结核分枝杆菌融合蛋白Ag85B-Hsp16.3、Ag85B-ESAT6 和分泌蛋白 Hsp16.3 体外对人肝癌细胞HepG-2 的作用*

目的:探讨结核分枝杆菌融合蛋白Ag85B-Hsp16.3、Ag85B-ESAT6及分泌蛋白Hsp16.3对人肝癌细胞HepG-2的作用。方法:将已构建的含3种目的基因的表达载体pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6和pProEXHTb-Hsp16.3,分别转入宿主菌E.coliDH5α中,诱导表达后分别获得Ag85B-Hsp16.3、Ag85B-ESAT6和Hsp16.3三种蛋白,采用Ni2+亲和层析柱进行纯化,并用透析方法进行目的蛋白的复性。复性的蛋白按照不同浓度和作用时间分别与肝癌细胞HepG-2反应,用MTT法检测细胞生长情况。结果:三种蛋白被成功纯化并复性。MTT数据统计分析显示,终浓度10μg/ml的三种蛋白对HepG-2细胞生长没有明显作用,当三种蛋白的终浓度分别为20、40、80μg/ml时均能够抑制HepG-2细胞的生长,并且抑制作用随着蛋白终浓度的增大以及作用时间的延长而增强。不同类别的蛋白抑制作用没有明显差别。结论:结核分枝杆菌的部分分泌蛋白能够抑制肝癌细胞HepG-2的生长。     

结核分枝杆菌分泌蛋白Ag85B、Hsp16.3和ESAT6在血清学检测中的初步应用

目的：探讨结核分枝杆菌分泌蛋白Hsp16.3、Ag85B以及融合蛋白ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6用于TB病人血清学检测的意义。方法：将已构建的含5种目的基因的表达载体（pProEXHTb-Hsp16.3、pProEXHTa-Ag85B、pProEXHTb-ESAT6-CFP10、pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6）,分别转入宿主菌E.coli DH5α中,诱导表达后分别获得Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6五种蛋白,采用Ni2＋亲和层析柱进行纯化,并用透析方法进行目的蛋白的复性。将经过复性的5种蛋白分别作为抗原,采用间接ELISA方法检测待测的血清样本,经OPD显色,测定各孔OD490值并判定结果。结果：五种蛋白被成功纯化并复性,通过ELISA方法共检测了22例TB病人血清、10例非结核病人血清和6例正常对照血清,Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6这5种抗原的灵敏度分别为36.4%、90.9%、77.3%、95.5%、100%,特异性分别为100%、75%、100%、93.8%、93.8%。统计分析显示,ESAT6-CFP10和Ag85B、Ag85B-Hsp16.3、Ag85B-ESAT6这三种蛋白ELISA检测的结果无差异,而与Hsp16.3和痰涂片检测结果有显著差异。结论：Ag85B-Hsp16.3和Ag85B-ESAT6可作为结核分枝杆菌ELISA检测的初选抗原。     

结核分枝杆菌分泌蛋白Ag85B、Hsp16.3和ESAT6在血清学检测中 的初步应用

目的:探讨结核分枝杆菌分泌蛋白Hsp16.3、Ag85B以及融合蛋白ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6用于TB病人血清学检测的意义。方法:将已构建的含5种目的基因的表达载体(pProEXHTb-Hsp16.3、pProEXHTa-Ag85B、pProEXHTb-ESAT6-CFP10、pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6),分别转入宿主菌E.coli DH5α中,诱导表达后分别获得Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6五种蛋白,采用Ni2+亲和层析柱进行纯化,并用透析方法进行目的蛋白的复性。将经过复性的5种蛋白分别作为抗原,采用间接ELISA方法检测待测的血清样本,经OPD显色,测定各孔OD490值并判定结果。结果:五种蛋白被成功纯化并复性,通过ELISA方法共检测了22例TB病人血清、10例非结核病人血清和6例正常对照血清,Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6这5种抗原的灵敏度分别为36.4%、90.9%、77.3%、95.5%、100%,特异性分别为100%、75%、100%、93.8%、93.8%。统计分析显示,ESAT6-CFP10和Ag85B、Ag85B-Hsp16.3、Ag85B-ESAT6这三种蛋白ELISA检测的结果无差异,而与Hsp16.3和痰涂片检测结果有显著差异。结论:Ag85B-Hsp16.3和Ag85B-ESAT6可作为结核分枝杆菌ELISA检测的初选抗原。     

