结核病患者和结核病患者的共有蛋白以及非结核病患者的特异性蛋白；
38.非结核病患者和结核病患者的共有蛋白包括rv3768、rv1848、rv0028、rv0301、rv1022、rv0095c、rv2523c、rv0546c、rv1778c、rv0100、rv1724c、rv1875、rv3735、rv3284、rv0577、rv3849、rv2697c、rv2360c、rv0489、rv3246c、rv3841、rv3285、rv0764c、rv2837c、rv2907c、rv3756c、rv3118、rv3291c、rv2534c、rv0865、rv1876、rv3688c、rv3295、rv3283、rv1404、rv0036、rv1070c、rv1719、rv3855、rv2731、rv2324、rv0815c、rv2754c、rv3257c、mt2455、rv0025、rv3013、rv0009、rv1628c、rv3420c、rv2779c、rv3089、rv0187、rv1356c、rv3503c、rv2528c、mt0946、rv0130、rv2098c、rv0730、rv0887c、rv2883c、rv2564、rv3042c、rv3788、rv2158c、rv2258c、rv2102、rv0398c、rv0861c、rv0459、rv1742、rv3009c、rv1284、rv0384c、rv1860、rv0390、rv2426c、rv2243、rv2233、rv2889c、rv1654、rv1094、rv1692、rv2043c、rv1828、rv3117、rv1389、rv2558、rv0678、rv3315c、rv2619c、rv3050c、rv0333、rv0793、rv1059、rv1405c、rv3836、rv1046c、rv1710、rv2438c、rv0046c、rv2460c、rv1604、rv0640、rv0571c、rv0551c、rv1657、rv1794、rv1142c、rv3198c、rv1896c、rv3279c、rv1531、rv2461c、rv3154、rv1015c、rv3007c、rv2511、rv3677c、rv1463、rv0258c、rv2492、rv2614a、rv1034c、rv2514c、rv3322c、rv1112、rv0952、rv1919c、rv1636、rv0350、rv0119、rv0283、rv2996c、rv2862c、rv0984、rv2680、rv0351、rv0310c、mt3289、rv0396、rv3160c、rv1451、rv0056、mt1560.1、mt3268、rv3715c、rv0271c、rv0511、rv2478c、rv1413、rv2473、rv2540c、rv1957、rv0772、rv3309c、rv3301c、rv2841c、rv3799c、rv0684、rv2114、mt2330.1、rv2944、rv2026c、rv2505c、mt1547、rv2467、rv3395a、rv1292、rv2282c、rv1988、rv1716、rv3550、rv2914c、rv0582、rv2423、rv2525c、rv0078a、rv2471、rv0274、rv2291、rv3699、rv1852、rv1478、rv2096c、rv1717、rv2374c、rv2521、rv2851c、rv3099c、rv1677、rv3237c、rv1379、rv1377c、rv1347c、rv0491、rv2959c、rv2913c、rv2718c、rv1671、rv1019、rv1219c、rv2179c、rv2185c、rv1949c、rv2513、rv2149c、rv1683、rv3521、rv1003、rv1419、rv0726c、rv0970、rv0078、rv0821c、rv2689c、rv2825c、rv3442c、rv2436、rv3465、rv3748、rv0232、rv2477c、
rv0045c、rv2376c、rv3311、rv0687、rv0937c、rv3559c、rv2847c、rv2703、rv3280、rv1827、rv3168、rv1013、rv2499c、rv3213c、rv0956、rv0429c、rv1718、rv0534c、rv3609c、rv0041、rv1056、rv1264、rv2293c、rv3899c、rv3676、rv3717、rv2711、rv3248c和mt3269中的至少一种；
