旋毛虫不同发育时期排泄分泌物抗原在免疫学诊断中的应用研究

[目的]系统了解并比较旋毛虫不同发育时期排泄分泌物(ES)抗原的免疫学特性,探索可用于临床检测出栏猪旋毛虫感染的血清学诊断技术。[方法]分别以旋毛虫肠道期10 h肌幼虫(10 h ML)、肠道期30 h成虫(30 h Ad)、3 d成虫(Ad3)、6 d成虫与新生幼虫混合(Ad6+NBL)以及肌幼虫(ML)五个不同发育时期的ES作为包被抗原,应用ELISA方法,检测感染不同剂量、不同天数的猪血清中的抗旋毛虫抗体Ig M和Ig G水平,绘制抗体消长规律曲线并进行数据分析。[结果]10 h ML ES和ML ES作为包被抗原适合检测不同感染剂量、感染35 d之前的猪抗旋毛虫Ig M抗体,低剂量感染10 d左右可以检出,高剂量感染5 d也可以检出;Ad3 ES作为包被抗原对高剂量感染35 d之前的猪抗旋毛虫Ig M抗体检测敏感;Ad3和ML的ES作为包被抗原可检测不同剂量、感染35 d之后的猪抗旋毛虫Ig G抗体,其中Ad3 ES抗原检测低剂量感染的效果优于ML ES抗原。[结论]肠道期肌幼虫、成虫和肌幼虫的ES抗原可用于检测旋毛虫的早期感染,成虫和肌幼虫的ES抗原可用于检测出栏猪的旋毛虫感染。本研究为进一步合理有效利用旋毛虫不同发育时期的ES抗原,建立更有效的检测屠宰动物旋毛虫感染的方法提供了重要理论基础和参考。     

旋毛虫成虫丝氨酸蛋白酶抑制剂的原核表达及免疫原性研究

以旋毛虫成虫的总RNA为模板,应用RT-PCR扩增获得旋毛虫成虫丝氨酸蛋白酶抑制剂(TsAdSPI)基因。经克隆鉴定正确后,将TsAdSPI基因连接到pET-28a表达载体,转化至大肠杆菌BL21(DE3)中进行IPTG诱导表达,并将纯化后的目的蛋白免疫新西兰大白兔,制备多克隆抗体。应用Western-blot及ELISA方法对重组蛋白的免疫原性和抗体效价进行分析。SDS-PAGE结果显示,目的蛋白分子质量为44 k Da,以包涵体形式表达。经Western-blot和ELISA方法分析,制备的多克隆抗体效价可高达1∶51200,表明该蛋白具有较好免疫原性。表明TsAdSPI蛋白可作为旋毛虫感染早期血清学诊断方法的候选抗原,为新型的抗旋毛虫及相关寄生虫药物研发打下结实基础。     

旋毛虫丝氨酸蛋白酶抑制剂定位及免疫保护效果评估

探讨旋毛虫丝氨酸蛋白酶抑制剂(Ts-serpin)的抗原性、定位及免疫保护性。将本实验室前期构建的旋毛虫丝氨酸蛋白酶抑制剂的原核表达载体进行大量表达纯化。利用不同感染时间的猪旋毛虫阳性血清,通过Western blot方法对纯化后的重组丝氨酸蛋白酶抑制剂(rTs-serpin)进行反应原性鉴定,并制备多克隆抗体,用间接免疫荧光法检测Ts-serpin在旋毛虫肌幼虫中的定位。随后将rTs-serpin免疫小鼠进行免疫保护效果评估。结果显示:rTs-serpin可被不同感染时间的猪旋毛虫阳性血清特异性识别,表明rTsserpin是高反应原性抗原;Western blot结果显示旋毛虫排泄分泌物和旋毛虫虫体粗提取物均存在Ts-serpin,间接免疫荧光显示Tsserpin定位在旋毛虫肌幼虫表皮中;免疫保护试验结果显示rTs-serpin免疫后的小鼠旋毛虫肌幼虫减虫率约为32.2%。综上所述,Tsserpin主要定位在旋毛虫肌幼虫表皮,rTs-serpin具有较强的抗原性,且对小鼠具有免疫保护作用。     