结核分枝杆菌Ag85B-Esat6-HspX融合基因真核表达载体的构建

目的构建结核分枝杆菌Ag85B-Esat6-HspX融合基因,并对其在体外真核细胞中进行表达。方法用PCR法从结核分枝杆菌H37Rv株基因组中分别扩增Ag85B、Esat6、HspX基因,插入到pUC19-T载体,序列测定正确后,将融合基因再次克隆到真核表达载体pcDNA3.1（-）。重组质粒经酶切鉴定并测序正确后,用MegaTran1.0转染293T细胞,并用Western-Blot检测目的蛋白的表达。结果 Western-blot检测到分子量大小约65 kDa的目的蛋白。结论成功地构建了结核分枝杆菌Ag85B-Esat6-HspX融合基因的真核表达载体,且该重组载体可在体外真核细胞中获得特异性的表达。     

小鼠对结核分枝杆菌Ag85B-Esat6-HspX融合基因的免疫反应

目的研究结核分枝杆菌Ag85B-Esat6-HspX融合基因的免疫原性。方法将40只BALB/c小鼠随机分成4组,NS组、BCG组、pcDNA-HspX组和pcDNA-Ag85B-Esat6-HspX组,每组10只。BCG组只在0周时皮内注射卡介苗1次。NS组、pcDNA-HspX组及融合基因组分别于0、2、4周肌肉注射生理盐水和重组质粒DNA,共免疫3次。在免疫的第2周,4周及最后一次免疫后2周检测体血清中的总IgG水平。同时,最后一次免疫后2周,取脾细胞检测细胞免疫反应。结果构建的融合基因重组质粒DNA免疫动物后能产生针对结核杆菌特定抗原的特异性细胞免疫和体液免疫应答,具有较强的免疫原性。结论结核分枝杆菌Ag85B-Esat6-HspX融合基因可作为DNA疫苗进行保护作用的研究。     

结核分枝杆菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表达载体的构建与表达

CFP10、ESAT6、Ag85A和Ag85B是结核分枝杆菌的主要免疫优势抗原。为了构建一种可同时表达这4种抗原的真核多基因共表达载体pcDNA-CFP10-ESAT6-Ag85A-Ag85B(pcDNA-CEAB),并利用HEK 293T细胞对其体外表达进行检测。采用酶切、连接的方法,将CFP10和ESAT6编码基因以(Gly4Ser)3蛋白Linker连接,插入至质粒pcDNA3.1(+)多克隆位点CMV启动子与加尾信号BGH pA之间,使两者融合表达,将Ag85A和Ag85B编码基因以内部核糖体进入位点(Internal ribosome entry site,IRES)序列连接,并赋予RSV启动子和加尾信号BGH pA,使两者在RSV启动子作用下独立表达。重组质粒经酶切及测序验证后转染至HEK 293T细胞中进行体外表达实验,48 h后提取总蛋白,利用CFP10、ESAT6、Ag85A和Ag85B特异性抗体进行Western blotting检测。结果显示多基因共表达载体pcDNA-CEAB在真核细胞HEK 293T中得到表达,且CFP10、ESAT6、Ag85A和Ag85B抗原能被相应的特异性抗体所识别,表明质粒pcDNA-CEAB构建正确,这为进一步研究其免疫原性和免疫保护效果奠定了基础。     

用小鼠模型分析可溶性表达结核分枝杆菌Ag85A的免疫原性

【目的】可溶性表达结核分枝杆菌Ag85A蛋白,并评价其免疫原性。【方法】利用冷休克表达质粒和含有伴侣质粒的大肠杆菌对Ag85A蛋白进行可溶性原核表达,并进行纯化与鉴定,通过C57BL/6小鼠模型对Ag85A蛋白的免疫原性,包括诱导机体特异性体液免疫应答和细胞免疫应答水平进行分析。【结果】重组菌诱导后裂解上清中检测到可溶性Ag85A蛋白的表达,经过亲和层析纯化收获了纯度在90%以上的Ag85A蛋白,Western blot鉴定显示其具有较好的免疫反应性。Ag85A蛋白免疫小鼠后,血清中可以检测到高水平的Ig G抗体效价,其中Ig G2b水平要高于Ig G1。通过特异性多肽、蛋白刺激脾脏和腹股沟淋巴结细胞可分泌高水平的IFN-γ、TNF-α等Th1型细胞因子。【结论】实现了Ag85A蛋白的可溶性表达,免疫特性评价显示Ag85A蛋白可诱导机体产生强烈的特异性体液免疫应答及Th1型的细胞免疫应答,从而为其进一步免疫学功能的研究奠定了重要基础。     

