旋毛虫肌肉幼虫ES和S_3抗原诱发小鼠保护性免疫的研究

迄今为止,国外对旋毛虫抗原诱发保护性免疫及其抗原生化分析的报道较多,但国内有关旋毛虫幼虫排泄物分泌物抗原(ES)和杆细胞颗粒相关抗原(S_3)诱发保护性免疫的研究报道尚少。我们于1990年5月进行了本实验,初步探索旋毛虫幼虫ES和S_3抗原接种小鼠后诱发保护性免疫的情况,为旋毛虫病的免疫预防打下基础。     

旋毛虫肌幼虫排泄分泌物中特异性诊断抗原的研究

目的　寻找旋毛虫肌幼虫排泄分泌(ES)物中的特异性诊断抗原。　方法　应用SDSPAGE和Western印迹对旋毛虫肌幼虫体外培养18、30h后的ES抗原中的蛋白组分进行研究。　结果　旋毛虫肌幼虫培养18、30h后得到的ES抗原组分大致相同,两种ES抗原中主要蛋白带的分子量为112、110、108、97、53、49、45、42、35、23和16kDa。18hES抗原中的102、97、95和53kDa以及30hES抗原中的53、49、45和43kDa均与并殖吸虫病、华支睾吸虫病、日本血吸虫病及囊尾蚴病患者血清发生明显的交叉反应。ES抗原中的23kDa蛋白组分只与旋毛虫感染的大鼠、小鼠及患者血清反应,而不与上述其它寄生虫感染者、正常大鼠和小鼠及正常人血清发生交叉反应。　结论　旋毛虫肌幼虫ES抗原中的23kDa蛋白组分为旋毛虫肌幼虫的特异性抗原,可用于旋毛虫病的血清学诊断及血清流行病学调查。     

人体旋毛虫病的免疫诊断及旋毛虫特异性抗原识别

用检查肌肉组织中幼虫的方法诊断人体旋毛虫病有敏感性低,活检所致副作用,费时,检查者视觉疲劳等不足,因此,免疫诊断方法尤其是ELISA以其较高的敏感性和重现性被广泛运用。在免疫学检测中常用两种旋毛虫抗原,即由成虫、新生幼虫或感染期幼虫(IL)制备的虫体抗原及排泄分泌(ES)抗原。本研究试图用感染性幼虫的粗制虫体抗原(CLE)作人体旋毛虫病的免疫诊断抗原,检测其敏感性和特异性,并寻找和识别CLE中旋毛虫的特异性抗原组分。     

旋毛虫重组抗原的体外表达及其影响因素

旋毛形线虫(Trichinella spiralis)简称旋毛虫,成虫和幼虫分别寄生于同一宿主的小肠和肌肉内。由旋毛虫引起的旋毛虫病(trichinellosis)是一种人兽共患寄生虫病,目前在我国及其他国家仍有爆发流行。由于旋毛虫病的病原学诊断比较困难,免疫诊断则显得尤为重要。但旋毛虫虫体抗原成份复杂,用于免疫诊断时常和其它寄生虫病发生交叉反应而出现假阳性。随着分子生物学技术的发展,基因重组抗原具有可大量制备,特异性好等优点而越来越受到了人们的重视[1-3]。然而,表达载体的种类、目的基因的长度及诱导剂等诸多因素均可影响重组抗原的表达。一、…     

旋转卧式两性电解质柱分离旋毛虫特异性抗原

当前用于旋毛虫病诊断的抗原是直接从培养基中提取的排泄/分泌(ES)抗原,含120000条左右幼虫的75ml培养基中仅提取约1mg的抗原。本文采用旋转两性电解质柱作等电聚焦,直接从肌期幼虫粗抗原中提取ES抗原,并就其在诊断旋毛虫病的特异性和敏感性与体外培养肌期幼虫的ES抗原进行了比较。从猪体内分离旋毛虫,并在1CR小鼠和     

