肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

肝片吸虫天冬氨酰内肽酶的原核表达及反应原性分析

为了获得肝片吸虫天冬氨酰内肽酶(Fasciola hepatica legumain, FhLEG)基因的重组蛋白,分析其作为诊断抗原的可能性,试验根据GenBank中公布的FhLEG基因序列(登录号为AJ314846)设计特异性引物,以肝片吸虫总RNA为模板通过RT-PCR方法获得FhLEG基因,经克隆后测序并利用DNAStar 7.1、ExPASy和SWISS MODEL软件进行生物学分析。构建表达重组质粒pET-28a(+)-FhLEG,在大肠杆菌Rosetta (DE3)感受态细胞中诱导表达,对表达后的LEG蛋白进行SDS-PAGE检测,纯化后进行Western-blot分析。结果表明:扩增得到的FhLEG核苷酸序列大小为1 260 bp, A+T含量为56.9%,与GenBank中收录的肝片吸虫LEG核苷酸和氨基酸序列的同源性分别为99.6%和99.3%。LEG蛋白主要由6个α-螺旋、3个β-折叠和1个β-转角构成,具有较好的抗原表位,推测该蛋白可能具有较好的抗原性。经SDS-PAGE电泳,在51 ku处检测到目的条带,重组蛋白FhLEG与肝片吸虫阳性血清发生反应,而与阴性血清不发生反应。说明肝片吸虫天冬氨酰内肽酶具有良好的反应原性。     

绵羊肝片吸虫谷胱甘肽硫转移酶基因的克隆与序列分析

本研究扩增肝片吸虫(Fasciola hepatica,Fh)谷胱甘肽硫转移酶(GST)基因,并进行同源性分析。根据GenBank发表的部分Fh GST基因序列设计并合成一对特异性引物,利用RT-PCR方法扩增出Fh GST基因完整的开放阅读框(open reading frame,ORF),测定序列,使用分子生物学软件进行同源性分析。获得Fh GST基因全长682 bp,编码218个氨基酸,与澳大利亚分离的肝片吸虫GST同源性较高,与大片吸虫和卫氏并殖吸虫的GST也有较高的同源性。不同虫株GST基因具有较高的同源性,因此Fh GST蛋白不适合用作诊断抗原,但由于其存在交叉反应,GST基因作为分子疫苗的候选基因具有重要意义。肝片吸虫GST基因的克隆,为进一步研究GST蛋白的功能和作用奠定了基础。     

肝片吸虫MeCatL-B融合基因的构建、表达及其间接ELISA检测方法的建立

为分析组织蛋白酶的抗原性及评价其作为肝片吸虫(Fh) ELISA诊断抗原的潜力,本研究采用RT-PCR技术扩增FhCatL1D和CatB4基因的优势抗原表位集中区段,利用重叠延伸PCR技术融合CatL1D和CatB4基因片段获得约810 bp的Fh CatL-B融合基因(MeCatL-B),构建原核重组表达载体pET-MeCatL-B,并将其转化入BL21 (DE3)感受态细胞中诱导表达,经SDS-PAGE和western blot检测,结果显示表达的重组蛋白MeCatL-B (rMeCatL-B)约46 ku,可以被Fh阳性血清特异性识别。以纯化的rMeCatL-B为包被抗原,经优化各反应条件后建立检测Fh抗体的间接ELISA方法,该检测方法的特异性强、敏感性高、重复性好。利用该方法对97份绵羊临床血清样品进行检测,并与商品化FhELISA抗体试剂盒检测结果对比,结果显示二者符合率达95.88%。本研究为进一步研发Fh血清学诊断试剂盒奠定了前期基础。     

基于GAPDH重组蛋白建立羊肝片吸虫病间接ELISA检测方法

为了建立早期羊肝片吸虫病的血清学快速检测方法，本试验成功克隆并表达肝片吸虫GAPDH重组蛋白，Western blot结果显示，该重组蛋白具有良好的抗原活性。用肝片吸虫GAPDH重组抗原包被，建立肝片吸虫病血清抗体的间接ELISA方法；随后优化反应的最佳血清稀释度和抗原包被量，同时筛选其他反应条件。结果显示，间接ELISA方法批内和批间重复试验的最大变异系数均小于10%,GAPDH重组抗原与华枝睾吸虫、日本血吸虫和捻转血矛线虫阳性血清均无交叉反应。对来自黑龙江省各地区的96份羊血清进行检测，阳性率为16.67%(16/96)。结果表明本试验建立的方法具有良好的敏感性和特异性，能够为早期诊断羊肝片吸虫病提供参考。     