真核表达编码Ag85A基因重组体的构建

抗原85复合体（Ag85）是BCG合成的能够刺激机体产生细胞免疫和体液免疫的多种成分之一,Ag85A是抗原85复合体组成成分之一,可显著刺激细胞免疫功能增强。为研究经口接种Ag85A的DNA疫苗的免疫效应,根据结核分枝杆菌Ag85A的基因序列自行设计了一对PCR引物,以人型结核杆菌H37Rv标准毒力株的DNA为模板,经过PCR扩增出Ag85A目的基因,纯化PCR产物TA克隆入载体pUCm-T载体,蓝白斑筛选将回收的PCR产物用限制性核酸内切酶Xhol和BamHI双酶酶切后,经T4DNA连接酶作用,与真核表达载体pCDNA3．1^＋连接,筛选得到的阳性克隆经DNA测序鉴定证实为Ag85A基因,且被克隆到载体pCDNA3．1^＋中的CMV启动子的下游,成功构建并鉴定的真核表达载体pCDNA3．1^＋携带Ag85A基因的重组体,命名为pCDNA3．1^＋／Ag85A。将其转化大肠埃希菌并使之大量扩增,并采用无内毒素提取质粒方法收集此重组质粒DNA．即为可经口途径喂饲小鼠的结核杆菌Ag85A的DNA疫苗,为口服DNA疫苗的临床应用研究奠定基础。     

用小鼠模型分析表达牛结核分枝杆菌Ag85B重组腺病毒的细胞免疫特性

【目的】构建表达牛结核分枝杆菌抗原Ag85B重组腺病毒rAd-Ag85B,并用小鼠模型分析其细胞免疫特点。【方法】采用PCR方法,扩增结核分枝杆菌Ag85B的编码基因fbpB,测序后构建pDC516-Ag85B重组质粒。利用脂质体将pDC516-Ag85B与pBHGfrtΔE1,3FLP共转染Ad293细胞包装成重组病毒rAd-Ag85B。空斑纯化后用电镜负染、目的基因转录和蛋白表达进行rAd-Ag85B验证。同时将rAd-Ag85B和rAd(wtAd)分别经皮下注射免疫BALB/c小鼠,二免二周后取小鼠脾脏细胞,进行CD69表面分子表达、淋巴细胞增殖和ELISPOT实验分析。【结果】在电镜下能观察到包装的重组病毒粒子,且在转录和蛋白水平上验证了rAd-Ag85B构建成功。免疫试验结果显示,rAd-Ag85B能激活CD4+T和CD8+T细胞表面分子CD69的表达,并引起淋巴细胞增殖。ELISPOT表明rAd-Ag85B呈现Th1免疫特点。【结论】成功构建的rAd-Ag85B能引起机体针对PPD蛋白或Ag85B多肽的Th1免疫应答。     

新型抗结核蛋白亚单位疫苗Ag85A-γ干扰素的免疫效果评价

本研究旨在评价新型抗结核融合蛋白亚单位疫苗Ag85A-γ干扰素(Ag85A-IFN-γ)的免疫效果.在成功表达并纯化了去除Ag85A信号肽的融合蛋白Ag85A-IFN-γ后,将其与免疫佐剂二甲基三十六烷基铵(DDA)混合,皮下免疫C57BL/6小鼠3次,每次间隔2周,末次免疫2周后进行效果评价.采用酶联免疫吸附试验(ELISA)检测血清中IgG、IgG1和IgG2c水平.结果显示,融合蛋白Ag85A-IFN-γ组的IgG水平均高于单独抗原组,且融合蛋白组IgG2c/IgG1 比值显著高于对照组,表明融合蛋白能刺激宿主产生更强烈的体液免疫反应,更倾向于激发T辅助细胞1型(Th1型)免疫反应.流式细胞术检测特异性CD4+和CD8+ T细胞比例,发现Ag85A-IFN-γ组CD8+/CD4+最高,显示该融合蛋白能显著增强CD8+ T细胞增殖.上述结果表明,融合蛋白Ag85A-IFN-γ有望成为有效的新型抗结核融合蛋白亚单位疫苗.     

Attempted immunotherapy for Mycobacterium tuberculosis with viral and protein vaccines based on Ag85B-ESAT6 in a mouse model

The increasing threat of drug-resistant strains of Mycobacterium tuberculosis (M. tb) and co-infection with human immunodeficiency virus (HIV) has worsened the international public health crisis and challenged conventional chemotherapy. Therapeutic vaccines, which possess the capacity to stimulate the immune system and affect the disease progression, deserve reconsideration to aid chemotherapy. Vaccines based on Ag85B-ESAT6 fusion protein were tested as potential immunotherapeutic vaccines against ongoing intravenous infection in a mouse model. Therapeutic efficacy was evaluated by enumeration of bacilli in infected tissues and by histological examination of the lungs. Ag85B-ESAT6 with the adjuvant dimethyl dioctadecylammonium bromide (DDA) - monophosphoryl lipid A (MPL) did not reduce bacterial load, however induced a sharp weight loss and worsened pathology. Recombinant virus-based vaccines failed to protect mice against tuberculosis either. More efforts should be taken to search for protective candidates and elucidate the mechanism for immunotherapy.     