39.非结核病患者的特异性蛋白包括rv1080c、rv3002c、rv0191、mt2625、mt1196、rv2909c、mt3780、rv1667c、rv1202、rv0030、rv0113、rv0110、rv2765、rv1829、rv1414、rv1416、rv1685c、rv2310、rv0801、rv3678a、mt1364、rv0099、rv3709c、rv2202c、rv1045、rv1606、rv2333c、rv1331、rv3838c、rv3406、mt3103、rv2818c、rv3039c、rv3672c、rv1816、rv0518、rv1159a、rv2557、rv3791、rv2358、rv0831c、rv3567c、rv3705c、rv0737、rv1448c、rv0957、rv2546、rv1147、rv3592、rv0146、rv0158、rv0076c、rv1882c、rv1245c、rv2238c、rv3061c、rv2400c、rv3561、rv2626c、rv0164、rv0733、rv0195、mt0085.1、rv0138、rv2134c、rv3557c、rv0855、rv0583c、rv0361、rv1820、mt2068、rv3241c、rv1680、rv2927c、rv1725c、rv2712c、rv3573c、rv2234、rv3588c、rv2498c、rv0552、rv0422c、rv0385、rv2384、rv1894c、rv1428c、rv2133c、rv0364、rv2683、rv1025、rv3076、rv0566c、rv0178、rv1109c、mt0812、rv0302、rv1911c、rv2368c、rv0905、rv2503c、rv1337、rv3755c、rv2763c、rv3472、rv1837c-1、rv3427c、rv2912c、rv2299c、rv2226、rv1299、rv2696c、rv0273c、rv2407、rv0857、rv2115c、rv2592c、rv3121、rv1262c、rv1932、rv2535c、rv1395、rv0072、rv1766、rv1439c、rv1536、rv2495c、rv0585c、rv1168c、rv3835、rv3341、mt1342、rv2937、rv1698、rv3437、rv1362c、rv3024c和rv0155中的至少一种。
40.结核分枝杆菌蛋白组的应用包括：
41.将结核分枝杆菌蛋白组用于制备结核分枝杆菌的检测试剂或结核免疫功能状态的检测试剂；试剂包括结核分枝杆菌蛋白组；或编码结核分枝杆菌蛋白组的dna分子；或由含有dna分子的重组菌产生的重组蛋白；
42.将结核分枝杆菌蛋白组用于制备结核治疗试剂；治疗试剂包括以结核分枝杆菌蛋白组作为免疫原获得的多克隆抗体和/或识别结核分枝杆菌蛋白组抗原蛋白的单克隆抗体；
43.结核分枝杆菌蛋白组用于研究结核分枝杆菌的作用机制，例如是结核分枝杆菌可能具有的免疫逃逸机制。
44.实施例1：
45.将研究对象根据已知情况分为两组：
46.(1)非结核病患者组(hv，39人)：该组无任何结核病及相关分枝杆菌病症状及体征，且tst试验阴性(排除变态反应前期；免疫系统受干扰：应用糖皮质激素等免疫抑制剂或营养不良及麻疹、百日咳等；免疫功能低下：严重结核病、各种危重患者、淋巴细胞系统免疫缺陷等情况)；
47.(2)结核病患者组(tb，32人)：初诊为结核病且未进行治疗的结核病患者组；依据肺结核诊断标准(ws288-2017)标准，经过临床、实验室及影像学检查确诊为肺结核的患者。初诊肺结核病患者指初次发现、并未接受任何抗结核药物治疗或发现肺结核后虽经不规则、不合理抗结核治疗化疗，但疗程不足1个月的患者；复发肺结核患者指初治失败或治愈但再次复发的患者(去除确诊肺结核后接受不规则、不合理化疗超过1个月的患者)。
48.t细胞蛋白的获得：
49.1、外周血单个核细胞(peripheral blood mononuclear cell，pbmc)的分离：
50.(1)将采集的新鲜抗凝血5ml(肝素抗凝剂)，转移到15ml离心管中；
51.(2)每个离心管中加入等量的pbs，混匀；
52.(3)另取新的15ml离心管，加入5ml淋巴细胞分离液；
53.(4)将稀释后的血液缓慢加入含有淋巴细胞分离液的离心管中；
54.(5)2000rpm，室温，离心30min；
55.(6)吸取第二层云雾状细胞(pbmc)到15ml离心管中；
56.(7)加入适量pbs，洗涤pbmc，1000rpm，室温，离心10min；
57.(8)弃上清，重复步骤(7)；
58.(9)弃上清，用适量pbs重悬pbmc，计数。
59.2、流式细胞仪检测和分选t细胞：