两种旋毛虫丝氨酸蛋白酶抑制剂对巨噬细胞炎性细胞因子mRNA表达的影响

丝氨酸蛋白酶抑制剂在寄生虫入侵宿主的过程中发挥着关键作用,能够参与到入侵宿主、免疫逃避、炎症反应、凝血系统和细胞迁徙等过程。为了探讨两种丝氨酸蛋白酶抑制剂(Ts Ad SPI和Ts Ka SPI)对巨噬细胞所分泌炎性细胞因子的调节作用,应用实时荧光定量PCR技术对两种SPIs分别处理的小鼠巨噬细胞进行TNF-α、IL-1β、IL-6、IL-12、IL-10、TGF-βmRNA表达水平的检测。结果显示,Ts Ad SPI和Ts Ka SPI均可不同程度的抑制LPS活化的小鼠巨噬细胞促炎性细胞因子TNF-α、IL-1β、IL-6和IL-12 mRNA的表达水平。并且两种SPI均可不同程度的促进巨噬细胞抗炎性细胞因子IL-10和TGF-β的表达。表明这两种旋毛虫SPI可通过调节宿主巨噬细胞影响入侵时宿主的免疫应答。     

旋毛虫ES抗原对小鼠腹腔巨噬细胞NOD1受体通路影响的研究

为了解旋毛虫ES抗原对宿主巨噬细胞NOD1受体通路的影响,本研究通过体外实验将旋毛虫ES抗原作用于小鼠腹腔巨噬细胞,观察NOD1受体及其信号通路中关键分子及相关细胞因子的表达动态。应用荧光定量PCR监测细胞内NOD1、RIP2和NF-κB m RNA的转录水平,western blot测定NOD1、RIP2、NF-κBp65、NF-κB p-p65蛋白表达量,ELISA测定细胞培养上清中TNF-α、IL-1β和IL-6含量变化。结果显示,在一定的ES抗原作用浓度和时间范围内,巨噬细胞中各目的基因的转录水平均先增高,当作用时间和作用浓度超过一定值时则开始下降,作用24 h时NOD1 m RNA转录水平显著低于对照组(p0.01);ES抗原浓度15μg/mL作用时间9 h时,巨噬细胞中NOD1、RIP2和NF-κB p-p65蛋白量显著增加(p0.01),此时巨噬细胞培养上清中TNF-α、IL-1β、IL-6含量显著增高(p0.01)。结果表明,旋毛虫ES抗原在一定的作用时间和浓度范围内,可以激活并调节小鼠腹腔巨噬细胞中NOD1受体通路,上调NOD1受体通路中关键分子RIP2和下游分子NF-κB的表达,可以促进细胞因子TNF-α、IL-1β和IL-6的分泌;并且超过一定时间和浓度的ES抗原刺激可以导致小鼠腹腔巨噬细胞出现免疫耐受。本研究证明了宿主NOD1受体参与了旋毛虫引起的宿主免疫应答,为旋毛虫病防治和理解旋毛虫对宿主的感染机制提供新的思路。     

旋毛虫Serpin基因在不同发育时期的mRNA表达水平变化及虫体表达特性

为研究旋毛虫丝氨酸蛋白酶抑制剂(Serpin),基因在不同发育时期的mRNA表达水平变化,本研究应用实时荧光定量PCR技术对不同发育时期的虫体的Serpin在mRNA表达水平进行检测。同时利用鼠抗重组蛋白血清对Serpin在旋毛虫成虫、新生幼虫、成囊前期幼虫及肌幼虫中进行定位,以鉴定该Serpin基因的表达特性。研究结果显示,在成虫时期,3 d时表达量明显高于6 h和5 d的表达量(P﹤0.05);成囊前期表达量很低,其中18 d时表达量最低(P﹤0.05);肌幼虫时期在26 d、38 d和48 d均有大量表达(P﹤0.05)。免疫荧光染色结果表明,Ts Serpin蛋白在5 d成虫期的体表表达;在新生幼虫和14 d成囊前期幼虫的体内体表均有表达;38 d肌幼虫时期明显可见表皮及体内均有表达。本实验为探究旋毛虫在宿主体内免疫逃避机制奠定基础。     