肝片吸虫Fh8标签融合表达口蹄疫O型病毒结构蛋白

口蹄疫病毒(foot and mouth disease virus,FMDV)的结构蛋白VP1难以实现体外的高效表达。本研究为获得良好免疫原性的VP1蛋白,选用肝片吸虫Fh8小肽段作为新型融合表达标签,构建重组Fh8-VP1免疫原,以期获得大肠杆菌中的高效表达。分别合成VP1的20~40aa和129~169aa之间的核苷酸片段,并用2个甘氨酸(GG)连接2个片段,合成的核苷酸片段(ΔVP1)克隆入Fh8标签之后,构建重组菌BL21(DE3)/pColdⅠ-Fh8-ΔVP1,SDSPAGE分析蛋白表达形式,Western blot方法检测重组His-Fh8-ΔVP1的抗原性。结果显示,成功构建重组质粒BL21(DE3)/pColdⅠ-Fh8-ΔVP1,并在大肠杆菌BL21(DE3)中获得高效表达,且以包涵体形式存在于菌体蛋白,可与猪源FMDV阳性血清发生特异性反应。     

大片吸虫磷酸丙糖异构酶基因的克隆表达和免疫原性分析

磷酸丙糖异构酶(TPI)是生物体糖酵解的一种关键酶,对大片吸虫获取能量而赖以生存有着十分重要的作用。本试验根据肝片吸虫TPI (GenBank:KC164346.1)的基因序列,设计带有BamHⅠ和XhoⅠ作为酶切位点的引物,以大片吸虫的cDNA为模板,扩增大片吸虫磷酸丙糖异构酶(FgTPI)基因。结果：FgTPI基因开放阅读框为762 bp,编码253个氨基酸,理论等电点pI为8.07;构建的重组表达质粒FgTPI-pET-28a(+)成功在大肠杆菌中表达,重组蛋白的分子量约为32 kDa;重组蛋白免疫BALB/c小鼠,收取多克隆抗体,ELISA检测结果显示,rFgTPI能诱导较高的IgG抗体水平;用感染大片吸虫动物的血清检测重组蛋白的免疫原性,Western blot分析表明,重组蛋白rFgTPI具有良好的免疫原性;生物信息学预测显示,FgTPI主要以α螺旋和β折叠为主,β转角较少,有3个较长的无规则卷曲;通过α-磷酸甘油脱氢酶偶联法测定其活性,发现FgTPI酶促反应最佳pH值为7.5,最适温度为35℃。本试验结果为深入研究FgTPI的生物学功能、评价其作为抗大片吸虫病疫苗候选分子...     

细粒棘球绦虫成虫表膜抗原特异性基因的筛选及克隆

为筛选细粒棘球绦虫(Echinococcus granulosus,Eg)表膜抗原基因或具有诊断性的特异性基因,提取Eg成虫表膜抗原,经ELISA、Western blot对该抗原的免疫学特性进行初步研究.以Eg成虫表膜抗原高免鼠血清为探针,筛选Eg成虫cDNA文库,将阳性噬菌斑的PCR产物和pGEM-T载体连接,转染到DH5α,对得到的重组子进行测序,并进行同源性分析.ELISA检测显示,表膜抗原免疫鼠诱导产生了特异性抗体,Westem blot鉴定该抗体能识别Eg成虫表膜抗原、原头蚴、Eg发育不成熟、Eg发育成熟抗原.筛选出6个阳性克隆,DNA片段大小在1～2kb之间.对阳性克隆进行同源性分析,结果与EgP-29mRNA同源性为99%,与Eg14-3-3蛋白mRNA同源性80%-99%.筛选Eg成虫cDNA文库所获得的基因,证明了Eg虫体表膜抗原的存在,有望成为犬细粒棘球绦虫的诊断抗原以及犬抗细粒棘球绦虫的候选基因.     

牛布鲁氏杆菌外膜蛋白Omp22的原核表达及反应原性鉴定

为了制备牛布鲁氏杆菌外膜蛋白Omp22的抗原,试验采用PCR技术扩增Omp22基因;通过PCR和双酶切技术鉴定重组质粒pET28a(+)-Omp22;应用SDS-PAGE对重组质粒pET28a(+)-Omp22的表达进行鉴定;采用Western-blot方法对表达的重组蛋白Omp22与布鲁氏杆菌阳性血清的反应原性进行分析。结果表明:PCR扩增得到Omp22基因,其大小为639 bp,共编码213个氨基酸,并成功构建重组质粒pET28a(+)-Omp22;该重组质粒在大肠杆菌BL21(DE3)感受态细胞中获得表达,表达的重组蛋白Omp22能与布鲁氏杆菌阳性血清发生特异性反应。说明重组蛋白Omp22具有良好的反应原性,为后续开发诊断抗体的试剂盒奠定了基础。     

