敲除pckA基因的结核杆菌引起的免疫反应的研究

研究结核杆菌pckA基因编码的磷酸烯醇型丙酮酸羧激酶(PEPCK)诱导机体产生的保护性免疫反应。用敲除pckA基因的牛结核杆菌BCG和野生型BCG分别感染小鼠,取肝、肺、脾进行病理分析,并进行脾细胞培养,检测CD4 、CD4 /CD8 、细胞因子IFNI-γI、L-12和TNF等。用敲除pckA基因的BCG感染的小鼠比野生型BCG感染的小鼠体内产生的结核结节少且不典型,炎性程度低。野生型BCG感染的小鼠脾脏内的CD4 T细胞和CD4 /CD8 、细胞因子IFN-γ、IL-12、TNF均明显高于敲除pckA基因BCG感染的小鼠。pckA基因为结核杆菌生长所必需,其编码产物PEPCK能够刺激机体产生免疫反应,是一种很好的疫苗候选分子。     

鸭病毒性肝炎病毒VP1基因表达及其抗体检测ELISA方法的建立

[目的]研究DHV VP1基因在大肠杆菌中的表达,并以纯化的重组蛋白为抗原建立鸭病毒性肝炎抗体检测ELISA方法.[方法]采用RT-PCR技术,扩增VP1基因,与Pmd18-T载体进行连接,构建DHV VP1基因克隆重组质粒.然后定向插入到Pet-32a( )表达载体,筛选原核表达载体Pet-32a-VP1,进行ITPG诱导表达分析.以纯化的重组蛋白为抗原建立间接ELISA方法并初步应用于临床.[结果]DHV VP1基因可在大肠杆菌中稳定、高效地表达.Western blot检测表明,表达的重组蛋白能与鸭肝炎阳性血清发生特异性反应.确定间接ELISA方法的抗原最佳包被浓度为5 ug/孔,血清最佳稀释度为1:100,临界值为OD450值≥0.302,建立的ELISA方法具有较好的敏感性、特异性和重复性.通过对80份血清样品的检测表明,该方法与中和试验的符合率为97.5%,初步临床应用结果表明该方法可用于雏鸭母源抗体和免疫后抗体的消长变化的检测.[结论]以大肠杆菌表达的DHV VP1重组蛋白为抗原建立的间接ELISA方法可用于鸭病毒性肝炎抗体的检测.     

肺炎嗜衣原体蛋白酶样活性因子免疫优势区基因 重组蛋白在早期诊断中的应用

[目的]克隆和表达肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)蛋白酶样活性因子(CPAF)免疫优势区基因,评价重组蛋白在早期感染诊断中的应用价值.[方法]挑选并克隆出Cpn CPAF免疫优势区基因,构建原核表达载体,诱导表达并纯化重组蛋白,分析其抗原特异性;间接ELISA法检测Cpn参考血清、临床血清标本中的特异性IgM抗体,以及呼吸道感染患者痰咽拭子中的Cpn抗原;检测沙眼衣原体(Chlamydia trachomatis,Ct)临床阳性血清和泌尿生殖道分泌物.[结果]高效表达和纯化出一相对分子量约51.3kDa的重组蛋白;Western blot证明其只与人抗Cpn抗血清发生特异性反应;间接ELISA法检测40份Cpn IgM参考血清,阴性和阳性结果的一致率均为100%(40/40);与"金标准"方法MIF对照,检测300例临床血清标本中的IgM抗体,符合率为98.3%;与PCR试剂对照,检测120份呼吸道感染患者痰咽拭子中的Cpn抗原,符合率为88.3%;检测Ct阳性血清和泌尿生殖道分泌物,与Ct没有交叉反应.[结论]制备的CPAF免疫优势区基因重组蛋白具有良好的抗原性,在Cpn感染早期诊断中具有较高的利用价值.     

