【作者】 宇芙蓉；

【导师】 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2011， 硕士

【摘要】 目的猪囊尾蚴病是由猪带绦虫的幼虫寄生中间宿主而引起的一种重要的人畜共患寄生虫病,是我国重点防治的寄生虫病之一。它不仅严重地危害着广大人民群众的健康,也给畜牧业生产造成极大的损失。加强猪囊尾蚴病的诊断研究对其防治具有重要意义。本课题利用从猪囊尾蚴cDNA文库中扩增出来的cC1基因,克隆至原核表达载体,利用所得的重组蛋白,免疫25w产蛋海兰母鸡,制备抗猪囊尾蚴特异性鸡卵黄免疫球蛋白(IgY)并鉴定。动态监测IgY效价,大量纯化效价较高阶段的IgY,通过间接ELISA试验和Dot-ELISA试验探讨cC1蛋白和IgY用于诊断猪囊尾蚴病的价值,为猪囊尾蚴病的免疫学诊断提供一种新的方法。方法重组质粒pET28a-cC1阳性重组子转化宿主菌E.coli BL21,提质粒,双酶切鉴定(BamH I、Xhol I)阳性克隆,并送上海生工生物工程技术服务有限公司测序鉴定,IPTG诱导表达,SDS-PAGE和Western blot鉴定,纯化cC1蛋白。购买未经免疫的海兰母鸡雏鸡,养至25W产蛋。每只母鸡以200μg/只进行皮下加肌肉多点注射免疫,共免疫4次,首次免疫后28d进行加强免疫3次,每次间隔时间为10d。收集免疫前、后鸡蛋,标记后4℃保存备用。水稀释法粗提抗猪囊尾蚴IgY抗体,盐析法纯化抗体,SDS-PAGE和Western-blotting进行鉴定。间接ELISA法测定抗体效价,选取效价最高时间段的鸡蛋,EGGstractTM IgY Purification System纯化试剂盒大量提取纯化IgY抗体4℃保存备用。利用cC1蛋白与111份患者血清(正常人血清30例,猪囊尾蚴感染者血清20例,血吸虫感染者血清30例,钩虫感染者血清16例,华支睾吸虫感染者血清15例)反应,进行敏感性、特异性和交叉反应性分析,检测重组抗原活性;同时利用制备的抗猪囊尾蚴IgY抗体与111份患者血清(猪囊尾蚴感染者血清20例,正常人血清30例,血吸虫感染者血清30例,钩虫感染者血清16例,华支睾吸虫感染者血清15例)反应,进行敏感性、特异性和交叉反应性分析,观察制备的抗猪囊尾蚴IgY抗体用作诊断试剂的潜能。结果重组质粒pET28a-cC1限制性内切酶双酶切鉴定与目的基因大小(1056bp)相符,同时测序结果证实重组表达质粒pET28a-cC1构建成功。Lowry法测定制备的纯化的重组cC1蛋白浓度为3.5g/l,相对分子量约40ku。免疫海兰母鸡后,间接ELISA法测定抗体效价,初次免疫后5d左右产生抗体,随着时间推移,抗体滴度逐渐上升,至免疫后55d达到最高峰,效价达到1:105以上。不同产蛋母鸡之间抗体滴度存在个体差异。用EGG stractTM IgY Purification System纯化试剂盒大量纯化IgY,经考马斯亮蓝测定试剂盒测得的IgY浓度为4.96 g/l。SDS-PAGE检测IgY分子量与理论值一致。Western-blotting显示,免疫后的IgY与cC1蛋白可以相互识别,而免疫前的鸡蛋的抗体提取物则不能识别cC1蛋白。以cC1包板,成功建立间接ELISA体系,检测20例猪囊尾蚴病患者血清中抗体,敏感性为85%,特异性为92.20%;假阳性率为15% ;假阴性率为7.8%;阳性预测值为68%;阴性预测值为96.5 %;Youden指数为0.772(真实性较好),总一致率90.1%;期望一致率:Pe为67.57%;Kappa值: K = 0.6947(一致性好);阳性似然比9.67%;阴性似然比0.16;初步实验表明用cC1诊断猪囊尾蚴血清中抗体有着较为满意的结果。Dot-ELISA检测20例猪囊尾蚴病患者血清中循环抗原,敏感性为95%,特异性为98.9%,假阳性率为5%,假阴性率为1.1% ,阳性预测值为95%,阴性预测值为98.9%,Youden指数:YI为0.939(真实性很好),总一致率P0为98.2%,期望一致率Pe为70.46%,Kappa值为0.939,一致性好;与华支睾吸虫病患者血清、血吸虫病患者血清及钩虫虫病患者血清无交叉反应。结论本研究成功大量制备了抗猪囊尾蚴特异性IgY抗体,具有良好的安全性和稳定性,易于保存。成功建立间接ELISA体系和Dot-ELISA体系,分别用于猪囊尾蚴病人血清中抗体和循环抗原的检测,初步试验表明,有较为满意的结果,有望为猪囊尾蚴病诊断提供了新的试剂。更多还原

