病原生物学-结核病防治基础研究(下)

1、.临床样本是否需要分子诊断?是否有强的结核病临床症状?是否有严重的肺部感染?进一步确定和药物敏感检测耐药性分子诊断MTB及其耐药性诊断结核菌阳性?YesYesYes培养阳性？YesNoYesNot TBNo培养 (6-8 weeks)镜检镜检阳性?No等培养结果No结核菌快速诊断方法结核菌快速诊断方法.PCR实验室工作平面图实验室工作平面图试剂准备间（试剂准备间（Reagent Preparation Room）样品准备间（样品准备间（Specimen Preparation Room）核酸扩增室（核酸扩增室（Amplification Room）产物分析间（产物分析间（Product Ana

2、lysis Room）工作流程工作流程实验人员.Hain GenoType MTBDR plusMTB IDRpoBkatGinhAHK $300 per test商业化试剂.Line Probe assay.FluoroquinolinesAminoglycosidesethambutolMTBDRsl for second line drugsHK $ 500 per test.2061349 TCG(TTG) S(L) 5313225 GCT(GCC) A(A) 823CTAnalysis of rpoB of RIF-R strains.1261200 ACC(ACT) T(T) 48

3、11349 TCG(TTG) S(L) 5311508 TTC(TCC ）F(S) 5843225 GCT(GCC) A(A) 823CCTT.Andersen p, Nature Review Immunology, 2005; 3:656Live MycobacteriumLive Mycobacterium（recombinant BCG etcrecombinant BCG etc）Subunit vaccineSubunit vaccine.Cited from Agger EM. Vaccine, 2002; 21:7-14Model of interaction between

4、environmental Mycobacteria and BCG.Sensitisation with environmental Mycobacteria blocks the activity of the BCG vaccine but not subunit vaccines.新型疫苗发展策略新型疫苗发展策略Andersen p, Nature Review Immunology, 2005; 3:656结核活菌苗（重组结核活菌苗（重组BCGBCG）亚单位疫苗亚单位疫苗（蛋白多肽；（蛋白多肽；DNADNA疫苗；病毒载体）疫苗；病毒载体）.减毒活疫苗减毒活疫苗.卡介苗免疫保护效果卡介

5、苗免疫保护效果.引自Mustafa AS. Molecular Immunology, 2002; 39:113.重组重组BCGBCG疫苗的优点疫苗的优点.连接连接酶切酶切BamHEcoRHindPPPBCG转化转化rBCG.pMV261pMV261（染色体外）（染色体外） 与与 pMV361pMV361（染色体内）（染色体内）oriEattpintPhsp60AphMCSpMV361Phsp60AphoriEoriMpMV261MCS.attPattL标志标志基因基因intattR选择标志选择标志外源基因外源基因P整合表达载体整合表达载体X分支杆菌分支杆菌基因组基因组attB整合、插入整合、

6、插入X X切割、连接切割、连接选择选择外源外源P.salineBCGBCG:RD1-2F9saline BCG BCG:RD1-2F9.引自Pym AS. Nature Medicine, 2003; 9: 533.重组重组Mtb Ag85B BCGMtb Ag85B BCG疫苗疫苗(rBCG30) (rBCG30) 免疫活性增强免疫活性增强DTHDTH反应增强反应增强引自Horwitz MA. PNAS, 2000; 97(25):13853.rBCG30rBCG30免疫保护性增强免疫保护性增强体重变化体重变化.rBCG30 rBCG30 免疫免疫保护性增强保护性增强肺脏和肝脏组肺脏和肝脏组

7、织学观察织学观察.Yin-Yang Model Zhang Ying, Clin Pharmacol Ther. 2007;82(5):595-600. Traditional model Resuscitation, Replication, growth, death, DormancyESAT-6Ag85BHspXPersistersRevertsDormant.30kD30kD分泌蛋白分泌蛋白(Ag85)(Ag85)的免疫原性的免疫原性T T细胞增殖反应细胞增殖反应引自引自Sinha RK. Vaccine, 1997; 15: 689Sinha RK. Vaccine, 1997;

8、15: 689.引自引自Mustafa AS. Molecular Immunology, 2002; 39:113T T细胞表位细胞表位.Evans JT. Vaccine, 2004; 22:1964Mtb8.4 微球体刺激脾细胞分泌微球体刺激脾细胞分泌IFN-.胞内细胞胞内细胞因子染色因子染色显示显示CD8+ IFN-+ T细胞被细胞被激活激活.单一剂量单一剂量Hsp65-TDM-PLG微球微球体诱导产生较强的免疫保护力体诱导产生较强的免疫保护力. 佐佐 剂剂 蛋白疫苗蛋白疫苗DNA疫苗疫苗效果效果DDACFP +MPLCFP DDA+MPLESAT-6, Ag85B +DDA+treh

9、alose dibehenateESAT-6, Ag85B +polystyrene microsphereCFP +PLG microsphereMtb8.4 +PLG microsphere+ TDM Hsp65+.细胞因子增强亚单位疫苗细胞因子增强亚单位疫苗的免疫保护力的免疫保护力.(a) The translation product of AMM (Ag85BMPT64(190198)Mtb8.4) with the molecular weight of 46.7 kDa. (b) Purification of AMM produced in E. coli. AMM was p

10、urified from inclusion bodies by NiNTA His Bind column. M, protein molecular weight marker; 13, washed with buffer C with pH 6.3; 4 and 5, AMM was eluted with buffers D and E with pH 5.9 and 4.5, respectively.djuvant Dimethyl- dioctyldecyl ammonium bromide (DDA) , one kind of cationic liposomes BCG

11、polysaccharide nucleic acid (BCG PSN).powder. Ultraviolet absorption spectrum of BCG PSN.BCG PSN prepared was scanned by ultraviolet absorption spectrum from 190 nm to 360 nm. The scan showed that BCG PSN adjuvant had special absorption at peak of 206 nm and 260 nm. .LungSpleen* p0.05 compared to Ag85B boost; * * p0.05 compared to PBS; * *p0.05 compared to BCG .* p0.05 compared to PBS; * p0.05 compared to BCG . AMM b