结核分枝杆菌ESXB-ESXA融合蛋白的表达及其作为检测抗原的初步应用

目的:探索一种大量表达结核分枝杆菌ESXB-ESXA融合蛋白的方法,分析其抗原性,评价其在结核抗体检测中的初步应用。方法:采用PCR方法从结核分枝杆菌基因组中分别扩增esxB和esxA基因,用Linker(G4S)3连接2条目的片段,连接pET-32a(+)载体,构建ESXB-ESXA融合蛋白表达载体;重组表达载体转化宿主细胞大肠杆菌BL21(DE3),IPTG诱导表达、纯化,Western印迹分析重组蛋白的抗原性;建立以融合重组蛋白为抗原的ELISA和胶体金检测方法,分析其在结核病抗体检测中的应用。结果:构建了ESXB-ESXA融合蛋白的表达载体,重组ESXB-ESXA在大肠杆菌中得以高效表达,表达量占菌体总蛋白的70%以上;Western印迹分析表明该重组蛋白具有较好的抗原性;ELISA和胶体金检测结果显示,用重组ESXB-ESXA抗原检测临床结核病患者有较好的特异性。结论:重组ESXB-ESXA融合蛋白表达量高并具有较好的抗原性,可作为结核病抗体检测的备选抗原。     

低盐度可诱导鲈鱼胞浆型PEPCK基因表达

磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达.     

微小核糖核酸106b对肝细胞葡萄糖异生的影响及机制*

目的: 探讨微小核糖核酸106b(miR-106b)对肝细胞葡萄糖异生作用及其机制。方法: 正常人L02肝细胞培养于含10%胎牛血清的DMEM中,利用miR-106b模拟物和抑制剂(mimics和antagomiR,分别20 nmol/L)处理L02肝细胞24 h,Western blot法检测蛋白和磷酸化蛋白的表达,定量RT-PCR检测mRNA的表达,葡萄糖试剂盒检测培养液中葡萄糖含量。结果: miR-106b模拟物可明显增加磷酸烯醇式丙酮酸羧激酶(PEPCK)和葡萄糖-6-磷酸酶(G6Pase)的蛋白表达(P均＜0.01)、增加磷酸烯醇式丙酮酸羧激酶1(PCK1)的mRNA表达(P＜0.01)、降低葡萄糖激酶(GCK)的mRNA表达(P＜0.01)。miR-106b抑制剂可显著降低PEPCK和G6Pase的蛋白表达(P均＜0.01)、降低PCK1的mRNA表达(P＜0.01)、增加GCK的mRNA表达(P＜0.01)。此外,miR-106b模拟物或抑制剂可显著降低或增加信号转导和转录激活子3(STAT3)的蛋白表达(P均＜0.01)。STAT3特异性抑制剂可显著拮抗miR-106b抑制剂对肝细胞葡萄糖异生的抑制作用。结论: miR-106b通过抑制STAT3信号通路而增加肝细胞葡萄糖异生。     

PEPCK不同启动子调控报告基因表达效率的比较

目的构建两种不同的磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子调控报告基因表达质粒,比较该启动子在各种细胞系中的表达效率。方法采用PCR克隆大鼠PEPCK启动子1323 bp和560 bp两条片段,将两个PEPCK启动子片段插入pGL4.26-Luc2报告质粒中,将重组质粒转染到肝脏细胞系和脂肪细胞系,以非脂肪或肝脏细胞系做对照,比较PEPCK驱动荧光素酶表达效率。结果通过酶切鉴定及基因测序,证明pGL4.26-PEPCK-Luc2荧光素酶表达质粒构建成功,且能够在体外表达荧光素酶。结论两种同片段的PEPCK启动子在各细胞系中的表达效率差异无显著性。     

结核分枝杆菌重组38kD蛋白用于新疆南疆维吾尔族和 湖南湘中汉族血清学诊断的研究

目的：对结核分枝杆菌38kD 蛋白编码基因进行克隆表达及纯化,建立基于重组38 kD 蛋白的酶联免疫吸附法(ELISA)检测 结核病人血清标本,评价重组38 kD蛋白用于结核病血清学诊断抗原的价值。并比较分析其在汉族和维吾尔族人群中的血清学 诊断的差异。方法：用PCR方法扩增38 kD 蛋白的编码基因, 构建重组质粒, 转化到大肠杆菌BL21 中,经IPTG诱导表达, 得到纯 化的38 kD 蛋白,建立以38 kD蛋白为包被抗原的ELISA,并检测临床确诊的结核病人血清标本。结果：ELISA 检测结核病患者 血清标本的维吾尔族阳性率为34%（52/153）,汉族为52.4%（65/124）,两者对比有统计学差异（X2＝9.538,P＜0.005）。在阴性对照 中的维吾尔族特异度为96.4%（159/165）,汉族为98. 8%（130/133）,结果无统计学意义（X2＝0.111,P＞0.5）。结论：重组38kD 蛋白 用于血清学诊断的敏感度在维吾尔族和汉族中有差异,而其诊断特异度无差别。     