【Abstract】 Objective: Cysticercosis is an important zoonotic diseases caused by The larvae of Taenia solium ,is a focus on prevention and control of parasitic diseases. It is not only seriously endangering the health of the masses, but also causing great loss of livestock production. Therefore, strengthening the diagnosis of swine cysticercosis is important for its prevention and treatment. In this study, we clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli. 25-weeks old hens was immunized by subcutaneous injection and intramuscular injection with purified Cysticercus cellulosae cC1 protein. Then the anti-cysticercus specific yolk immunoglobulin (IgY) was prepared and identified from by cC1. We monitored the dynamic a antibody titer of IgY,and purified the antibody at higher titer. We explored cC1 protein and IgY in the the role of diagnosing swine cysticercosis by indirect ELISA and Dot-ELISA test .Methods: The recombinant plasmid pET28a-cC1 presented by Professor Fang Jiang of Bengbu Medical College was transformed into E. coli BL21. The recombinant plasmid pET28a-cC1 digested with BamH I and Xho I ,The results sent to Shanghai Sangon Biotech Technical Services Limited sequencing . The fusion expression of cC1 was induced by IPTG. The expression was subjected to SDS-PAGE followed by identification with Western blotting. The 25 week-old egg-laying HY-line hens were immunized with 200μg cC1 by subcutaneous injection and intramuscular injection with purified Cysticercus cellulosae cC1 protein for 4 times. Each hen was immunized with 200μg antigen each time, the second injection were performed 28 days later, Subsequent injection were performed at 10-day interval. The eggs layed before and after immunization were collected to make anti-cC1 IgY antibody(Storage Temperature is 4℃).IgY was extracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDS-PAGE and Western-blotting. Indirect ELISA was used to determine the titer of the antiserum. Large amount IgY was purified by EGGstractTM IgY Purification System while the antibody had a high titer. with indirect ELISA method , to examine the sera from 111 cases, including 30 normal human controls, 20 Cysticercosis patients, 30 schistosomiasis patients, 16 hookworm patients, and 15 clonorchiasis patients, and sensitivity, specificity and cross reactivity were tested respectively. and with Dot-ELISA method , to examine the sera from 111 cases, including 30 normal human controls, 20 Cysticercosis patients, 30 schistosomiasis patients, 16 hookworm patients, and 15 clonorchiasis patients, and sensitivity, specificity and cross reactivity were tested respectively.Result: The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, The concentration of cC1 was 3.5g/l and the molecular weight was 40ku . The molecular size of IgY detected by SDS-PAGE was equal to its predictive value. Western blotting results showed that the cC1 could be recognized by IgY collected from immunizsed hens while not by IgY collected before immunization. Using indirect ELISA, the facts were observed that the IgY antibody began to produce by hens on the fifth day after the initial immunization, and the antibody titer increased gradually over time while reached the peak with a titer overtoped 1:105 on the 55th day after immunization. There is a discrepancy on IgY antibody titer between different hens. The IgY was purified heavy by EGG stractTMIgY Purification System and the concentration was 4.96g/l by Coomassie brilliant blue . The primary detection of antibodies to cC1 in the patients with cysticercosis showed a promising sensitivity and specificity with indirect ELISA. We successfully established an indirect ELISA system to detect 20 cases of cysticercus antibodies in serum of patients with a sensitivity of 85%,specificity of 92.20%, false positive rate was 15%,false negative rate was 7.8%, positive predictive value was 68%, negative predictive value was 96.5%, Youden index was 0.772,the total agreement rate of was 90.1%, Pe was 67.57%; Kappa value was 0.6947, positive likelihood ratio was 9.67%, negative likelihood ratio was 0.16,It show that the diagnosis of cysticercosis with cC1 serum antibody has a satisfactory results. We use the Dot-ELISA to detect 20 cases of cysticercosis patients with circulating antigen in serum, the sensitivity was 95%, specificity was 98.9%, false positive rate of 5%, false negative rate of 1.1%, positive predictive value of 95 %, negative predictive value was 98.9%, Youden index was 0.939, P0 the total agreement rate was 98.2%, expected agreement rate Pe was 70.46%, Kappa value was 0.939, and clonorchiasis patients with serum, schistosomiasis hookworm disease patients infected serum and serum-free cross-reaction.Conclusion: We have successfully prepared the IgY antibody specific to cysticercus cellulosae with good security and stability. The yield of IgY is generous and easy to store. We successfully established an indirect ELISA system and the Dot-ELISA system, respectively, for the serum Cysticercus antibodies and circulating antigen detection.The preliminary tests showed that there is a satisfactory result, diagnosis of cysticercosis is expected to provide a new reagent .更多还原