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.     

血清学诊断中结核杆菌磷酸烯醇型丙酮酸羧激酶的应用

[目的]在原核系统中表达结核杆菌磷酸烯醇型丙酮酸羧激酶(phosphoenolpyruvate car-boxykinase PEPCK),并研究该蛋白在诊断结核病人血清抗体中的应用价值.[方法]应用基因重组技术表达重组蛋白结核杆菌磷酸烯醇型丙酮酸羧激酶,经亲和层析法纯化表达产物.用表达的重组蛋白免疫小鼠,研究其免疫学特性.间接酶联免疫吸附试验(Enzyme link immunosorbent assay,ELISA)检测结核病人血清中特异性IgG抗体,并与结核杆菌抗体胶体金法诊断试剂盒检测结果对比.[结果]试验表明转化入大肠杆菌中的重组质粒能够表达并纯化出相对分子量为72 kDa的重组蛋白;Western blot证实重组蛋白能够与小鼠抗BCG血清发生特异性反应;重组蛋白免疫小鼠后,小鼠血清中的抗体滴度可达1∶1280以上;重组蛋白用作ELISA包被抗原检测病人血清阳性率为17.3%(30/173),其中排菌病人的阳性率为32.5%(13/42),不排菌病人的阳性率为12.9%.该方法结果与结核杆菌抗体胶体金法诊断试剂盒的检测结果相比,敏感性为51.0%,特异性为96.7%.[结论]结核杆菌PEPCK具有较好的免疫原性和抗原性,有可能作为结核病血清学诊断的一组抗原之一.     

肺炎衣原体单克隆抗体的研制和应用

目的:以杂交瘤技术制备抗肺炎衣原体(Cpn)单克隆抗体,用于衣原体感染的诊断及相关疾病的研究。方法:以进口Cpn抗原免疫BALB/c小鼠,将免疫小鼠的脾脏细胞与SP2/0细胞融合,用间接ELISA法筛选抗体阳性杂交瘤细胞。收集接种过杂交瘤细胞的小鼠腹水,分别用ELISA法检测抗体效价、用免疫琼脂扩散试验鉴定单抗的类别、用微量荧光免疫试验(MIF)检测单抗的种属特异性。用克隆表达的主要外膜蛋白(MOMP)通过Dot-ELISA法分析单抗的特异性。通过建立直接免疫荧光法(DIF)检测病人和正常人外周血单核细胞(PBMC)标本,并进行统计处理。结果:小鼠脾脏细胞与SP2/0细胞的融合率为61.46%(236/384),最终获得4株稳定分泌Cpn单抗的细胞株。用ELISA法检测小鼠腹水,效价高者可达1∶100000。免疫琼脂扩散试验鉴定为IgG类单抗,扩散效价达1∶128。自制单抗能与重组MOMP发生结合反应,表明其为抗CpnMOMP抗体。自制单抗与进口单抗类似,即与鹦鹉热衣原体(Cps)出现一定程度的交叉反应,而与沙眼衣原体(Ct)则无交叉反应。对240份PBMC标本用自制单抗和进口单抗同时检测Cpn抗原,2种单抗检测均阳性的共86份,经SPSS软件分析两者具有较好的一致性。DIF检测显示,心血管疾病和呼吸道疾病Cpn抗原阳性检出率分别为69.34%(95/137)和72.06%(49/68),与正常人标本Cpn抗原阳性率相比,均具有显著性差异。结论:获得IgG类抗CpnMOMP单抗,自制Cpn单抗的特异性和敏感性均与进口单抗具有较好的一致性。PBMCCpn抗原检测的统计分析证实,对于动脉粥样硬化等某些疾病的发生和发展,Cpn感染可能是重要的原因之一,但其中的因果关系还有待深入研究。     

布鲁菌bp26基因的原核表达及间接ELISA方法的临床初步应用

构建诊断人布病的优势抗原BP26原核表达载体,并建立以该蛋白作为包被抗原的间接ELISA方法。人工合成bp26基因片段,构建原核表达载体pET30a-bp26,并转化入大肠埃希菌BL21(DE3)中,IPTG诱导表达,Ni柱亲和纯化BP26重组蛋白,间接ELISA检测其抗原活性,并优化间接ELISA方法的各个参数,检测临床大量样本,对该方法性能进行评价。PCR鉴定及DNA测序证实pET30a-bp26重组质粒构建成功,并在0.8 mmol/L IPTG 20 ℃过夜诱导的条件下大量表达,经亲和纯化获得高纯度的可溶性BP26重组蛋白,间接ELISA结果显示该重组蛋白具有抗原活性,优化参数后,该方法的敏感度为70.00%,特异性为94.74%。成功获得高抗原活性的原核表达可溶性BP26重组蛋白,并建立用于临床特异性诊断人类布病的间接ELISA方法。     

