猪囊尾蚴低分子质量抗原基因Ag1V1蛋白原核表达条件的优化

<正>猪囊尾蚴病是由猪带绦虫(Taenia solium)幼虫引起的一种重要的人畜共患寄生虫病[1]。目前,囊尾蚴诊断中影像学诊断效果较好,但价格昂贵[2],免疫学诊断方法成本低于影像学诊断,适于在感染地区推广应用。囊尾蚴病的免疫学诊断方法主要包括抗体检测和抗原检测。无论抗原检测还是抗体检测均需要筛选敏感性好、特异性强的抗原。本研究在成功克隆猪     

猪囊尾蚴头节单克隆抗体的制备及特性鉴定

为制备抗猪囊尾蚴头节单克隆抗体(MAb),本研究采用纯化的猪囊尾蚴头节(Cysticercus Cellulosae Scolex)作为抗原免疫BALB/c小鼠,通过杂交瘤细胞技术,获得3株分泌抗猪囊尾蚴头节MAb的杂交瘤细胞系(分别命名为2C6、3A5和4G10)。对这3株MAb进行生物学特性鉴定,其染色体平均记数为92±10对,经间接ELISA检测腹水效价分别为1:12800、1:6400和1:25600。亚类鉴定证实,这3株亚类均为IgG1型。Westernblot检测表明,3株纯化的MAb均可与猪囊尾蚴头节抗原发生特异反应;而与猪囊尾蚴囊壁和囊液抗原均不发生反应。本研究获得的3株MAb为猪囊尾蚴病免疫学诊断研究奠定技术基础。     

猪囊尾蚴特异性单克隆抗体的制备及其识别抗原成分的鉴定

通过SDS-PAGE方法对猪囊尾蚴囊液抗原成分进行了分离鉴定,并分别分离纯化了其中的16 000和10 000特异性蛋白质成分;将16 000和10 000纯化蛋白质成分分别做成佛氏佐剂苗,分组免疫BALB/c小鼠,超免后,无菌取脾脏制备脾细胞,分别与NS0骨髓瘤细胞进行融合,经阳性筛选和特异性鉴定获得2株分泌猪囊尾蚴特异性单抗的杂交瘤细胞,命名为TSCF-1611H12B8和TSCF-1012G5B5。免疫学鉴定结果表明,单抗TSCF-1611H12B8和TSCF-1012G5B5与猪细颈囊尾蚴、旋毛虫、住肉孢子虫和蛔虫抗原间不存在交叉反应;单抗TSCF-1611H12B8识别囊液中16 000和10 000蛋白质抗原条带,而单抗TSCF-1012G5B5识别囊液中的10 000蛋白质条带。     

兔豆状囊尾蚴囊液蛋白的纯化与抗原性鉴定

为了找出抗原性强、免疫原性好的兔豆状囊尾蚴抗原蛋白,本研究对兔豆状囊尾蚴的头节、体节和囊膜等结构性抗原与囊液中代谢性抗原进行了比较,发现囊液代谢性抗原的免疫效果优于结构性抗原。通过SDS-PAGE和微孔超滤分离法对囊液蛋白进行纯化,再用纯化的蛋白免疫小鼠,制备高免血清,并对免疫鼠的免疫器官进行病理学和免疫组织化学检查。结果证明用从囊液中纯化的63 000蛋白免疫效果最好。用纯化的63 000蛋白免疫小鼠可获得高效价抗血清,小鼠免疫器官活化,巨噬细胞、滤泡树突细胞、淋巴细胞和浆细胞等各种免疫细胞增多,用免疫组织化学染色,免疫器官中的CD4、CD8T细胞和CD20B细胞呈强阳性反应。总之,从囊液中分离纯化的63 000蛋白抗原性强、免疫原性,可用于对豆状囊尾蚴病的免疫学诊断。     

羊绦虫蚴病免疫学诊断研究进展

羊绦虫蚴病是以羊为中间宿主、犬为终末宿主的绦虫蚴病,常见的有羊棘球蚴病、羊囊尾蚴病、羊脑多头蚴病、羊细颈囊尾蚴病。这些羊绦虫蚴病免疫学诊断方面如ELISA取得一些进展,但仍然缺乏高敏感性和特异性的诊断抗原,尸体剖检依然是临床诊断的金标准。     

猪囊尾蚴抗原的免疫化学研究

应用琼脂双扩散(DID),免疫电泳(IEP),聚丙烯酰胺凝胶电泳(PAGE)对猪囊尾蚴的匀浆抗原和囊液抗原进行分析。结果表明,两种抗原间存在相同的抗原成份,囊液的免疫化学性质优于匀浆抗原,并从免疫学和生物化学角度初步探索了囊液抗原和匀浆抗原中蛋白质之间的相互关系及主要蛋白质组分。     

旋毛虫ES抗原特异性成分的分离和鉴定

在旋毛虫病的免疫学诊断中,目前常用的抗原是虫体粗制抗原(CWE)、排泄—分泌抗原(ES)及其纯化物。研究表明,CWE易出现假阳性反应,其诊断准确率不高;而ES易出现假阳性反应较少,特异性较强,因此愈来愈受到国内外学者的重视。但在ES中究竟存在几种特异性蛋白成分?它们的分子     