旋毛虫重组35.5ku蛋白免疫诊断价值的研究

目的研究旋毛虫重组35.5ku蛋白的免疫诊断价值。方法通过RT-PCR扩增旋毛虫35.5ku蛋白基因TspSP-1.2,构建重组质粒pUC18-TspSP-1.2,测序正确后亚克隆至表达载体pET-30a(+)中,构建重组表达载体pET-30a(+)-TspSP-1.2,转化大肠埃希菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,用SDS-PAGE和Western blot鉴定重组蛋白的抗原性。结果构建的重组表达载体pET-30a(+)-TspSP-1.2能够表达旋毛虫35.5ku蛋白,该重组蛋白可被旋毛虫感染小鼠血清、旋毛虫ES抗原免疫鼠血清及旋毛虫患者血清识别,与日本血吸虫、并殖吸虫、华支睾吸虫患者及正常人血清均无交叉反应,但与猪囊尾蚴患者血清有交叉反应。结论旋毛虫重组35.5ku蛋白可作为旋毛虫病免疫诊断的候选抗原。     

一种可诱导高水平抗旋毛虫保护力的40-mer合成多肽疫苗

旋毛虫分泌型抗原(ES)接种近交系小鼠大多可获得高水平的保护力。几种纯化的肌肉期幼虫蛋白亦可诱导保护性免疫,其中的一种分子量为43000(43k)糖蛋白为旋毛虫幼虫组1抗原(TSL-1)的成员,存在于肌肉期幼虫的分泌物及其表面,具有高度的免疫原性,能诱导保护性免疫力。作者根据该分子序列,合成了40—80、81—120和121—160三种40-mer的合成多肽(SP),用于旋毛虫疫苗的研究试验。     

一种免疫显性的53kDa旋毛虫肌肉幼虫排泄-分泌抗原的分子克隆和表达

应用基因工程技术对旋毛虫排泄-分泌(ES)抗原进行分子克隆,并对表达蛋白的性质和相应基因进行了分析。人工感染小鼠获得旋毛虫成虫和肌肉内幼虫,新生幼虫由体外培养成虫中收集。用异硫氰酸胍分离虫体DNA和总RNA,Poly(A)mRNA经Oligo-dT层析纯化。用Poly(A)mRNA合成双链cDNA,插入λgtll噬菌体,体外包装     

旋毛虫热激蛋白的研究进展

旋毛虫热激蛋白(heat shock protein,HSP)是一个高度保守的蛋白质家族,具有特殊的生物学作用,开展HSP的研究对旋毛虫病的免疫诊断和免疫预防具有重要意义.该文对旋毛虫与宿主HSP水平的变化、旋毛虫HSP的分离纯化、克隆表达及其检测方法等方面的研究进展进行了综述.     

以重组蛋白为抗原的ELISA法检测旋毛虫抗体

目的 寻求特异性强、敏感性高的旋毛虫病诊断抗原。 方法 以旋毛虫新生幼虫期特异性T668基因在E.coli高效表达的重组蛋白为抗原，分别以兔、猪和健康者血清及旋毛虫病阳性血清为一抗，以辣根过氧化物酶（HRP）标记的山羊抗兔IgG、山羊抗猪IgG和山羊抗人IgG为二抗，建立检测旋毛虫抗体的间接ELISA方法，并以旋毛虫肌幼虫排泄?鄄分泌（ES）抗原作为检测对照。 结果 以T668重组蛋白为抗原，对兔、猪和人旋毛虫病血清进行检测，阳性检出率为100 ％，且敏感性高（0.016 μg / 孔），与ES抗原检测结果完全一致。 结论 T668重组抗原有望替代ES抗原检测旋毛虫抗体。     

Parasite excretory-secretory antigen and antibody to excretory-secretory antigen in body fluids and kidney tissue of Dirofilaria immitis infected dogs

Excretory-secretory (ES) antigens of adult Dirofilaria immitis were detected by enzyme-linked immunosorbent assay (ELISA) in serum and urine from each of 12 experimentally infected dogs. Excretory-secretory antigens in serum were first detected 154 days postinfection. Serum antibodies directed against parasite ES antigens were detected by ELISA in all dogs. Kidney tissue elution studies were performed in 10 dogs, and antibody and parasite ES antigens were demonstrated in each case. Antibody or parasite antigen was not detected in serum, urine, or kidney eluates from uninfected dogs. At peak concentrations of the ES antigens in serum, there were correlations with the number of adult D. immitis present in the dogs (r2 = 23.8, P less than 0.05) and with the antigen concentration in kidney eluates (r2 = 73.5, P less than 0.001). Peak serum antibody concentrations were not correlated with either the number of adult worms or the antibody concentrations in kidney eluates. This study suggests that detection of parasite antigens in urine may be an important diagnostic aid. In addition, the correlation between the concentration of D. immitis ES antigens in kidney tissue and in serum without a similar correlation between serum and kidney antibody concentrations suggests that D. immitis ES antigens adhere to kidney tissue.     