旋毛虫抗原基因T668的定点突变及原核表达

旋毛虫抗原基因T668是本实验室发现的1条高反应原性的特异性抗原基因,该基因编码旋毛虫丝氨酸蛋白酶。本试验利用重叠PCR的方法对旋毛虫抗原基因T668成功实现酶活性中心重要位点定点突变改造,将酶活性中心的结合位点259位亲水性丝氨酸和催化三联体中240位的丝氨酸分别突变为疏水性丙氨酸,并构建、纯化了可溶性表达的双位点突变的重组蛋白,后经Western blot分析表明所表达的重组蛋白能够与旋毛虫感染26d阳性血清发生特异性反应,具有良好的反应原性。本试验为后续开展对该丝氨酸蛋白酶的研究及应用奠定了坚实的基础。     

旋毛虫丝氨酸蛋白酶基因的克隆与分析

以旋毛虫感染猪血清为抗体探针,对旋毛虫3日龄成虫cDNA文库进行免疫筛选,将获得的阳性克隆进行测序,用分子生物学软件对所得cDNA序列进行了分析。序列分析结果显示,阳性克隆Zh68cDNA全长为1372bp,含有1个1287bp的完整的开放阅读框架,编码由429个氨基酸组成的多肽,理论分子质量为47．5ku,等电点为8．45。SMART分析表明,1～18位氨基酸为信号肽序列,37～277位氨基酸为典型的丝氨酸蛋白酶胰蛋白酶类结构域,其中His88、Asp142、Ser233为催化中心的3个主要氨基酸残基,78～80位氨基酸为N-糖基化位点（NCS）,另外还有6个保守的Cys形成二硫键。BLASTn同源性分析表明,与其他生物丝氨酸蛋白酶的基因序列无明显的同源性,为1个新的cDNA分子。BLASTp蛋白质同源性分析表明,与丝氨酸蛋白酶的同源性为30％左右。Southern—blot杂交表明,此基因在旋毛虫基因组中属于多基因家族,具有基因多态性。cDNA的PCR结果表明,此基因在旋毛虫肌幼虫、新生幼虫及成虫期均有表达。     

旋毛虫新生幼虫p46 000抗原基因的克隆及序列分析

应用旋毛虫感染猪血清，对旋毛虫新生幼虫cDNA文库进行了免疫筛选。对阳性克隆pBK-cMV-WN10的序列分析结果表明。cDNA全长为1352bp。含有1个1218bp的完整的开放阅读框架(ORF)，编码的多肽由406个氨基酸残基组成，其相对分子质量理论推导值为45900，等电点为5．43，N末端的信号肽及糖基化位点(NCS)表明其可能为分泌性糖蛋白，氨基酸序列19～156与158～295为重复区域，相似性为74％．C末端有1个半胱氨酸蛋白酶抑制剂结构域，但旋毛虫p46000抗原与其他线虫的半胱氨酸蛋白酶抑制蛋白结构有很大差异，可能已经失去半胱氨酸蛋白酶抑制蛋白的功能。PCR结果显示。从旋毛虫新生幼虫、肌幼虫、3日龄成虫和5日龄成虫cDNA中均扩增出此基因，表明此基因在旋毛虫各个时期均有表达。     

旋毛虫成虫DNaseⅡ—T3223-7基因的克隆及其表达特性鉴定

利用RT-PCR技术从旋毛虫成虫得到T3223-7基因,并进行扩增克隆到原核克隆载体pMD18-T中,将重组质粒转入克隆菌DH5α,提取质粒进行酶切和测序鉴定,连接至pET-28a表达载体,最后转入表达菌Rosetta(DE3)。用1mmol/L IPTG诱导培养重组表达菌,对菌体裂解物进行SDS-PAGE分析,发现重组蛋白以包涵体的形式表达,约为40 000,与理论值相符。将纯化后的重组蛋白免疫家兔,制备兔抗T3223-7蛋白多克隆抗体,间接ELISA测定多抗效价达1∶320 000,Western blot检测表明制备的多克隆抗体能与T3223-7抗原发生特异性反应,并能识别和结合旋毛虫成虫排泄分泌物(ES)。     