Ag85A真核表达重组体的构建及稳定转染细胞系的建立

构建结核杆菌抗原85A（AgS5A）的真核表达重组体,转染L929细胞,建立稳定转染细胞系。从质粒V1 Jns．tPA—Ag85A中经PCR扩增出Ag85A基因,利用DNA重组技术将其插入到真核表达载体peDNA3．1／myc—HisA中,经酶切和测序鉴定后,脂质体转染法转染L929细胞,通过G418选择培养,建立稳定转染细胞系,Western Blot检测Ag85A的表达。成功构建pcDNA3．1／mye—HisA—Ag85A真核表达载体并稳定转染L929细胞,成功表达了目的基因。为进一步研究Ag85ADNA疫苗对结核杆菌的免疫防护作用奠定了基础。     

Immune responses and protective efficacy of the gene vaccine expressing Ag85B and ESAT6 fusion protein from Mycobacterium tuberculosis

Genetic immunity is a new promising approach for the development of novel tuberculosis vaccines. In this study, it is shown that DNA vaccines expressing the fusion protein of antigen 85B (Ag85B) and early secreted antigenic target 6-kDa antigen (ESAT6) can induce high levels of specific IgG2a antibody subtype in the mice. With the prolongation of postimmunization time, the levels of IgG2a antibody decrease gradually. Although a high-level specific IgG2a antibody subtype is also elicited by classical BCG, the ratio of antibody subtypes IgG2a to IgG1 changes 4 weeks after immunization, and IgG1 is gradually shifted to the main antibody subtype. DNA vaccines also elicit cellular immunity as shown by specific spleen lymphocytes proliferation to Ag85B or ESAT6 protein and the production of high levels of IFN-gamma and IL-2, which is similar to that elicited by BCG. Vaccination of mice with DNA vaccines expressing the fusion protein Ag85B-ESAT6 results in a significant level of protection against the subsequent high-dose challenge with virulent Mycobacterium tuberculosis (MTB) H37Rv. Dramatic reduction in the number of MTB colony-forming units in the spleens and lungs is observed. Pathological examination showed that recombinant plasmid and BCG groups have only minor damage and organizational structures that are kept relatively complete, while in the control group, spleens and lungs are damaged seriously. Therefore, although the reducing degree of mycobacterial loads in the organ of mice immunized with recombinant plasmid is not more than that of BCG, through the analysis of pathological changes, we may conclude that the protective effect provided by DNA vaccine expressing the Ag85B-ESAT6 fusion protein is equivalent to that afforded by the classical BCG.     

共表达结核杆菌Ag85A-ESAT-6融合抗原与IL-21核酸疫苗的构建及免疫效果研究

目的：构建结核杆菌Ag85A-ESAT-6融合抗原与细胞因子IL-21共表达核酸疫苗pIRES-IL-21-Ag85A-ESAT-6,研究其对小鼠免疫功能的影响。方法：用PCR方法分别扩增出Ag85A和IL-21编码基因,并先后插入质粒pIRES-ESAT-6中,构成共表达核酸疫苗pIRES-IL-21-Ag85A-ESAT-6;免疫小鼠后,通过CTL和NK细胞杀伤活性和脾细胞增殖试验,以及小鼠血清抗体、IFN-γ和IL-4的检测,评价该核酸疫苗的免疫效果。结果：pIRES-IL-21-Ag85A-ESAT-6核酸疫苗免疫鼠的CTL活性、脾细胞增殖反应、IFN-γ水平及血清抗体产生明显高于对照组,差异有显著性意义。结论：pIRES-IL-21-Ag85A-ESAT-6核酸疫苗能够诱导小鼠产生有效的免疫反应,可作为新型结核疫苗的候选组分。     

The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B-DNA vaccine into a potent but non-protective Th1 immune response in mice

Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B-encoding plasmid DNA (DNA-85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B-specific CD4+ T cells producing IFN-gamma and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4+ T cell proliferation and IFN-gamma production in response to Ag85B protein, despite restraint of Th1 response by CD8+ T cell-dependent mechanisms and activation of CD4+ T cell-dependent IL-10 secretion. Importantly, these Ag85B-responding CD4+ T cells lost the ability to produce IFN-gamma and control MTB intramacrophagic growth in coculture with MTB-infected macrophages, suggesting that the protein-dependent expansion of non-protective CD4+ T cells determined dilution or loss of the protective Ag85B-specific CD4+ induced by DNA-85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA-priming, protein-booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective anti-tuberculosis immune response, and actually concur to disease progression.     

Serum antigen 85 levels in adjunct testing for active mycobacterial infections in orangutans

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.