Immunohistochemical localization of excretory/secretory antigens in adult Ancylostoma caninum using monoclonal antibodies and infected human sera

Human eosinophilic enteritis (EE) may result from hypersensitivity to the excretory I secretory (ES) antigens of adult Ancylostoma caninum. The origin of several antigens were identified by probing adult A. caninum with mouse monoclonal antibodies (MoAbs), sera from mice vaccinated with ES antigens and sera from human EE patients. Six MoAbs (AC/ES1–6) were produced against ES antigens, two being IgG3 and four IgM. Western blots demonstrated four different antigen specificities: MoAb AC/ES 1 bound strongly to an ES product at about 30kDa; AC/ES 2 recognized a broad band ranging from 50–200kDa; AC/ES 3, AC/ES 5 and AC/ES 6 reacted at about 68 kDa, and AC/ES 4 at about 97kDa. Sections of formalin-fixed, paraffin embedded adult A. caninum were then incubated with these MoAbs and immunostained by the peroxidase-anti-peroxidase (PAP) technique. The target epitope of MoAb AC/ES 1 was found mainly in the oesophageal, amphidial and excretory glands; AC/ES 2 reacted weakly with many structures in the sections; AC/ES 3, AC/ES 5 and AC/ES 6 were specific for excretory glands only, and AC/ES 4 bound to amphidial glands. Sera from immunized mice reacted with all three (especially the excretory) glands and the cuticle. In an indirect assay, worm sections probes with three human EE patient sera demonstrated maximal staining in the amphidial glands. Our findings confirm that ES products of A. caninum include immunogenic glandular secretions which may be involved in the pathogenesis of human EE.     

A comparison of cellular and humoral immune responses to trichuroid derived antigens in human trichuriasis

Individuals, residing in a region highly endemic for Trichuris trichiura, were examined for cytokine and proliferative responses to T. trichiura worm homogenate (TtAg), T. trichiura excretory/secretory products (TtES) and the equivalent antigenic preparations from the murine whipworm, Trichuris muris. Serum antibody levels against TtAg, T. muris worm homogenate and T. muris ES products were also studied. Measurable levels of immunoglobulin (Ig)G1, IgG4, IgA and IgE against T. muris antigens were detected, indicating a degree of conservation of epitopes between antigens derived from both species. Although levels of interleukin (IL)-4, IL-10, IL-13, tumour necrosis factor (TNF)-alpha and proliferative responses produced were comparable between homogenate antigens of either species and ES antigens of either species, a markedly different cellular response was observed in cultures stimulated with homogenate antigens compared to ES antigens. ES antigens preferentially induced IL-10 (P > 0.001) and TNF-alpha (P > 0.001) production, whereas levels of IL-4 (P > 0.001), IL-13 (P > 0.001) and proliferative responses (P > 0.001) were greater in cultures stimulated with whole worm extracts. Our findings suggest that T. muris preparations could be used as an alternative to T. trichiura proteins as a source of antigens in ex vivo cultures and that ES products stimulate a distinct immune response compared to somatic antigens.     

旋毛虫排泄分泌抗原对小鼠的保护性免疫

目的比较旋毛虫成虫排泄分泌抗原(ES抗原)、肌幼虫ES抗原、成虫和肌幼虫ES混合抗原对小鼠的免疫保护作用。方法用生理盐水培养法从培养液中提取成虫ES抗原、肌幼虫ES抗原,分别用成虫ES抗原、肌幼虫ES抗原、成虫和肌幼虫ES混合抗原免疫小鼠,同时设佐剂组和对照组,间隔7d共免疫3次。末次免疫后7天,每只小鼠用200条旋毛虫感染期幼虫经口进行攻击感染。感染后7天和30天检查各组小鼠肠道成虫数和肌幼虫数。结果旋毛虫成虫ES抗原组、肌幼虫ES抗原组、成虫和肌幼虫ES混合抗原组的成虫减虫率分别为87.95%、69.48%、84.34%,肌幼虫减虫率分别为74.79%、87.97%、86.87%。成虫ES抗原组、成虫与肌幼虫ES抗原混合组的成虫减虫率均高于肌幼虫ES抗原组(P均<0.05)。肌幼虫ES抗原组、成虫与肌幼虫ES抗原混合组的肌幼虫减虫率均高于成虫ES抗原组(P均<0.01)。结论旋毛虫成虫和肌幼虫ES混合抗原均能诱导小鼠产生抗成虫及肌幼虫较强的免疫力。     