乙肝病毒X蛋白结合蛋白通过下调PEPCK的表达抑制肝脏糖异生

乙肝病毒X蛋白结合蛋白（hepatitis B X-interacting protein,HBXIP）可以调节乳腺癌中糖代谢重编程. 为了研究HBXIP在生理条件下对糖代谢的调节作用及机制,本研究利用Cre/loxP重组酶系统成功构建了肝脏组织中HBXIP特异敲除小鼠. 当小鼠接受刺激后,与正常组小鼠相比,肝脏HBXIP敲除小鼠表现基础糖代谢功能异常,如葡萄糖、丙酮酸;相对于对照小鼠,肝脏HBXIP敲除小鼠对糖异生和胰岛素耐受性减弱. RT-PCR、Western blot实验和免疫组化实验结果表明,HBXIP敲除小鼠肝脏组织中糖异生关键酶磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxykinase,PEPCK)表达显著增加. QRT-PCR 分析30例临床肝组织中HBXIP mRNA和PEPCK mRNA表达水平发现,HBXIP与PEPCK表达水平呈负相关. 荧光素酶报告基因实验和ChIP实验结果表明HBXIP可以在基因转录水平调节PEPCK表达. 以上结果表明,HBXIP通过调节糖异生关键酶PEPCK的表达参与调控小鼠肝脏糖异生.     

结核分枝杆菌Rv3425蛋白的原核表达纯化及其血清学诊断价值

目的：用原核系统表达结核分枝杆菌Rv3425蛋白并纯化,评价该重组蛋白在结核病血清学诊断方面的价值。方法：以结核分枝杆菌H37Rv株基因组为模板,PCR扩增得到Rv3425基因序列,克隆至表达载体pET-28a中,转入大肠杆菌BL21（DE3）进行诱导、表达后纯化,用Western印迹和ELISA法进行抗原性初步评价。结果：在原核系统内经IPTG诱导表达后,Rv3425蛋白主要以包涵体形式存在,经复性和镍柱层析纯化后,纯度达95%以上;Western印迹和ELISA结果证明重组Rv3425具有较强的抗原活性;用纯化的Rv3425蛋白做抗原,临床诊断结核病人血清,阳性率达50%。结论：高纯度的Rv3425蛋白在结核病诊断方面具有很高的应用价值,可作为结核病诊断的备选抗原。     

Effects of chromium on the immune system

The performance of integral membrane antigens (IMAs) of Mycobacterium habana TMC 5135 in detecting antimycobacterial antibodies in serum and body fluids of patients mainly of extrapulmonary tuberculosis was evaluated. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana. Antimycobacterial antibodies were detected by ELISA using IMAs in serum and body fluids of 42 patients and 62 control subjects. As authentic adjunct Mycobacterium tuberculosis antigens were also detected (by ELISA) in body fluids and circulating immune complexes using anti-M. tuberculosis H37Ra antibodies. Anti-M. habana IMA antibody detection increased the positivity rate from 26.% (11/42) and 10% (4/42) obtained by culture and smear microscopy, respectively, to 86% (36/42). M. tuberculosis antigens were also found in 29 out of 36 anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also positive for anti-M. habana IMA antibodies. The mean antigen titres in 23 cases, positive for antigens in body fluids, were 2.34 times higher in those who were also positive for anti-IMA antibodies in serum than in those negative for these antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.     

