结核分枝杆菌Rv0081基因编码蛋白的生物信息学分析

运用生物信息学分析软件预测结核分枝杆菌（Mycobacterium tuberculosis, Mtb）Rv0081蛋白的生物学特征及筛选潜在的优势抗原表位。 从NCBI数据库获取Mtb Rv0081蛋白的氨基酸序列，利用生物信息学分析软件ProtParam、ProtScale及TMPRED分析Rv0081蛋白的理化性质及亲疏水性；TMHMM、SignalP-5.0 Server预测蛋白的跨膜区及信号肽；NetNGlyc-1.0 Server、NetPhos 3.1 Server分别预测蛋白的糖基化位点及磷酸化位点；STRING预测能与Rv0081相互作用的蛋白；分别运用SOPMA、SWISS-MODEL预测蛋白的二、三级结构；综合运用softberry、WoLF PSORT预测蛋白的亚细胞定位；运用DNAStar预测蛋白的B细胞抗原表位；综合运用SYFPEITHI、NetCTL 1.2 Server、Net MHC pan 4.1 server预测蛋白的CTL细胞抗原表位；综合运用SYFPEITHI、Net MHCII pan 4.0 server预测蛋白的Th细胞抗原表位。 结果表明，Rv0081蛋白由114个氨基酸组成，相对分子质量为12 356.32，亚细胞定位于细胞质中，为稳定的疏水性蛋白，无跨膜区和信号肽，含有1个糖基化位点及9个磷酸化位点；二级结构主要由α-螺旋和无规则卷曲构成，结构较松散；与hycE、hycP、Rv0088、Rv0083、hycD、hycQ、Rv0082、devR、Rv0080及Rv0079蛋白存在相互作用关系；综合分析各软件预测结果筛选出6个优势B细胞抗原表位、6个优势CTL细胞抗原表位及7个优势Th细胞抗原表位。Mtb Rv0081蛋白具有较多潜在的候选B、T细胞抗原表位，可作为研发新型结核疫苗的候选抗原。     

结核分枝杆菌Rv1385的抗原表位预测分析

结核分枝杆菌生长缓慢,难以获得足量的天然蛋白抗原。而利用基因工程技术制备重组蛋白抗原,存在着表达量低、不易纯化,以及特异性较低等不利因素。随着生物信息技术的发展,研究者可根据生物信息学软件预测,选择合成优势抗原肽段,而不需克隆整个蛋白,具有简便、经济、特异性高等特点。因此,本研究应用生物信息学方法,预测结核分枝杆菌Rv1385蛋白的抗原表位并分析其优势表位,对了解Rv1385蛋白的免疫学特性及其与结核分枝杆菌致病的关系具有重要意义。首先,从NCBI数据库下载Rv1385的氨基酸序列,然后采用生物信息学软件PSIPRED Server预测蛋白质二级结构;BepiPred 1.0 Server和ABCpred预测该蛋白的B细胞抗原表位;BIMAS、SYFPEITHI及NetCTLpan 1.1 Server预测该蛋白的CTL表位;SYFPEITHI和NetMHCIIpan 3.2 Server预测该蛋白的Th细胞表位。最后综合分析预测结核分枝杆菌Rv1385的优势候选抗原表位。结果显示,Rv1385蛋白二级结构中,α螺旋占51.8%,β折叠占41.6%,无规卷曲占6.6%;共有4个B细胞抗原位点,位于109～124、152～169、178～192、198～209氨基酸区段;3个CTL表位,位于263～271、87～95、240～248氨基酸区段;5个Th表位,位于77～91、87～101、234～247、70～84、46～60氨基酸区段。生物信息学分析显示Rv1385含有潜在的B细胞和T细胞抗原表位,为该蛋白抗原表位的进一步研究及应用奠定了基础。     

T细胞抗原表位预测的研究方法进展

确定T细胞所识别抗原分子上的短肽序列对T细胞表位进行定位,对于研究特异性免疫应答有着重要意义。综述了近年来实验确定和理论预测T细胞蛋白质抗原袁位的常用方法,以及T细胞抗原表位分析的研究方法。     