猪瘟病毒表面抗原E2基因的克隆与原核表达

为获得可用于诊断的猪瘟病毒(Classical swine fever virus,CSFV)E2蛋白重组抗原,对CSFV-E2的多个B细胞抗原表位进行重构和表达。将人工合成的E2蛋白多抗原表位基因与p ET-28a(+)载体连接后,转化至宿主菌BL21(DE3),IPTG诱导表达,SDS-PAGE电泳分析蛋白可溶性,His亲和层析柱纯化蛋白并进行抗原性检测。重构后的猪瘟病毒E2基因多抗原表位基因大小约500 bp,PCR、双酶切鉴定结果显示与预期相符;重组蛋白为可溶性表达,大小约23 k Da;Western blot结果显示,重组蛋白可与猪瘟阳性血清发生特异性结合,具有良好的免疫反应性,可作为诊断抗原用于猪瘟病毒感染动物的血清抗体检测。     

Vaccination of mice against schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.     

Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.     

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.     

The addition of a new immunomodulator with the adjuvant adaptation ADAD system using fatty acid binding proteins increases the protection against Fasciola hepatica

Fatty acid binding proteins (FABP) have shown protective immune response against Fasciola hepatica infection. We evaluated the protection induced by the Fh12 FABP from F. hepatica (Fh12) combined with the new immunomodulator the lipidic aminoalcohol OA0012 in the ADAD system in mice and sheep. In this work we introduced a lipidic aminoalcohol OA0012 as immunomodulator alone or in combination with the hydroalcoholic extract of Phlebodium pseudoaureum; PAL. Mice vaccinated with ADAD containing OA0012+Fh12 or OA0012+Qs+Fh12 had survival rates of 40-50%. Sheep ADAD-vaccinated with OA0012+Qs+Fh12 showed lower fluke recovery, less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Sheep ADAD-vaccinated with OA0012 combined PAL and Qs+Fh12 showed lower fluke recovery (42%), lower adult worms count (57%) lower faecal egg count (38%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the addition of a new immunomodulator of synthesis to ADAD system with FABPs increased the protection against F. hepatica.     

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.     

Vaccination of mice and sheep with Fh12 FABP from Fasciola hepatica using the new adjuvant/immunomodulator system ADAD

We evaluate the ability of a Fasciola hepatica FABP native antigen (Fh12) with a new vaccination system called ADAD to protect mice and sheep against an experimental F. hepatica infection. The vaccination protocol consists of a set of two injections. The first injection contains a micelle in which two components are included, saponin from Quillaja saponaria (Qs) and/or Anapsos (A) a Polypodium leucotomos hydroalcoholic extract, both emulsified in a non-mineral oil (Montanide) in a water/oil emulsion (30/70). This is subcutaneously injected to achieve the "adaptation" of the immune system to subsequent stimuli. The second injection contains in addition the Fh12 antigen. Two different experiments were carried out using two mouse strains (BALB/c and CD-1). Mice vaccinated with Qs+A+Fh12 presented a survival rate of 40%, when compared with control groups. Furthermore, we evaluated the efficiency of the vaccination in sheep against an experimental F. hepatica challenge. The vaccinated sheep presented lower fluke recovery (24.5%), number of eggs in bile fluid (58.1%) and faeces (40.3%) than control groups. The recovered flukes were shorter (32.7%), immature (34.0%) and with lower body mass (31.6%) than non-complete vaccinated sheep. Thus, the new ADAD system could be a good alternative for future vaccination experiments against fasciolosis.     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

大片吸虫硫氧还蛋白过氧化物酶基因的原核表达及免疫原性分析

构建大片吸虫硫氧还蛋白过氧化物酶(Fg.TPx)原核表达质粒,经大肠埃希菌Rosetta表达融合蛋白(Fg.TPx/His)并对其进行纯化和初步鉴定。PCR扩增Fg.TPx的基因片段,亚克隆至pET32a(+)中构建重组表达载体pPET32a-Fg.TPx。IPTG诱导表达,经镍离子亲和层析分离纯化后,进行SDS-PAGE和Western blot分析。重组质粒经限制性内切酶双酶切和测序分析表明构建成功,表达产物以可溶性和包涵体形式存在,纯度为90%,Wsetern blot证实该蛋白可与抗His单克隆抗体发生特异性结合反应,分子质量为TPx和His分子质量之和,表明是融合蛋白。重组蛋白可以被大片吸虫感染水牛阳性血清特异性识别。免疫家兔产生的抗体效价最高可达1∶4 000。成功构建了p ET32a-Fg.TPx原核表达质粒,重组蛋白得到高浓度表达,具有抗原性,为进一步研究其在大片吸虫病诊断中的应用奠定了基础。