Prokaryotic Expression of Thymidine Kinase Gene of Duck Plague Virus and Establishment of an Indirect ELISA Based on the Protein

In the study,an indirect ELISA was developed using the purified thymidine kinase (TK) protein as antigens to detect the antibody of duck plague virus (DPV).The TK gene of DPV was amplified by PCR using specific primers designed according to the sequence of TK gene in GenBank.Using gene recombination technology,the TK gene was cloned and inserted into prokaryotic expression vector pET-32a and the recombinant plasmid was identified by PCR,double enzyme digestion and sequence analysis.The positive recombinant plasmid was then transformed into E.coli BL21 (DE3) and induced by IPTG.The expressed protein was purified by gel extraction and analyzed by Western blotting.Then an indirect ELISA was established to detect DPV antibody by using the purified recombinant TK as the coating antigen.The other assay conditions were also optimized.The recombinant plasmid was constructed successfully and Western blotting detected the target protein,TK recombinant protein could be recognized by positive serum,and the result indicated that the recombinant protein had good antigenicity.The optimum reaction conditions were as follow:The optimal dilution of enzyme labelled antibody was 1:200,the optimal dilutions of antigen and antibody were 1:400 and 1:200,reaction time was 60 min,most prefer blocking solution was 5% BSA,closed for 60 min,chromogenic time was 10 min.The method had good stability and high sensitivity,and it could provide a reliable method for the clinical detection,immunization surveillance,and prevention and cure of DP.     

鸭瘟病毒胸苷激酶基因的原核表达及其蛋白间接ELISA检测方法的建立

本研究旨在以表达纯化的鸭瘟病毒胸苷激酶(thymidine kinase,TK)重组蛋白作为包被抗原,建立检测鸭瘟病毒抗体的间接ELISA方法。根据GenBank上收录的鸭瘟病毒TK基因序列设计出1对引物,采用PCR扩增出鸭瘟病毒TK基因。将TK基因与原核表达载体pET-32a连接,通过PCR、双酶切和测序鉴定,将阳性重组质粒转化至大肠杆菌BL21(DE3)工程菌,IPTG诱导表达。采用切胶纯化的方法纯化蛋白,Western blotting分析鉴定表达产物。用纯化的TK重组蛋白作为包被抗原,并优化其他条件,最终建立检测鸭瘟病毒抗体的间接ELISA方法。结果显示,重组质粒构建成功,经Western blotting检测得到目的蛋白,TK重组蛋白能被阳性血清识别,说明该重组蛋白具有较好的抗原性。确定了最佳反应条件:酶标二抗的最佳稀释度为1:200,最佳抗原、抗体的稀释度分别为1:400和1:200,抗原抗体作用时间为60 min,最佳封闭液为5% BSA,最佳封闭时间为60 min,最佳显色时间为10 min。结果表明,该方法具有良好的稳定性及较高的敏感性,为鸭瘟的检疫、监测和防制工作提供了一种有效的手段。     

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.     

捻转血矛线虫ES24抗原基因的克隆与表达特性分析

根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。     

Proteolytic enzymes from Trichinella spiralis larvae.

Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytic enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containing gelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.     