结核分枝杆菌Rv3425特异性蛋白片段与CFP10-ESAT6融合蛋白在结核抗体检测中的应用价值

目的:对结核分枝杆菌Rv3425蛋白进行生物信息学分析及抗原表位预测,评价Rv342519-176重组蛋白与CFP10-ESAT6融合蛋白在结核抗体检测中的应用。方法:PCR扩增结核杆菌Rv3425蛋白19～176位的编码基因片段,原核表达并纯化Rv342519-176重组蛋白(rRv342519-176)和CFP10-ESAT6融合蛋白(rCFP10-ESAT6),建立以重组蛋白为抗原的ELISA方法,评价2种蛋白在结核抗体检测中的联合应用价值。结果:具有抗原表位的rRv342519-176与rCFP10-ESAT6在大肠杆菌中高效表达;ELISA结果显示,rRv342519-176的敏感性为39%,特异性为97.5%;rCFP10-ESAT6的敏感性为37%,特异性为95%;2种蛋白联合检测,敏感性提高到57%,特异性为95%。结论:生物信息学预测Rv3425的优势抗原表位,表达并纯化的rRv342519-176和rCFP10-ESAT6在结核抗体检测中具有一定的抗原互补性,在保持特异性的基础上可显著提高检测敏感性。     

猪流行性腹泻病毒S蛋白主要抗原表位区的原核表达及间接ELISA检测方法的建立

【目的】建立一种快速、特异、敏感的检测血清中猪流行性腹泻病毒(PEDV)抗体的方法。【方法】利用生物学软件对PEDV S蛋白进行抗原位点分析,选择S蛋白的主要抗原表位区进行原核表达。采用SDS-PAGE和Western-blot对重组蛋白进行鉴定及抗原性分析。用纯化的重组蛋白作为包被抗原,经过条件优化、特异性和重复性试验,建立一种针对血清中PEDV抗体的间接ELISA检测方法。【结果】表达了重组S蛋白,重组的S蛋白能与PEDV阳性血清发生特异性反应,并建立一种基于重组S蛋白的间接ELISA检测方法。组内及组间变异系数均小于10%,重复性较好。建立的间接ELISA检测方法分别与商品化PEDV抗体检测试剂盒和Western-blot鉴定结果相比,两者符合率分别为86.67%和88.89%。【结论】建立的间接ELISA方法可以用于PEDV抗体的检测。     

T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein

The immune response to mycobacterial pathogens comprises a significant percentage of T cells with specificity for a 65-kDa heat shock protein (hsp) which is highly conserved in bacteria and man. PBMC were activated in vitro with killed Mycobacterium tuberculosis and afterward tested for CTL activity on autologous target cells primed with 1) killed M. tuberculosis, 2) intact recombinant 65-kDa hsp of Mycobacterium bovis/M. tuberculosis; or 3) tryptic fragments of the recombinant 65-kDa hsp. Strong CTL activity was observed on targets primed with killed M. tuberculosis or with tryptic fragments of the 65-kDa hsp, but not on those primed with the intact 65-kDa hsp. M. tuberculosis activated T cells from 2/13 donors tested exerted killer activity against unprimed targets. To assess whether T cell responses were directed against self-epitopes shared by the mycobacterial and human 65-kDa hsp, four peptides of at least 10 amino acids length were synthesized corresponding to fully or almost identical regions of these molecules. Peripheral blood T cells from 8/9 individuals tested, after activation with killed M. tuberculosis, expressed strong CTL activity toward autologous targets primed with one or more of these synthetic peptides. By using HLA-DR transfected murine L cells we found that the epitopes were recognized in the context of histocompatible HLA-DR (class II) molecules. We conclude that the demonstration of T cells with specificity to self-epitopes in vitro is not indicative for autoimmune disease. However, if at certain stages of infection such T cells are activated by crossreactive microbial epitopes they could cause autoimmune responses.     