猪囊虫病快速免疫诊断技术的研究与应用

用纯化的猪囊尾蚴囊液蛋白作诊断抗原,吸附于硝酸纤维素膜片上,经４０％乙醇固定,以斑点酶联免疫吸附法检测猪囊虫病抗体。结果发现：在一抗,二抗和底液中加入１０ｍＭ ＥＤＴＡ,四甲基联苯胺作显色剂,１ｈ内即可完成诊断,并显著提高免疫诊断的灵敏性和特异性。     

猪囊尾蚴排泄分泌抗原Ts8B2基因的克隆、表达及纯化

利用RT-PCR技术克隆了猪囊尾蚴排泄分泌抗原Ts882蛋白基因,测序结果表明：序列长度为270bp,包含258bp的编码区,编码85个氨基酸,与GenBank中西班牙分离株的核苷酸和氨基酸序列的同源性均为99％;将该基因成熟肽部分亚克隆至原核表达载体pGEX-6p-1,转化大肠杆菌BL21（DE3）,IPTG诱导后,进行SDS-PAGE初步分析,在34ku位置有1条蛋白条带,与预期结果一致。通过GST亲和纯化获得了较高纯度的重组融合蛋白,Westernblot结果表明,纯化后的重组融合蛋白可与兔抗全囊囊尾蚴血清发生特异性反应。排泄分泌抗原Ts882的成功表达、纯化为进一步研究该蛋白在抗体检测中的应用奠定了基础。     

细粒棘球蚴病诊断抗原研究进展

细粒棘球蚴病严重危害人畜健康,给畜牧业造成巨大经济损失。该病的早期诊断有很重要的意义。囊液抗原、原头节抗原和囊壁抗原等天然抗原是用于该病诊断的主要抗原,但存在敏感性差、特异性弱等缺点。近年来,人们合成了AgB、Ag5、EpC1、EgTPx等重组抗原,将其用于诊断,并获得了较高的敏感性和特异性。论文将用于诊断的天然抗原和重组抗原进行了综述。     

猪囊尾蚴重组诊断抗原的研究进展

猪囊虫病是一种重要的人畜共患寄生虫病,严重危害了人类的健康,对囊虫病的有效诊断成为控制该病的关键。目前,囊虫病的诊断主要依靠免疫学检测,所用抗原多由虫体制备,存在虫源短缺而不易大量获得的问题。利用基因重组技术可以解决以上问题。本文拟对猪囊尾蚴重组诊断抗原的研究进展做一综述。     

Countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats

The procedure of countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats was carried out for antemortem diagnosis of T. hydatigena cysticercosis in experimentally and naturally infected goats. The antigens of cyst fluid, scolex and membrane of T. hydatigena metacestodes were purified and compared. The sensitivity of the test in experimentally and naturally infected goats was 57.1 and 52.5%, respectively, whereas its specificity using antisera raised against T. solium cysticercosis, hydatid cyst and Fasciola gigantica was 66.7 and 83.4% with partially purified and fractionated antigens, respectively. Of all three antigens, the cyst fluid antigen was found to be most reactive. The test could be employed for antemortem diagnosis of T. hydatigena cysticercosis using purified antigen.     

Performance of the ELISA test for swine cysticercosis using antigens of Taenia solium and Taenia crassiceps cysticerci

Studies were conducted to evaluate antigens of Taenia solium (Tso) and Taenia crassiceps (Tcra) cysticerci in the ELISA test for the diagnosis of swine cysticercosis. The samples analyzed were cysticercosis positive and negative control sera and heterologous sera. Four antigens were assayed: vesicular fluid (VF) and crude (T) Tcra and scolex (S) and crude (T) Tso. All antigens showed good performance, but VF-Tcra was the best followed by T-Tcra. Sensitivity rates of ELISA were respectively, in 2nd and 3rd standard deviation cut-offs, 96.0 and 80.0% for the VF antigen and specificity of 97.5 and 100.0%. Cross-reactivity was verified only for hidatidosis and ascaridiosis. Due to the high performance observed, the ELISA test using Tcra antigens should be recommended for the diagnosis of swine cysticercosis.     

猪囊尾蚴重组抗原和基因工程疫苗的研究进展

猪囊尾蚴病（Cysticercosis）是由猪带绦虫的幼虫囊尾蚴寄生于人或猪等而引起的人畜共患寄生虫病,是公认的世界经济病之一。严重威胁着人体健康,并给畜牧业造成重大经济损失,猪囊尾蚴病的免疫防治势在必行。然而在猪囊尾蚴病疫苗研究中,疫苗抗原的选择和来源一直困扰着兽医工作者。该文就近年来猪囊尾蚴病诊断重组抗原和基因工程疫苗的分子生物学研究进展进行了综述。     