60.(1)将pbmc转移到1.5ml的ep管中，分别加入荧光染料alexa fluor647mouse anti-human cd3(5μl alexa抗体/100μl细胞悬液)及阴性同型对照抗体，室温避光孵育20min；
61.(2)加入适量pbs混匀，1000rpm，4℃，离心5min；
62.(3)弃上清，加入1ml pbs轻轻吹洗，除去未结合的抗体，1000rpm，4℃，离心5min，重复2次；
63.(4)弃上清，加入1ml pbs重悬荧光抗体标记的pbmc上机检测；
64.(5)调整流式细胞仪(bd，facs aria iii)至最佳工作状态，先上荧光标记抗体的阴性同型对照组，再上荧光标记抗体的实验组。检测细胞免疫型为cd3
+
t淋巴细胞百分比并进行分选。
65.3、t细胞蛋白提取及检测：
66.(1)每1
×
106个细胞加入100μl的细胞裂解液，振荡5-10s，冰上静置10min，重复3次；12000g，4℃，离心30min，上清即为t细胞蛋白溶液；
67.(2)bca法测蛋白浓度hv组的蛋白浓度为141ng/μl，tb组的蛋白浓度为494ng/μl，所有样本浓度均满足后续实验要求；
68.(3)sds-page电泳，考马斯亮蓝染色后检测蛋白条带，如图1和图2所示，所有样本均为总蛋白。
69.实施例2：
70.筛选与人t细胞蛋白相互作用的结核分枝杆菌蛋白：
71.mtbprot
tm
结核分枝杆菌蛋白质组芯片共有4262种结核分枝杆菌重组蛋白质，包括结核分枝杆菌标准株h37rv基因编码的3829个蛋白质和结核分枝杆菌致病菌cdc1551基因编码的433个蛋白质，整体覆盖率高达91％，是目前世界上第一张结核分枝杆菌蛋白质组芯片。
72.1、样本标记及检测：
73.(1)根据试剂盒cydye protein labellingcy3 mono 5-pack(ge，pa23001)的操作说明，使用cy3荧光素标记样本；
74.(2)western blot及dot blot检测样本荧光标记效果，所有样本标记均合格；样本
信号与样本梯度呈正相关，最低检测限度为0.5ng，检测结果满足后续实验要求；
75.(3)封闭：将蛋白芯片从-80℃冰箱中取出，4℃复温，每孔加入300μl的封闭液，置于侧摆摇床，4℃封闭3h；
76.(4)样本孵育：弃除封闭液，迅速加入预先制备好的样本孵育液(终浓度为1μg/ml)，200μl/孔，置于侧摆摇床，4℃孵育过夜(从此步骤开始，注意避光操作)；
77.(5)清洗：将芯片置于水平摇床，用清洗液室温清洗3次，5min/次，完成后用超纯水，室温清洗2次，5min/次；
78.(6)干燥：用吸水纸从芯片边缘吸取残留的水分，不要接触芯片表面；
79.(7)扫描：按照扫描仪的操作规范和使用说明扫描干燥过的芯片，如果未及时进行扫描，可将芯片存放于避光的载玻片盒中，-20℃保存，且三天内完成扫描；
80.杂交结果如图3和图4所示，图4放大图中，标号1为阳性点，标号2为阳性对照点(cy3-bsa)。除阳性对照点外，非结核病患者和结核病患者的t细胞蛋白与结核分枝杆菌蛋白杂交后芯片上均有阳性位点，说明筛选到对应的互作蛋白；
81.(8)数据提取：扫描所得图像通过genepix pro v6.0软件分析获取原始数据，然后以芯片上所有蛋白的归一化信噪比(snr)值为计算对象，判断该位点阳性水平，以snr≥1.5为阈值筛选出结核分枝杆菌蛋白组。
82.通过研究蛋白质-蛋白质功能的相互作用，可以将mtb作为一个系统分析其功能，并识别该系统的模式和属性，可以进一步区获得性免疫和先天免疫之间结核分枝杆菌蛋白的特异性相互作用。实验研究已经发现并鉴定了一些mtb和人之间的蛋白-蛋白相互作用(protein-protein interactions，ppi)。酵母双杂交(east two-hybrid，y2h)体系、affinity pull-down(ap)与质谱(ms)联合分析是发现这些相互作用的两种主要方法。然而，y2h的灵敏度仅为20％，ap/ms虽能检测复合物中的蛋白质，但当同时纯化多个蛋白时，却无法区分蛋白间的直接和间接的相互作用。此外，这些方法不仅耗时且成本昂贵，特别是在采用高通量模式时。近年来，基于基因序列间的同源性、互序性、相互作用域和结构，已经开发出多种计算方法来预测宿主与病原体间的ppi。然而，这些方法大多还有待进行严格的验证。因此，预测宿主-病原体间的ppi的计算方法的准确性在很大程度上还是未知的。
83.实施例将流式细胞术与蛋白芯片技术相结合，筛选过程具有一定的技术难度，且在自然条件下，以高通量的方式对蛋白质-蛋白质相互作用进行客观的分析，与原有方法相比，不仅节约了时间且降低了成本，有一定的优越性。