基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价

【目的】猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)是近年来新发现的一种猪肠道冠状病毒,2014年首次暴发于美国,随后亚洲多个国家也相继报道,对养猪业构成了巨大威胁。以大肠杆菌表达纯化的PDCoV重组核衣壳(N)蛋白为包被抗原,建立检测PDCoV抗体的间接ELISA方法,为PDCoV的血清抗体检测和流行病学调查提供工具。【方法】以PDCoV CHN-HN-2014株的基因组RNA为模板,通过RT-PCR扩增PDCoV核衣壳蛋白(N)基因的全长cDNA,将其插入原核表达载体pET-30a中,构建原核表达质粒p ET30a-N,转化大肠杆菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,以纯化的重组N蛋白为包被抗原,建立PDCoV N-ELISA抗体检测方法,评估其特异性、敏感性、稳定性,并用于临床血清的检测。【结果】SDS-PAGE电泳检测证实表达的重组N蛋白主要以可溶性形式存在,Western blotting证实表达的重组蛋白具有反应活性。用纯化的重组蛋白建立的N-ELISA具有良好的特异性、敏感性、稳定性。与中和试验同时检测148份免疫猪血清和102份临床血清,两种方法的阳性符合率为88.99%,阴性符合率为92.90%,总符合率为91.20%。用建立的ELISA方法检测267份临床血清,PDCoV抗体阳性血清的比率为66.67%。【结论】建立的猪德尔塔冠状病毒N-ELISA抗体检测方法与中和试验的符合率高,可用于PDCoV血清抗体检测和流行病学调查。     

猪δ冠状病毒(PDCoV)N蛋白的原核表达及多克隆抗体制备

旨在表达和纯化猪δ冠状病毒(PDCoV)N蛋白并制备该蛋白的多克隆抗体。以RT-PCR扩增PDCoV N基因并与表达载体pET-28a构建重组质粒,转化Transetta(DE3)菌株诱导表达,SDS-PAGE鉴定融合蛋白表达,以纯化的N蛋白免疫家兔制备多克隆抗体,Western blot验证兔抗血清特异性,间接ELISA测定抗血清效价。利用间接免疫荧光试验(IFA)、免疫荧光试验(IF)、流式细胞术(FCM)鉴定其诊断应用价值。重组N蛋白为可溶性表达,大小约为44 kD,制备的兔抗N蛋白抗体效价可达1∶204 800。IFA与FCM试验证实该抗体能与PDCoV特异性结合,与PEDV及TGEV无交叉反应,IF试验表明该抗体可用于检测小肠组织中的PDCoV。     

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。     

肺炎支原体P1重组蛋白的提取纯化及应用研究

提取纯化肺炎支原体(Mycoplasma pneumoniae,Mp)P1重组蛋白,建立酶联免疫吸附实验(ELISA)的方法,协助临床肺炎支原体感染的诊断。以GST融合蛋白层析柱提取、纯化Mp P1重组蛋白做抗原,以全肺炎支原体菌体成分做抗原对照,建立间接ELISA实验方法,检测40份正常献血者血清标本和51份疑似MP感染临床血清标本的IgG抗体。重组蛋白经SDS-PAGE可见诱导表达的样品在分子量大约59 ku处有明显条带,经Western blotting可与肺炎支原体免疫血清发生反应。ELISA实验检测51份临床标本,由P1重组蛋白抗原检测阳性31份,阳性率为60.78%。Mp检测阳性20份,阳性率为39.22%。实验精确度检测阳性混合血清的变异系数(CV值)为5.40%,阴性混合血清变异系数为1.10%。用Mp P1重组蛋白抗原建立的ELISA检测方法,其敏感性高于全肺炎支原体抗原,可用于临床肺炎支原体感染的诊断。     

鸭疫里氏杆菌多种血清型间接ELISA检测方法的建立

【目的】鸭疫里氏杆菌(Riemerella anatipestifer,RA)是一种重要的禽病病原,分为21个血清型。但一直缺乏一种针对多种血清型广泛适用的抗体检测方法。前期的研究表明,外膜蛋白A (Outer membrane protein A,OmpA)广泛存在于多种血清型的RA菌株中,是一种重要的免疫原性蛋白,并且其基因序列在RA血清型之间具有高度的保守性,提示其可以作为RA感染血清抗体检测的靶点分子。以重组蛋白OmpA建立间接酶联免疫吸附试验检测RA的抗体。【方法】通过诱导表达条件的摸索及蛋白纯化,获得适用于ELISA包被的重组OmpA抗原。通过Western-blot证明重组蛋白OmpA是否与RA多种血清型发生免疫学反应。进行方阵试验以确定ELISA抗原的最佳包被浓度、被检测血清的反应浓度。重复性、特异性和敏感性试验检查该方法的实用性。【结果】实验证实加入1%乙醇的诱导培养基有利于重组蛋白的可溶性表达。Western-blot结果表明,重组蛋白OmpA可以与1、2、6、10、11、13、14和17型多种RA主要流行血清型有良好的免疫反应性。经方阵试验确定抗原的最佳包被浓度为8 mg/L,待检血清的最佳稀释度为1:160。所建立检测方法具有良好的重复性、特异性和敏感性。【结论】实验建立的鸭疫里氏杆菌多种血清型间接ELISA检测方法可以用于免疫后抗体消长以及感染性抗体的检测。     