Improvements in the serodiagnosis of helminthic zoonoses

Remarkable progress has been achieved in developing improved serodiagnostic assays for a group of diseases for which other diagnostic methods are often lacking. Toxocariasis, trichinellosis, dirofilariasis, Taenia solium cysticercosis and the cystic and alveolar forms of hydatid disease are occult infections in humans and sometimes in lower animal hosts. Although Strongyloides stercoralis achieves patency in humans, parasitologic diagnosis is often very difficult. Efforts to develop reliable immunodiagnostic methods have spanned several decades but progress had been slow until recently. The complexity and nonspecificity of helminth antigens were major problems which prevented the full realization of the benefits of the highly sensitive assay systems now available. Modern immunologic methods including hybridoma technology, immunoaffinity chromatography and immunoblotting, however, have yielded improved reagents and the means to characterize their nature and function. The outcome of this research has been more sensitive and specific serologic tests based on measurement of both circulating antigens and antibodies as well as improved understanding of the nature of host-parasite interactions. Although much remains to be done, many improved immunodiagnostic procedures are already being applied in clinical diagnosis, epidemiologic studies and control programs directed against the helminthic zoonoses.     

A sero-epidemiological study of bovine cysticercosis in Zambia.

A sero-epidemiological study of Taenia saginata cysticercosis was carried out in adult cattle in Zambia to determine the prevalence and study the influence of the farming system on the infection rate. Serum samples were examined for circulating parasite antigen by a monoclonal-based sandwich ELISA. Thirty-eight of 628 serum samples were found positive (prevalence 6.1%). Cysticercosis was significantly more prevalent in feedlots and in traditional farming systems than in dairy farms. It is suggested that the continuous man to animal contact and the use of casual workers in feedlots may be factors that are conductive to T. saginata transmission.     

五种旋毛虫抗原对猪的免疫保护作用研究

本实验研究了旋毛虫肌幼虫可溶性粗抗原、排泄分泌抗原（ＥＳ）、表面抗原（ＳＡ）及成虫ＥＳ、ＳＡ５种抗原对猪的免疫保护作用。结果５种抗原对猪均具有一定程度的免疫原性，可诱导猪体产生对攻击感染的抵抗力（减虫率），其中肌幼虫粗抗原为５５．２０％；肌幼虫ＥＳ为４２．５６％，肌幼虫ＳＡ为７２．２１％；成虫ＥＳ为３２．９２％；成虫ＳＡ为４２．１７％。免疫５种抗原后用肌幼虫“Ｂ”抗原、新生幼虫可溶性抗原及成虫可溶性抗原进行ＥＬＩＳＡ检测，均可测出血清抗体应答反应，其中以相应抗原测出的抗体应答较强烈。免疫５种抗原后猪外周血液中Ｂ淋巴细胞减少，Ｔｈ及Ｔｓ增加，Ｔｈ／Ｔｓ比值降低，呈暂时的细胞免疫抑制现象。     

Detection of Taenia solium Cysticercosis in Pigs by ELISA with an Excretory-Secretory Antigen

Serodiagnosis of Taenia solium cysticercosis in pigs was conducted by an enzyme-linked immunosorbent assay with an excretory-secretory antigen. The antigen was obtained by in vitro cultivation of the cysticerci in a synthetic medium RPMI 1640. The sensitivity and specificity of the ELISA in detecting infection in pigs reared on free range was 92% and 100%, respectively. In addition, 33.33% of pigs in which infection could not be detected at meat inspection were found positive by ELISA. However, none of the sera from a group of farm-reared pigs were positive. No cross reactions were observed in pigs that contained either the cysticerci of Taenia hydatigena or hydatid cysts.     

Serodiagnosis of porcine cysticercosis by enzyme-linked immunosorbent assay (ELISA) using fractionated antigens

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Taenia solium cysticercosis was evaluated in experimentally and naturally infected pigs, using T. solium larval scoleces and its fractionated 1st and 2nd peaks on Sephadex G-200 as antigens. First peak antigen gave maximum sensitivity and highest antibody titres. The overall sensitivity of this test was found to be 91.5, 95.8 and 70.8% with scolex, 1st and 2nd peak antigens, respectively. False positive reactions occurred in 9.09% of uninfected pigs with scolex and 1st peak antigens and cross-reactions occurred in 25% of Taenia hydatigena-infected animals using scolex and 2nd peak antigens. No cross-reaction was observed using 1st peak antigen. The specificity of the test was 92.3, 96.2 and 92.3% with scolex, 1st and 2nd peak antigens, respectively.     

Evaluation of the ELISA test for the antibody detection in cattle naturally and experimentally infected with Cysticerccus bovis

The ELISA test was evaluated for the diagnosis of bovine cysticercosis using heterologous antigens from the larvae of T. solium and T. crassiceps, by using different types of positive and negative control sera, to allow a broader analysis of the results. The ELISA test showed low sensitivity under natural conditions of bovine cysticercosis manifestation, but high rates (up to 90%) under experimental conditions. The high specificity of the test (81-100%) made evident its capacity to differentiate cysticercosis from other bovine diseases. No difference in performance was found among the antigens studied. It was concluded that the ELISA test has deficiencies in detecting anti-cysticercosis antibodies of animals at slaughterhouse. However, it can be useful in detecting experimentally infected animals and differentiating cysticercosis from other bovine diseases.