84.对筛选的结核分枝杆菌蛋白进行异同性分析并绘制venn图；
85.结核分枝杆菌蛋白组含结核分枝杆菌蛋白共390个，非结核病患者和结核病患者的共有蛋白253个，包括rv3768、rv1848、rv0028、rv0301、rv1022、rv0095c、rv2523c、rv0546c、rv1778c、rv0100、rv1724c、rv1875、rv3735、rv3284、rv0577、rv3849、rv2697c、rv2360c、rv0489、rv3246c、rv3841、rv3285、rv0764c、rv2837c、rv2907c、rv3756c、rv3118、rv3291c、rv2534c、rv0865、rv1876、rv3688c、rv3295、rv3283、rv1404、rv0036、rv1070c、rv1719、rv3855、rv2731、rv2324、rv0815c、rv2754c、rv3257c、mt2455、rv0025、rv3013、rv0009、rv1628c、rv3420c、rv2779c、rv3089、rv0187、rv1356c、rv3503c、rv2528c、mt0946、rv0130、rv2098c、rv0730、rv0887c、rv2883c、rv2564、rv3042c、rv3788、rv2158c、rv2258c、rv2102、rv0398c、rv0861c、rv0459、rv1742、rv3009c、rv1284、rv0384c、rv1860、rv0390、
rv2426c、rv2243、rv2233、rv2889c、rv1654、rv1094、rv1692、rv2043c、rv1828、rv3117、rv1389、rv2558、rv0678、rv3315c、rv2619c、rv3050c、rv0333、rv0793、rv1059、rv1405c、rv3836、rv1046c、rv1710、rv2438c、rv0046c、rv2460c、rv1604、rv0640、rv0571c、rv0551c、rv1657、rv1794、rv1142c、rv3198c、rv1896c、rv3279c、rv1531、rv2461c、rv3154、rv1015c、rv3007c、rv2511、rv3677c、rv1463、rv0258c、rv2492、rv2614a、rv1034c、rv2514c、rv3322c、rv1112、rv0952、rv1919c、rv1636、rv0350、rv0119、rv0283、rv2996c、rv2862c、rv0984、rv2680、rv0351、rv0310c、mt3289、rv0396、rv3160c、rv1451、rv0056、mt1560.1、mt3268、rv3715c、rv0271c、rv0511、rv2478c、rv1413、rv2473、rv2540c、rv1957、rv0772、rv3309c、rv3301c、rv2841c、rv3799c、rv0684、rv2114、mt2330.1、rv2944、rv2026c、rv2505c、mt1547、rv2467、rv3395a、rv1292、rv2282c、rv1988、rv1716、rv3550、rv2914c、rv0582、rv2423、rv2525c、rv0078a、rv2471、rv0274、rv2291、rv3699、rv1852、rv1478、rv2096c、rv1717、rv2374c、rv2521、rv2851c、rv3099c、rv1677、rv3237c、rv1379、rv1377c、rv1347c、rv0491、rv2959c、rv2913c、rv2718c、rv1671、rv1019、rv1219c、rv2179c、rv2185c、rv1949c、rv2513、rv2149c、rv1683、rv3521、rv1003、rv1419、rv0726c、rv0970、rv0078、rv0821c、rv2689c、rv2825c、rv3442c、rv2436、rv3465、rv3748、rv0232、rv2477c、rv0045c、rv2376c、rv3311、rv0687、rv0937c、rv3559c、rv2847c、rv2703、rv3280、rv1827、rv3168、rv1013、rv2499c、rv3213c、rv0956、rv0429c、rv1718、rv0534c、rv3609c、rv0041、rv1056、rv1264、rv2293c、rv3899c、rv3676、rv3717、rv2711、rv3248c和mt3269；