弓形虫速殖子细胞质、细胞膜、排泄-分泌抗原蛋白分子测定及特异性免疫反应的试验分析

目的　研究弓形虫速殖子各类抗原的蛋白组分 ,主要蛋白分子的抗原性和用于血清学诊断的价值。　方法　用聚丙烯酰胺凝胶电泳分析弓形虫速殖子各类抗原的蛋白组分和主要蛋白分子 ,用免疫印渍技术以弓形虫患者血清识别弓形虫各类抗原的免疫反应靶位。　结果　弓形虫速殖子细胞质抗原、细胞膜抗原和分泌代谢抗原的主要蛋白分子分别为 85、5 2、38ku;4 8、35、16、14 ku和 15 8、95、2 7、15、13ku及弓形虫患者特异性血清识别速殖子各类抗原的免疫反应靶位分别是 32、4 0和 2 7ku。　结论　弓形虫速殖子各类抗原间存在各自独特的抗原决定簇和可被特异性记忆抗体识别的免疫反应受体。此对于提高弓形虫感染的免疫学诊断及免疫学预防均具有一定的实用价值。     

抗旋毛虫肌幼虫ES抗原IgY的制备及鉴定

目的制备抗旋毛虫肌幼虫排泄-分泌(excretory-secretory,ES)抗原的鸡卵黄免疫球蛋白(IgY),测定其效价及用于检测抗原的敏感性。方法 4只24w龄罗曼母鸡用旋毛虫肌幼虫ES抗原经大腿外侧与胸部肌肉免疫4次(首次剂量为500μg/只,加强剂量为250μg/只),每次间隔10d。取免疫前和首次免疫后42d的鸡蛋卵黄,用水稀释法提取IgY,考马斯亮蓝法测定蛋白含量,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)及间接荧光抗体试验(IFAT)对IgY进行分析,ELISA检测纯化后IgY的效价及检测抗原的敏感性。结果罗曼鸡经ES抗原免疫后,每枚鸡蛋经提纯后均可得到约70mg抗体,SDS-PAGE表明纯化的IgY有2条主要蛋白带,分子量为67kDa、23kDa,Western blot与IFAT发现提纯的IgY可识别肌幼虫虫体与ES抗原。IgY的抗体效价为1∶107,IgY-夹心ELISA检测旋毛虫抗原的敏感性为1.17ng/mL。结论制备的抗旋毛虫ES抗原的IgY具有较高的效价与敏感性。     

Excretory/secretory antigens (ES) from in-vitro cultures of Taenia crassiceps cysticerci,and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis

Antigens were obtained from cysticerci of the ORF strain of Taenia crassiceps, by culture of cysts in protein-free hybridoma medium (PFHM). Budding of new vesicles was observed after 24-48 h. Excretory/secretory (ES) antigens (peptides of <20 kDa) were recovered in the medium after culture for 48 h. SDS-PAGE analysis of vesicular-fluid (VF) antigens (obtained by rupturing T. crassiceps cysticerci in PFHM) and the ES antigens indicated partial homology between the two preparations. ES peptides of 18- and 14-kDa were recognized by polyclonal antibodies produced in rabbits immunized either with the VF antigens or with a total-antigen preparation of T. solium cysticerci. Antibodies present in samples of serum or cerebrospinal fluid (CSF) from patients with neurocysticercosis also reacted with ES peptides. An anti-ES monoclonal antibody detected antigens in the CSF from 10 patients with neurocysticercosis, showing the antigenic homology of the ES antigens with those of T. solium cysticerci in human infections.     

Sandwich ELISA detection of excretory-secretory antigens of Toxocara canis larvae using a specific monoclonal antibody

We produced a new monoclonal antibody (mAb) to the excretory-secretory (ES) antigens of Toxocara canis larvae. The mAb (IgG1) reacts specifically with the 120 kDa protein of many ES molecules and does not have any cross-reactivity with adult T. canis antigens. Sandwich ELISA to detect the ES antigens was performed using the mAb and rabbit polyclonal antiserum. The lower limit for the detection of ES antigen was 4 ng/ml; assay was proportional within a concentration range of 4 ng/ml to 1 microg/ml of ES antigen. This assay system may prove valuable when seeking to quantify parasite burden early in infection and when determining the efficacy of anthelmintic treatment.     