猪细小病毒NS1抗原表位的真核表达及ELISA方法的建立

以猪细小病毒NS1抗原表位的真核表达及ELISA建立为目的,以猪细小病毒基因组DNA为模板,扩增获得PPVNS1主要抗原表位基因,将其插入到真核表达载体pPICZα-A中,获得重组质粒pPICZα-A-NS1。电转化后将重组毕赤酵母菌株诱导表达,SDS-PAGE检测证实重组蛋白获得了高效表达,Western blot实验表明表达产物具有良好的反应原性。将纯化后的重组蛋白包被酶标板,经棋盘法优化,初步建立了检测猪细小病毒特异性抗体的间接ELISA方法。结果表明,抗原最佳包被浓度为1.9μg/mL,血清最佳稀释度为1∶80,阳性判断标准定为OD待检血清>0.4且OD待检血清/OD标准阴性值>2.0。用该方法对猪血清样品进行检测,结果显示本方法与HI试验的符合率达91.2%,与国外同类试剂盒的符合率为92.1%,该间接ELISA方法特异性强、敏感性高,可用于猪细小病毒病感染抗体的检测。     

沙眼衣原体L2型主要外膜蛋白的原核表达及其抗原性分析

克隆表达沙眼衣原体(Ct)L2血清型的主要外膜蛋白(MOMP)基因,并鉴定重组MOMP(rMOMP)的抗原性,为进一步研究Ct感染的诊断和预防技术奠定基础。应用PCR技术对CtL2型标准株的MOMP基因进行特异性扩增,将扩增产物克隆入表达载体pET-32a(+),成功构建了rMOMP-pET-32a(+)表达质粒,转化大肠杆菌BL21(DE3)后摇菌进行rMOMP的诱导表达、鉴定和纯化,免疫印迹和ELISA法分析显示rMOMP可与兔源抗CtL2多克隆抗体发生特异性反应,表明rMOMP具有良好的抗原性。     

猴B病毒被膜糖蛋白gC的原核表达及诊断价值评价

目的:原核表达猴B病毒糖蛋白gC胞外区片段,评价其在猴B病毒血清学检测中的特异性和敏感性,为猴B病毒检测奠定基础。方法:利用PCR方法扩增gC胞外区基因片段,同时合成该片段的优化密码子序列,将其插入表达载体pET-28b(+)形成重组质粒,转化至大肠杆菌BL21(DE3)并进行诱导表达;纯化蛋白先以Western印迹进行验证,再以ELISA方法和标准阳性、阴性血清评价其诊断价值。结果:直接从病毒DNA模板上获得的基因片段未能表达,而优化后的基因片段表达水平较高,表达蛋白的相对分子质量约为48×103,为包涵体形式,占菌体总蛋白的30%左右;Western印迹显示,重组蛋白能与猴B病毒阳性血清和His单克隆抗体发生免疫反应;34份标准阳性血清和25份阴性血清的ELISA检测结果显示,重组蛋白的敏感性和特异性分别为94.1%(32/34)和100%(25/25)。结论:利用原核表达系统制备的gC胞外区重组蛋白,可作为猴B病毒血清学检测抗原,其特异性和敏感性都较好。     

Outer membrane complex proteins of Chlamydia pneumoniae

Abstract The protein composition of the outer membrane complex (OMC) of Chlamydia pneumoniae strain AR-39 was analyzed by metabolic labeling with [³⁵S]methionine and [³⁵S]cysteine. Cysteine-rich proteins with molecular masses of 98, 60 doublet, 39.5 (MOMP) and 15.5 kDa were found in the OMC of C. pneumoniae . The cysteine-rich proteins of the OMCs of the threee Chlamydia species showed specific reaction patterns by immunoassay and autoradiography to rabbit or turkey immune sera. Recognition of the MOMP and 60-kDa proteins of the three species was cross-reactive. However, the C. pneumoniae 98-kDa protein was recognized by anti- C. pneumoniae (AR-39) and anti- C. psittaci (TT3) immune sera. None of the immunee sera recognized the 12-kDa cysteine-rich complex.     