86.非结核病患者的特异性蛋白137个，包括rv1080c、rv3002c、rv0191、mt2625、mt1196、rv2909c、mt3780、rv1667c、rv1202、rv0030、rv0113、rv0110、rv2765、rv1829、rv1414、rv1416、rv1685c、rv2310、rv0801、rv3678a、mt1364、rv0099、rv3709c、rv2202c、rv1045、rv1606、rv2333c、rv1331、rv3838c、rv3406、mt3103、rv2818c、rv3039c、rv3672c、rv1816、rv0518、rv1159a、rv2557、rv3791、rv2358、rv0831c、rv3567c、rv3705c、rv0737、rv1448c、rv0957、rv2546、rv1147、rv3592、rv0146、rv0158、rv0076c、rv1882c、rv1245c、rv2238c、rv3061c、rv2400c、rv3561、rv2626c、rv0164、rv0733、rv0195、mt0085.1、rv0138、rv2134c、rv3557c、rv0855、rv0583c、rv0361、rv1820、mt2068、rv3241c、rv1680、rv2927c、rv1725c、rv2712c、rv3573c、rv2234、rv3588c、rv2498c、rv0552、rv0422c、rv0385、rv2384、rv1894c、rv1428c、rv2133c、rv0364、rv2683、rv1025、rv3076、rv0566c、rv0178、rv1109c、mt0812、rv0302、rv1911c、rv2368c、rv0905、rv2503c、rv1337、rv3755c、rv2763c、rv3472、rv1837c-1、rv3427c、rv2912c、rv2299c、rv2226、rv1299、rv2696c、rv0273c、rv2407、rv0857、rv2115c、rv2592c、rv3121、rv1262c、rv1932、rv2535c、rv1395、rv0072、rv1766、rv1439c、rv1536、rv2495c、rv0585c、rv1168c、rv3835、rv3341、mt1342、rv2937、rv1698、rv3437、rv1362c、rv3024c和rv0155。
87.实施例3：
88.通过实施例2分别从2组研究对象中筛选出以非结核病患者和结核病患者的共有蛋白以及非结核病患者的特异性蛋白的结果来看，说明mtb侵袭宿主细胞的过程中，这些蛋白表达可能被选择性地抑制而发挥不同的调节作用，由此推测和mtb的免疫逃逸机制相关，所以结核分枝杆菌蛋白组有利于研究结核分枝杆菌的作用机制；特别是根据结核分枝杆菌蛋白组被分为非结核病患者和结核病患者的共有蛋白以及非结核病患者的特异性蛋白两
组的情况，可以进一步研究mtb的免疫逃逸机制。
89.实施例4：
90.基于mtbprot
tm
结核分枝杆菌蛋白质组芯片筛选与jurkat细胞系蛋白相互作用的结核分枝杆菌蛋白：
91.为验证实施例2的结果，本实施例选择jurkat细胞系作为对照。jurkat细胞系属急性t细胞白血病细胞系，是一种悬浮细胞，生长速度较快，最佳培养状态时细胞聚集成葡萄串状，细胞圆润透亮，似鱼卵。
92.1、jurkat细胞复苏：
93.(1)从液氮罐中取出冻存的jurkat细胞，快速将冻存管放入37℃水中，不断轻轻摇晃，直到细胞完全溶解；
94.(2)将细胞转移到15ml离心管中，加入2ml rpmi-1640培养液，1000rpm，离心5min，弃上清；
95.(3)用适量的完全培养基(rpmi-1640+10v/v％胎牛血清)重悬细胞；
96.(4)25cm2的细胞培养瓶中加入5ml预热的完全培养基，然后加入1ml重悬后的细胞，轻轻混匀。
97.(5)5v/v％co2，37℃培养。
98.2、jurkat细胞传代培：
99.(1)将培养瓶中的细胞转移到15ml离心管中，1000rpm，离心5min，弃上清；
100.(2)用适量的完全培养基(rpmi-1640+10v/v％胎牛血清)重悬细胞；
101.(3)25cm2的细胞培养瓶中加入5ml预热的完全培养基，然后加入1ml重悬后的细胞，轻轻混匀；
102.(4)5％co2，37℃培养；
103.刚复苏的细胞生长比较缓慢，约3-4d传一代，传过两代后，可2-3d传一代，每次传代后在倒置显微镜下观察细胞形态，在传过几代合适的时候可进行后续实验。
104.3、jurkat细胞蛋白提取及检测：
105.(1)每1
×
106个细胞加入100μl的细胞裂解液，振荡5-10s，冰上静置10min，重复3次；12000g，4℃，离心30min，上清即为jurkat细胞蛋白溶液；