半胱氨酸蛋白酶基因在家蚕杆状病毒表达系统中的表达

为了探讨杆状病毒的致病机理、改善杆状病毒表达系统的表达效率 ,将来源于家蚕核型多角体病毒ZJ8株的半胱氨酸蛋白酶 (cystenineprotease,CP)基因克隆到转移载体pVL 1393上 ,获得重组转移载体pVL cp ,与线性化的Bm BacPAK6病毒DNA共转染家蚕Bm 5贴壁细胞。通过蓝白斑筛选、纯化后得到的重组病毒经PCR鉴定证明 ,cp基因已被正确导入 ,注射感染家蚕 5龄幼虫 12 0h后表达产物活性达到最高。对表达产物进行SDS PAGE及酶活力分析 ,检测到半胱氨酸蛋白酶在家蚕生物反应器中的表达 ,其表达量约为 16 0 0U/mL。     

Assessing the agreement between Ostertagia ostertagi ELISA tests performed using the crude adult antigen and the adult and larval stage 4 excretory/secretory antigens

The objective of this study was to determine the agreement between ELISA tests conducted using three O. ostertagia antigens: crude adult worm, larval stage 4 (L4) excretory/secretory (ES) and adult ES. This study was carried out on 289 Holstein cows from five herds in Prince Edward Island and one herd in Nova Scotia. Composite milk samples of these cows were collected (between May and September 2002) from the respective provincial laboratories and sent to the Atlantic Veterinary College where each sample was tested for antibodies to O. Ostertagi using an indirect microtitre ELISA test. Results were expressed as optical density ratio (ODR) values. Each milk sample was tested with three ELISA tests, with each test using a different O. ostertagi antigen. There was a slight rise in ODR values of both adult antigens, between May and August, with higher values obtained using the adult ES antigen. L4 ES ODR values were generally higher than those for both adult antigens during the study period, except for May. There was a more dramatic rise in L4 ES ODR values between May and August. Rises in ODR in May and end of July coincided with periods of mass maturation of L4 to adult worms. The results of the study showed that the concordance correlation coefficient (CCC) between tests performed using both ES and the crude antigens were low (crude adult versus adult ES=0.31, crude adult versus L4 ES=0.30). The highest CCC was observed between tests done using both ES antigens (CCC=0.56). Generally, the study results suggest that the antibody response (detectable by the ELISA) is mainly directed against ES antigens (especially L4) than the crude adult worm antigen.     

Evaluation of excretory-secretory antigens for the serodiagnosis of swine trichinellosis

Groups of hog sera from endemic and non-endemic areas for swine trichinellosis in Yugoslavia were tested by ELISA using excretory-secretory (ES) antigens collected from T. spiralis muscle larvae maintained in vitro for 24, 48 or 72 h. The 24-h ES had the highest level of specificity for T. spiralis infection. Antigen preparations recovered after 48 or 72 h yielded an increasing rate of false-positive reactions. Additional antigens occurred in the 48- and 72-h ES preparations as determined by gel electrophoresis and monoclonal antibody binding. The occurrence of false-negative reactions was directly correlated with T. spiralis worm burdens. Hogs with muscle larvae densities greater than 10 larvae per gram were all positive by ELISA. Among 17 hogs with less than 10 larvae per gram, only one hog was negative by ELISA with 24-h ES antigen; the false-negative rate was higher with 48- and 72-h ES. These results show that ES antigen produced during the first 24 h of in vitro cultivation is highly specific for the immunodiagnosis of swine trichinellosis.     

Prokaryotic Expression and Identification of P66 Gene from Borrelia garinii SZ

In order to obtain the P66 protein from Borrelia garinii (B. garinii) SZ, the first strand cDNA was synthesized based on the total RNA extracted from B. garinii SZ, and then the targeted P66 gene was amplified by PCR. The fragment was linked into the pET-30a(+) vector, and transformed into Escherichia coli BL21(DE3). After identified by PCR, double restriction enzyme digestion, and nucleotide sequencing, the recombinant protein was expressed and purified. Polyclonal antibody was then prepared from New Zealand rabbit immunized with purified recombinant protein. The recombinant protein was about 70 ku in size confirmed by SDS-PAGE, and Western blotting analysis indicated that the recombinant P66 protein could recognize the mouse monoclonal anti-His-tag, positive sera of spirochete from mouse, and anti-P66 polyclonal antibody. Additionally, the anti-P66 polyclonal antibody could recognize native P66 protein. In this study, we successfully expressed the recombinant P66 protein and obtained the anti-P66 polyclonal antibody, which provided the foundation for further functional studies of P66 protein from B. garinii SZ.