Detection of excretory/secretory coproantigens in experimental hookworm infection

This report describes the detection of hookworm excretory/secretory (ES) antigens in soluble hamster fecal extracts by an enzyme-linked immunosorbent assay (ELISA). A rabbit polyclonal IgG antibody against Ancylostoma ceylanicum ES was used to capture hookworm coproantigens that were then detected using pooled, high-titer, infected hamster serum. The ELISA was capable of detecting ES proteins over a range of 10 ng/mL to 10 mug/mL when the antigens were diluted in buffer or uninfected fecal extract, and ES could be detected in infected hamster feces at dilutions up to 1:256. Examination of the kinetics of coproantigen production demonstrated that detectable amounts of ES were produced as early as four days after A. ceylanicum infection, whereas fecal eggs were not observed until day 17. Moreover, fecal ES levels correlated well with intestinal worm burden and could be detected in wet or dry stool samples stored for 14 days over a temperature range of -80 degrees C to 37 degrees C. The fecal ELISA was then adapted to analyze the excretion of AceES-2, a novel immunogenic ES protein recently cloned from A. ceylanicum cDNA. AceES-2 was found to be excreted in feces with kinetics similar to that of whole ES. Examination of individual hookworm antigens by this method will provide new insights into the molecular host-parasite interaction and may form the basis for future diagnostic methods.     

The influence of the procedure of excretory-secretory L1 trichinella spiralis antigen preparation on the efficiency of an ELISA test in pigs

BACKGROUND: Trichinellosis is a parasitic zoonosis transmitted to humans by consumption of raw or undercooked meat from animals infected by worms of the Trichinella genus. Every year seropositive cases are found among the human population and thus trichinellosis still remains an epidemiologically important disease in Poland. The usefulness of ELISA for anti-T. spiralis IgG detection in pigs is still limited by the nature of antigen. The objective in the present study was to compare the usefulness of excretory-secretory antigens of L1 T. spiralis for the serological detection of IgG antibodies in pigs. MATERIAL AND METHODS: The antigens were prepared in different laboratories: Ag ES L1 T. spiralis (N) in Germany, Ag ES L1 T. spiralis (W) in Italy and Ag ES L1 T. spiralis in Poland. Conventional, Iberian pigs were infected with 200, 1000 and 20 000 muscle larvae of T. spiralis. Serum samples were obtained at 5 and 1 dbi (day before infection), and 5, 10, 15, 20, 25, 30, 40, 50, 60 dpi (day post infection) and screened for specific IgG antibodies to excretory-secretory L1 T. spiralis antigens. Serum samples were obtained from the EU project TRICHIPORSE. The cut-off value of ELISA was determined on serum samples from 248 Trichinella-free pigs from Poznaii and Boza Wola, that were examined by artificial digestion. RESULTS: In pigs infected with 200 L1 T. spiralis larvae, specific IgG were detectable from 50 dpi, when the Ag ES L1 T. spiralis (N) was used, whereas when Ag ES L1 T. spiralis (W) and Ag ES L1 T. spiralis were used, the specific IgG were detectable from 40 dpi. In pigs infected with 1000 LI T. spiralis larvae, specific IgG was observed from 30 dpi when Ag ES L1 T. spiralis (W) and Ag ES L1 T. spiralis were used, but when Ag ES L1 T. spiralis (N) was used specific IgG were detectable from 40 dpi. In the group infected with the highest dose of T. spiralis larvae, specific IgG were detectable from 30 dpi when Ag ES L1 T. spiralis (N) and Ag ES L1 T. spiralis (W) were used, whereas when Ag ES L1 T. spiralis was used specific IgG were detectable from 20 dpi. The results strongly indicated that in the examined pigs, the specific IgG response against T. spiralis infection is dose dependent. Furthermore, it was shown that the high infectious dose induced earlier increasing of specific IgG response. Statistical analysis revealed a significant positive correlation between OD values obtained in procedures based on the three antigens. The results were statistically repeatable for procedures and for single pigs (P<0.01).