106.(2)bca法测蛋白浓度为3492ng/μl，所有样本浓度均满足后续实验要求；
107.(3)sds-page电泳，考马斯亮蓝染色后检测蛋白条带，如图5所示，所有样本均为总蛋白；
108.4、选与jurkat细胞系蛋白相互作用的结核分枝杆菌蛋白：
109.(1)样本标记及检测
110.1)根据试剂盒cydye protein labellingcy3 mono 5-pack(ge，pa23
111.001)的操作说明，使用cy3荧光素标记样本；
112.2)western blot及dot blot检测样本荧光标记效果，图6为western blot检测结果(包括hv组和tb组的结果)，所有样本标记均合格；图7-8为dot blot检测结果(包括hv组和tb组的结果)，样本信号与样本梯度呈正相关，最低检测限度为0.5ng，检测结果满足后续实验要求；
113.(2)封闭：将蛋白芯片从-80℃冰箱中取出，4℃复温，每孔加入300μl的封闭液，置
于侧摆摇床，4℃封闭3h；
114.(3)样本孵育：弃除封闭液，迅速加入预先制备好的样本孵育液(终浓度为1μg/ml)，200μl/孔，置于侧摆摇床，4℃孵育过夜(从此步骤开始，注意避光操作)；
115.(4)清洗：将芯片置于水平摇床，用清洗液室温清洗3次，5min/次，完成后用超纯水，室温清洗2次，5min/次；
116.(5)干燥：用吸水纸从芯片边缘吸取残留的水分，不要接触芯片表面；
117.(6)扫描：按照扫描仪的操作规范和使用说明扫描干燥过的芯片，如果未及时进行扫描，可将芯片存放于避光的载玻片盒中，-20℃保存，且三天内完成扫描；
118.(7)数据提取：扫描所得图像通过genepix pro v6.0软件分析获取原始数据，然后以芯片上所有蛋白的归一化信噪比(snr)值为计算对象，判断该位点阳性水平，以snr≥1.5为阈值筛选出潜在阳性蛋白。
119.5、结果：
120.(1)提取的jurkat细胞蛋白与结核分枝杆菌蛋白质组芯片杂交结果如图9所示，除阳性对照点外，jurkat细胞蛋白与结核分枝杆菌蛋白杂交后芯片上出现阳性位点，说明筛选到对应的互作蛋白。
121.(2)本发明筛选出与jurkat细胞蛋白互作的结核分枝杆菌蛋白共93个，分别为：rv3855、rv0937c、rv3768、rv1848、rv0028、rv0301、rv1022、rv0025、rv0095c、rv2523c、rv0546c、rv2293c、rv3609c、rv1778c、rv0100、rv1724c、rv1875、rv3735、rv3284、rv2847c、rv0577、rv1264、rv3849、rv2697c、rv2360c、rv0489、rv3246c、rv3676、rv3841、rv3285、rv0764c、rv2837c、rv2907c、rv1718、rv3756c、rv3118、rv3291c、rv2534c、rv0865、rv1876、rv3688c、rv3295、rv3559c、rv3283、rv1404、rv2499c、rv0036、rv1070c、rv1719、rv2731、rv2324、rv0815c、rv2754c、rv3257c、mt2455、rv3013、rv0009、rv1628c、rv3420c、rv2779c、rv3248c、rv3089、rv0187、rv1356c、rv3503c、rv2528c、rv3280、mt0946、rv0130、rv2098c、rv0730、rv0887c、rv2883c、rv2564、rv1827、rv3042c、rv3788、rv2376c、mt3269、rv2158c、rv2258c、rv2102、rv0398c、rv0861c、rv0459、rv3009c、rv1284、rv0390、rv2426c、rv2243、rv2233、rv2889c、rv3899c。
122.与健康人和结核病患者t细胞筛选到的潜在结核阳性蛋白进行异同性分析并绘制venn图，如图10所示，上述93个结核分枝杆菌蛋白均为非结核病患者、结核病患者t细胞和jurkat细胞共有。此外，与结核病患者t细胞相互作用的mtb蛋白数量远多于与jurkat细胞相互作用的mtb蛋白，表明细胞系在某些方面不能替代原代细胞；非结核病患者的特异性蛋白有137个，提示在mtb侵袭宿主细胞的过程中，宿主蛋白表达可能被选择性地抑制以发挥不同的调节作用
123.对于本领域的技术人员来说，可根据以上描述的技术方案以及构思，做出其它各种相应的改变以及变形，而所有的这些改变以及变形都应该属于本发明权利要求的保护范围之内。