结核分枝杆菌多表位诊断抗原的制备

将结核分枝杆菌(Mycobacterium tuberculosis,Mtb)多个B细胞预测表位串联表达(命名为B102),并初步评价其作为诊断抗原的血清学诊断价值。将MtbPstS1、ESAT6、CFP10、Ag85B、Ag85A及PPE54等6个蛋白的11个B细胞预测表位串联,加入合适的连接臂后全基因合成;将多表位片段插入带有TRX标签的表达质粒中,在大肠杆菌BL21(DE3)中诱导表达,并利用Ni~(2+)-Chelating亲和层析和DEAE阴离子交换层析纯化目的蛋白;利用Western blotting (WB)技术对目的蛋白抗原性进行鉴定,并建立Mtb抗体检测竞争法ELISA技术,初步评价此方法对阴阳血清样本的鉴别能力。目的蛋白以包涵体形式存在,其表达量约占菌体总蛋白的31.25%,经纯化及复性后蛋白B102可溶性存在,浓度为3.124mg/mL,纯度为96.71%;WB实验表明目的蛋白能与Mtb阳性血清相应抗体发生反应。对60份Mtb阳性血清及60份Mtb阴性血清进行检测得出其灵敏度为90.00%,特异性为93.33%,阳性预测值为93.10%,阴性预测值为90.32%,符合率为91.67%,McNemer检验的结果提示与"金标准"诊断结果无差异,Kappa=0.833,提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的Mtb多表位诊断抗原,作为诊断抗原可以应用于Mtb的血清学检测中。     

结核分枝杆菌Rv3871抗原优势肽段的克隆表达及抗原性鉴定

目的:为了提高结核病诊断试剂的特异性和敏感性,克隆表达结核分枝杆菌H37Rv株RD1区Rv3871抗原优势肽段,并应用ELISA法对其抗原性进行初步鉴定。方法:利用Biosun生物信息学软件对Rv3871抗原进行表位分析,通过PCR从结核分枝杆菌H37Rv基因组中扩增Rv3871抗原优势肽段编码基因,在大肠杆菌HB101中进行表达,采用间接ELISA法初步评价其抗原性。结果:Rv3871-1肽段检测的敏感性为32.5%(13/40),特异性为97.2%(35/36);Rv3871-2敏感性为45%(18/40),特异性为100%;Rv3871-3敏感性为37.5%(15/40),特异性为91.6%。结论:结核分枝杆菌RD1区Rv3871抗原优势肽段Rv3871-2有较高的特异性和敏感性,有望作为候选抗原用于结核病患者的血清学检测。     

结核分枝杆菌Rv3369基因的表达和纯化

在大肠杆菌中高效表达结核分枝杆菌Rv3369蛋白,获得纯化的重组蛋白rRv3369。通过聚合酶链反应(Polymerase chain reaction,PCR)扩增Rv3369基因;以质粒pET28a为表达载体,构建重组质粒,转化大肠杆菌BL21(DE3);以异丙基硫代半乳糖苷(IPTG)诱导表达目的蛋白,通过SDS-PAGE鉴定rRv3369在大肠杆菌中的表达,确定rRv3369在大肠杆菌中的表达形式;采用Ni-NTA His.Bind Resin来纯化重组蛋白。重组质粒pET28a-Rv3369中目的基因测序结果与报道序列相同。分子量约19.5kDa,表达量约占菌体总蛋白的18%,纯化后的重组蛋白样品经SDS-PAGE和激光密度扫描分析表明其纯度为90%以上,每100mL培养菌可获得1.56mg左右的重组蛋白。用亲和层析法纯化的重组蛋白纯度较好。     

结核分枝杆菌持续感染期抗原Rv1733c 的表达、纯化和鉴定

目的:克隆结核分枝杆菌持续感染期抗原Rv1733c基因,构建其原核表达载体,并在大肠杆菌中进行表达和纯化。方法:采用聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv基因组中扩增出Rv1733c基因片段,克隆入pMD18-T载体,序列测定正确后将其亚克隆入原核表达载体pPro-EXHTb,并在大肠杆菌DH5α中进行表达,表达蛋白经SDS-PAGE及Western-blot分析后,以Ni-NTA亲和层析纯化蛋白。结果:成功克隆了Rv1733c基因片段并构建了其原核表达载体pPro-EXHTb-1733c,转化E.ColiDH5α后能表达大小约30 KD的蛋白,Western-blot分析表明表达产物正确。通过亲和层析获得纯化蛋白。结论:成功构建结核分枝杆菌持续感染期抗原Rv1733c原核表达载体pPro-EXHTb-1733c,并获得纯化蛋白,为研究新型结核疫苗的靶抗原奠定了基础。     

范可尼贫血互补群E基本特性及抗原表位的生物信息学分析

范可尼贫血互补群E(FANCE)属于FA家族的重要成员之一,参与DNA链间交联损伤修复并在某些肿瘤中起重要作用。本研究运用生物信息学方法对FANCE的同源性、理化性质、亲/疏水性、信号肽、亚细胞定位、跨膜结构、蛋白结构、蛋白质互相作用、B/T细胞优势抗原表位以及肿瘤相关性进行预测。FANCE为无信号肽和跨膜区的疏水性蛋白,主要分布在细胞核和细胞质,具有较高的保守性。其二级结构以α-螺旋和无规则卷曲为主,含有2个N-糖基化、9个O-糖基化以及48个磷酸化位点,与FANCM、FANCD2、FANCC等蛋白发生相互作用。FANCE具有多个潜在的B/T细胞优势抗原表位和17个抗原决定簇,在急性髓性白血病（LAML）和皮肤黑色瘤（SKCM）中低表达,其低表达显著影响患者总生存率。这为深入研究FANCE在肿瘤中的分子机制提供理论依据,使FANCE可能成为新的治疗靶点。     

细胞毒性T细胞表位预测

细胞毒性T细胞表位是由8～10个连续的氨基酸残基构成的特殊肽段。本文对基于基序、基于结构、基于人工神经网络和隐马尔可夫模型的各类细胞毒性T细胞表位预测方法进行了综述。     

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.     