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.     

Systemic and mucosal antibody response in experimental Chlamydia pneumoniae infection of mice

Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.     

猪戊型肝炎病毒基因ORF2片段AG02的融合表达及间接ELISA方法的初步建立

采用巢式RT-PCR技术扩增猪戊型肝炎病毒（SHEV）结构蛋白基因ORF2部分片段AG02。将该片段插入pET32a表达载体,命名为PET-HEV,将重组表达质粒转化大肠杆菌BL21（DE-3）,经1mM IPTG诱导得到表达,进行SDS-PAGE分析,证明该片段得到融合表达,分子量为33KD。Western blot分析表明,重组蛋白可以与HEV阳性血清反应,表明该蛋白具有良好的抗原性。以纯化的重组蛋白建立了初步的间接ELISA方法,经方阵滴定确定最佳包被浓度为2.43μg/mL,血清最佳的稀释比为1:40,与万泰试剂盒相比较,证明我们建立的ELISA方法有较高的特异性和敏感性。     

梅毒螺旋体重组抗原的表达及在梅毒血清学诊断中的应用

目的：克隆、表达梅毒螺旋体（Treponema pallidum, Tp）膜蛋白TpN47、TpN17、TpN44.5和TpN15,建立双抗原夹心ELISA法,探讨其在梅毒血清学诊断中的应用。方法：应用聚合酶链反应法（PCR）从Tp全基因组中扩增4个目的片段,克隆到载体pET-HT-JKM中,在BL21(DE3)plysS菌中诱导表达,Ni-NTA亲和层析法纯化重组蛋白,辣根过氧化物酶标记,双抗原夹心酶联免疫吸附法检测人血清中的特异性IgM和IgG抗体。结果：成功表达了4种重组蛋白用作ELISA包被和酶标抗原检测国家Tp参比血清,特异性和灵敏度均为100％;检测385份临床血清样本,结果与TPPA法相比符合率达到99％（P＜0.01）,灵敏度和特异性分别为98.2%（162/165）和99.5%（219/220）。结论：表达的4种重组抗原具有很好的生物活性,为理想的梅毒的血清学诊断用抗原。以这4种表达蛋白为基础建立的双抗原夹心ELISA法灵敏度高,特异性好,而且方法简单,结果客观,易于自动化操作,值得临床大力推广。     

Serological response to chlamydial infection in sheep, studied by enzyme-linked immunosorbent assay and immunoblotting

Abstract Two enzyme-linked immunosorbent assays (ELISA) using highly purified elementary bodies (EB) of Chlamydia psittaci A/22 strain (ovine) or 6BC strain (psittacine) were set up for the detection of antichlamydial antibodies in sheep. No significant differences were observed between the two ELISAs, whereas these tests proved to be more sensitive than complement fixation test and showed a good correlation ( r = 0.75) with immunofluorescence assay. The periodate treatment of chlamydial antigens modified the results of serological responses studied by ELISA, depending on the sera. The average reduction of ELISA values by periodate was 28%, ranging between 5% and 65%. The immunoblot analysis of sheep sera showed high cross reactivity between the polypeptides of A/22 and 6BC strains. However, some differences were observed. The major outer membrane protein (MOMP) of 6BC strain was recognized at different molecular weight position (40 000 kDa) in comparison with the MOMP of A/22 strain (38 000 kDa). In addition, a clear band of 97 000 kDa was detected by all sheep sera tested with A/22 strain. This band was undetectable in the blots performed with 6BC strain.     

Serological response to chlamydial infection in sheep, studied by enzyme-linked immunosorbent assay and immunoblotting

Two enzyme-linked immunosorbent assays (ELISA) using highly purified elementary bodies (EB) of Chlamydia psittaci A/22 strain (ovine) or 6BC strain (psittacine) were set up for the detection of antichlamydial antibodies in sheep. No significant differences were observed between the two ELISAs, whereas these tests proved to be more sensitive than complement fixation test and showed a good correlation (r = 0.75) with immunofluorescence assay. The periodate treatment of chlamydial antigens modified the results of serological responses studied by ELISA, depending on the sera. The average reduction of ELISA values by periodate was 28%, ranging between 5% and 65%. The immunoblot analysis of sheep sera showed high cross reactivity between the polypeptides of A/22 and 6BC strains. However, some differences were observed. The major outer membrane protein (MOMP) of 6BC strain was recognized at different molecular weight position (40,000 kDa) in comparison with the MOMP of A/22 strain (38,000 kDa). In addition, a clear band of 97,000 kDa was detected by all sheep sera tested with A/22 strain. This band was undetectable in the blots performed with 6BC strain.