结核分枝杆菌抗体检测蛋白芯片的制备及应用

目的:用重组结核分枝杆菌ESAT6、CFP10、M16和M38抗原制备相应的抗体检测蛋白芯片。方法:将制备的结核分枝杆菌抗原ESAT6、CFP10、M16和M38及购买的LAM抗原点于醛基化修饰的玻片上,制备成结核抗体检测蛋白芯片;使用该芯片对130例临床结核病患者和50例健康体检者血液样品进行检测,分析其敏感性和特异性,以及5项结核抗体的构成比。结果:结核杆菌抗体检测蛋白芯片的敏感性为90.8%(118/130),特异性为90%(45/50),LAM的检出率最高为91.5%。结论:用ESAT6、CFP10、M16和M38及LAM抗原制备的结核杆菌抗体检测蛋白芯片用于结核病辅助诊断的敏感性和特异性较高,可用于结核分枝杆菌抗体检测蛋白芯片试剂盒的开发。     

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.     

Mucosal immunity in the brushtail possum (Trichosurus vulpecula): detection of antibody in serum and at female reproductive sites after intranasal immunization

Vaccination strategies for the brushtail possum, which rely upon stimulation of mucosal immunity, are being developed for biocontrol purposes. As little is known about how to stimulate possum immune responses via a mucosal site, groups of possums were immunized intranasally with keyhole limpet haemocyanin (KLH) alone or in combination with known or novel mucosal adjuvants. Antigen-specific antibody titres in female reproductive secretions were measured by ELISA and compared with antibody titres in the serum. Antigen-induced lymphocyte proliferative responses were measured as an indicator of cell-mediated responses. Intranasal immunization with KLH alone stimulated a weak serum antibody response that was significantly increased when KLH was given with cholera toxin subunit B (CTB), recombinant possum tumour necrosis factor alpha (TNF alpha) or live Mycobacterium bovis bacillus Calmette Guerin (BCG). Antibody titres in secretions from ovarian follicles and the uterus were very low in animals administered KLH alone. Significantly higher antibody titres to KLH were present in the reproductive secretions of possums immunized with KLH plus CTB, BCG or heat-killed Mycobacterium vaccae. Antibody titres were lower in mucosal secretions than in the serum, but there was a significant correlation between the two. In addition, coadministration of live BCG with KLH produced a strong antigen-specific cell-mediated response to KLH. This study has shown that an immune response to a protein antigen can be stimulated in possums by intranasal immunization and that antigen-specific antibodies can be detected in secretions from the female reproductive tract.     

结核分枝杆菌重组38kD蛋白用于新疆南疆维吾尔族和湖南湘中汉族血清学诊断的研究

目的：对结核分枝杆菌38kD蛋白编码基因进行克隆表达及纯化,建立基于重组38kD蛋白的酶联免疫吸附法（EusA）检测结核病人血清标本,评价重组38kD蛋白用于结核病血清学诊断抗原的价值。并比较分析其在汉族和维吾尔族人群中的血清学诊断的差异。方法：用PCR方法扩增38kD蛋白的编码基因,构建重组质粒,转化到大肠杆菌BL21中,经IPTG诱导表达,得到纯化的38kD蛋白,建立以38kD蛋白为包被抗原的ELISA,并检测临床确诊的结核病人血清标本。结果：ELISA检测结核病患者血清标本的维吾尔族阳性率为34％（52／153）,汉族为52．4％（65／124）,两者对比有统计学差异（x2=9．538,P〈O．005）。在阴性对照中的维吾尔族特异度为96．4％（159／165）,汉族为98．8％（130／133）,结果无统计学意义（x2=0．111,P〉0．5）。结论：重组38kD蛋白用于血清学诊断的敏感度在维吾尔族和汉族中有差异,而其诊断特异度无差别。     

利用rCFP-10/ESAT-6融合蛋白刺激γ-IFN体外释放测定替代结素皮试检出结核感染的可行性

评估rCFP-10/ESAT-6融合蛋白刺激γ-IFN体外释放测定与结素皮试检出结核感染的敏感性及特异性。对疑似结核病患者共229例进行随机、双盲、平行、对照、前瞻性试验,后经细菌培养证实患结核病的病人共129人,没有结核病史的非结核病患者共100人。以某一特定的γ-IFN体外释放水平及结素皮试反应硬结直径?10 mm为阳性切割值,rCFP-10/ESAT-6融合蛋白刺激γ-IFN体外释放测定的敏感性为96%,显著高于结素皮试(89%)(χ2=4.92;0.025相似文献