结核分枝杆菌Rv2628蛋白的免疫生物学特性

Rv2628蛋白是结核分枝杆菌Mycobacterium tuberculosis(M.tb)DosR调控的潜伏感染相关抗原。本研究对Rv2628蛋白进行了原核表达和纯化,并以巨噬细胞系和小鼠为研究模型,对其免疫生物学特性进行了分析。SDS-PAGE及Western blotting鉴定结果表明,Rv2628-His融合蛋白以包涵体形式表达,能与兔抗H37Rv多抗血清发生特异性反应,具有较好的免疫反应性。与巨噬细胞系RAW264.7的互作实验结果表明,在1–12 h内Rv2628蛋白能诱导前炎性因子IL-6的上调表达。将纯化的Rv2628融合蛋白皮下免疫BALB/c小鼠,夹心ELISA的测定结果表明,Rv2628蛋白免疫组诱导产生的特异性IFN-γ水平显著高于IL-4的水平(P0.000 1),呈现Th1型细胞免疫应答趋势;以Rv262811-30多肽作为包被抗原,通过间接ELISA测定的血清抗体效价能达到11 600,表明Rv2628也能诱导体液免疫应答。总之,Rv2628能促进巨噬细胞炎症反应的发生,激发小鼠产生强烈的Th1型细胞免疫应答和较好的体液免疫应答,具有作为亚单位疫苗的潜力,为M.tb与宿主之间的相互作用奠定了一定的理论基础。     

Mycobacterium smegmatis displays the Mycobacterium tuberculosis virulence-related neutral red character when expressing the Rv0577 gene

Neutral red staining is a cytochemical reaction that has been found to be related to Mycobacterium tuberculosis virulence and, therefore, the component involved in it is thought to be a virulence factor. To study the molecular basis of this reaction we constructed an M. tuberculosis cosmid library in Mycobacterium smegmatis and selected recombinant neutral red positive clones. Heterologous complementation identified Rv0577 as the gene responsible for this trait and we have also shown that it is expressed as a single polycistronic unit together with Rv0576 which could also be involved in the neutral red staining.     

Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes

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Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.     

结核分枝杆菌蛋白Rv3425对细菌表型及毒力影响的研究

为探索蛋白Rv3425在结核分枝杆菌(Mycobacterium tuberculosis,M. tuberculosis)中的功能,本研究以耻垢分枝杆菌(Mycobacterium smegmatis,M. smegmatis)为模式菌株,构建重组了耻垢分枝杆菌Ms-Rv3425。分别将构建菌株(Ms-Rv3425)、野生株(Ms)及空载对照(Ms-Pact)接种于7H9-OADC培养基中37 ℃培养,观察Ms-Rv3425与Ms及Ms-Pact之间在生长速率、菌落形态、生物膜以及聚集度方面的差异。分别用低pH值以及含有十二烷基磺酸钠(sodium dodecyl sulfate,SDS)、氨苄西林、异烟肼及利福平的培养基进行培养,计算存活率以分析抗逆和抗药能力;用上述压力条件培养结核分枝杆菌标准株H37Ra,分析Rv3425内源表达量的变化;进行THP-1细胞感染和BALB/c小鼠攻毒实验分析菌株的毒性变化。结果显示,与Ms及Ms-Pact相比,Ms-Rv3425的菌落形态更为粗糙且隆起,成膜及聚集能力增强;在压力条件下,Ms-Rv3425表现出更高的抗逆和抗药能力,H37Ra中Rv3425的表达量也显著上调;胞内存活率及小鼠致死率更高,各脏器病理损伤更为严重。综上所述,过表达Rv3425能够改变耻垢分枝杆菌的表型,提高抗逆性、抗药性和毒力。深入探讨PPE家族蛋白Rv3425的功能,将为结核病的防治带来新的视角。     

Rv1268c protein peptide inhibiting Mycobacterium tuberculosis H37Rv entry to target cells

Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease.The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells (¹⁴¹TGMAALEQYLGSGHAVIVSI¹⁶⁰) and other binding U937 monocyte-derived macrophages (²¹AVALGLASPADAAAGTMYGD⁴⁰). Such sequences’ ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure–function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine.