鸡传染性法氏囊病毒检测方法的建立
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摘要
本试验通过SPF鸡胚和CEF细胞增殖鸡法氏囊病病毒(IBDV)，吸取接毒后2～5d内死亡鸡胚的尿囊液和绒毛尿囊膜及细胞病变达到80％时的病毒细胞培养液，经反复冻融，利用氯仿沉淀病毒，聚乙二醇6000浓缩病毒，蔗糖梯密度低温超速离心和葡聚糖G—200凝胶柱过滤等方法纯化病毒，均得到了较高纯度的IBDV。将纯化的IBDV分别免疫家兔和小鼠，制备了兔抗IBDV、鼠抗IBDV的高免血清，用健康兔的血清免疫羊而获得羊抗兔免疫血清。经饱和硫酸铵、葡聚糖凝胶G—25脱盐后，获得了羊抗兔、兔抗IBDV及小鼠抗IBDV免疫球蛋白IgG的粗提物，经DEAE—32离子交换层析纯化了羊抗兔和小鼠抗IBDV的IgG。用改良的过碘酸钠法和柠檬酸三钠—鞣酸混合还原法分别制备了高纯度的羊抗兔辣根过氧化物酶标记抗体和胶体金标记抗体。依据方阵测定法，通过对兔抗IBDV IgG、小鼠抗IBDV IgG及标记抗体的最佳使用浓度的确定，建立了双夹心酶联免疫吸附试验(双夹心ELISA)、斑点酶联免疫吸附试验(Dot—ELISA)和斑点免疫金银染色法(Dot—IGSS)等3种用于检测IBDV抗原的免疫学方法。双夹心ELISA方法的包被抗体的最适浓度为1：100，兔抗IBDV的最佳反应浓度为1：200，酶标记抗体的最佳工作浓度为1：400。Dot—ELISA试验的兔抗IBDV的反应浓度为1：100，酶标记二抗的工作浓度为1：200。Dot—IGSS试验的兔抗IBDV的包被浓度为1：200，金标记抗体最适工作浓度为1：10。经重复性试验、特异性阻断及交叉性试验，结果表明3种检测方法具有特异性强、重复性高，均不与鸡新城疫、鸡传染性支气管炎和鸡产蛋减少综合症病毒抗原交叉反应的优点。通过敏感性的试验及其与琼脂扩散试验(AGP)的比较得到了以下结果，即双夹心ELISA检测法氏囊病毒抗原的敏感性最高，最低检出量为0.3192μg／ml，其次是Dot—IGSS，达到0.798μg／ml，最后是Dot—ELISA，其敏感性较低，只有1.596μg／ml。我们建立的这3种方法都比AGP的敏感性高，双夹心ELISA是AGP的100倍，Dot—IGSS是AGP的40倍，Dot—ELISA是AGP的20倍。通过比较鸡法氏囊病病毒不同毒株的编码A片段的基因序列，根据Genebank上经典毒株STC的A片段上编码VP2和VP5蛋白相互重叠部分处保守序列内合成一对寡核苷酸引物，建立了检测IBDV的反转录—聚合酶链式反应(RT-PCR)。经特异性、敏感性和重复性试验检测IBDV抗原，均扩增出了一大小为267bp的目的核苷酸片段。其敏感性达到了1.995ng／ml，是所有检测IBDV抗原的方法里最为敏感的方法。实验也证明PCR方法有快速、准确、操作简便、敏感性高、易于判读、特异性强、重复性好等优点，是检测IBDV抗原的最快和最有效的方法，可用于大量IBDV抗原样本的检测。
In this test, All Infectious bursal disease virus (IBDV) BC 6/85 F4 and IBDVHE BEI isolated strain were propagated on specific-pathogen-free (SPF) 10-day-old embryonated eggs and on chicken-embryo fibroblast (CEF) monolayer that were prepared from SPF embryonating chicken eggs. All IBDV BC 6/85 F4 and IBDV HE BEI isolated strain were obtained from allantochonon and allantoic fluid of died embryonated eggs in 2-5 days after inoculating and virus-infected cell fluid that were frozen and thawed three times after approximately 80% of the monolayer exhibited cytopathic effects. Highly purified IBDV growing on SPF embryonating chicken eggs was got respectively and essentially through freezing and thawing three times, precipitated virus with chloroform, concentrated by polyethylene glycol 6000 and ultracentrifugated in sucrose density gradient and IBDV propagated on CEF cell was concentrated by using polyethylene glycol 6000 and percolated with method of gel filtration with Sephadex G-200. The hyperimmune serum of
rabbit and mouse anti-IBDV was prepared by immunization in rabbit and mouse with purified IBDV. Mainly immunoglobulin G (IgG) of sheep anti-rabbit, rabbit and mouse anti-IBDV was obtained by ammonium sulphate precipitation subsequently unsalted with Sephadex G-25 and chromatography over diethylamino-ethyl-cellulose-32 (DEAE-32). The sheep anti-rabbit IgG conjugated to horseradish peroxidase(HRP) and labeled with colloidal gold was individually made by the method of sodium periodate and sodium citrate-tannic acid. Three immunology methods (indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISAX Dot-ELISA and Dot immunogold-silver staining (Dot-IGSS) ) were established by the optimal concentration fixation of rabbit, mouse anti-IBDV IgG and sheep anti-rabbit IgG according to the phalanx determination to detect IBDV antigen. The optimal incubating antibody concentration in sandwich ELISA method was 1 .' 100, the optimal rabbit anti-IBDV IgG concentration was 1 : 200 and in HRP labeled sheep
anti-rabbit antibody, optimal concentration was 1 : 400. In Dot-ELISA test reactive concentration of rabbit anti-IBDV was 1 I 100, the optimal working concentration of HRP labeled sheep anti-rabbit antibody was 1 : 200.The incubating concentration of rabbit anti-IBDV in Dot-IGSS test is 1 : 200 and sheep anti-rabbit labeled with
colloidal gold was 1 : 10.Three methods proved to be superior in terms of specificity and duplication through the specificity blocking test, cross test and duplication test and they had no antigen-crossing react with new castle virus (NDV). IBV and EDV. Comparing the result of sensitivity test with Agar Gel Precipitin Test (AGP) , sandwich ELISA had the highest sensitivity in detecting IBDV antigen and its lowest detective concentration was 0.3192@g/ml,then was Dot-IGSS test which detective concentration was 0.798@g/ml,and the last was Dot-ELISA which had the lowest concentration ,only 1.596@g/ml.All the established three methods were better then AGP in sensitivity. Sandwich ELISA was one hundred times than AGP, Dot-IGSS was forty times than AGP, Dot-ELISA was twenty times than AGP. Through the compare of sequence in genome segment A of IBDV different strains, a pair of oligonucleotide primers were synthesized in the overlapping area of VP5 and VP2 where the sequence is highly homologous according to the seq
uence of genome segment A of classic strains "STC" from Genebank and 267bp intent nucleotide fragment were amplified .The same size of intent gene fragment were got from the test of specificity, sensitivity and replicate detecting IBDV antigen and its sensitivity got to 1.995ng/ml.This method was most sensible one in whole method of detecting IBDV antigen. Our test also verified PCR method have the excellence of celerity, nicety, easy to performing, high sensitivity, easy to reading, high specificity and good repeating and this was a most nicety and most available method for detecting IBDV, which can be used for detecting a majority of IBDV specimens.
In this test, All Infectious bursal disease virus (IBDV) BC 6/85 F4 and IBDVHE BEI isolated strain were propagated on specific-pathogen-free (SPF) 10-day-old embryonated eggs and on chicken-embryo fibroblast (CEF) monolayer that were prepared from SPF embryonating chicken eggs. All IBDV BC 6/85 F4 and IBDV HE BEI isolated strain were obtained from allantochonon and allantoic fluid of died embryonated eggs in 2-5 days after inoculating and virus-infected cell fluid that were frozen and thawed three times after approximately 80% of the monolayer exhibited cytopathic effects. Highly purified IBDV growing on SPF embryonating chicken eggs was got respectively and essentially through freezing and thawing three times, precipitated virus with chloroform, concentrated by polyethylene glycol 6000 and ultracentrifugated in sucrose density gradient and IBDV propagated on CEF cell was concentrated by using polyethylene glycol 6000 and percolated with method of gel filtration with Sephadex G-200. The hyperimmune serum of
rabbit and mouse anti-IBDV was prepared by immunization in rabbit and mouse with purified IBDV. Mainly immunoglobulin G (IgG) of sheep anti-rabbit, rabbit and mouse anti-IBDV was obtained by ammonium sulphate precipitation subsequently unsalted with Sephadex G-25 and chromatography over diethylamino-ethyl-cellulose-32 (DEAE-32). The sheep anti-rabbit IgG conjugated to horseradish peroxidase(HRP) and labeled with colloidal gold was individually made by the method of sodium periodate and sodium citrate-tannic acid. Three immunology methods (indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISAX Dot-ELISA and Dot immunogold-silver staining (Dot-IGSS) ) were established by the optimal concentration fixation of rabbit, mouse anti-IBDV IgG and sheep anti-rabbit IgG according to the phalanx determination to detect IBDV antigen. The optimal incubating antibody concentration in sandwich ELISA method was 1 .' 100, the optimal rabbit anti-IBDV IgG concentration was 1 : 200 and in HRP labeled sheep
anti-rabbit antibody, optimal concentration was 1 : 400. In Dot-ELISA test reactive concentration of rabbit anti-IBDV was 1 I 100, the optimal working concentration of HRP labeled sheep anti-rabbit antibody was 1 : 200.The incubating concentration of rabbit anti-IBDV in Dot-IGSS test is 1 : 200 and sheep anti-rabbit labeled with
colloidal gold was 1 : 10.Three methods proved to be superior in terms of specificity and duplication through the specificity blocking test, cross test and duplication test and they had no antigen-crossing react with new castle virus (NDV). IBV and EDV. Comparing the result of sensitivity test with Agar Gel Precipitin Test (AGP) , sandwich ELISA had the highest sensitivity in detecting IBDV antigen and its lowest detective concentration was 0.3192@g/ml,then was Dot-IGSS test which detective concentration was 0.798@g/ml,and the last was Dot-ELISA which had the lowest concentration ,only 1.596@g/ml.All the established three methods were better then AGP in sensitivity. Sandwich ELISA was one hundred times than AGP, Dot-IGSS was forty times than AGP, Dot-ELISA was twenty times than AGP. Through the compare of sequence in genome segment A of IBDV different strains, a pair of oligonucleotide primers were synthesized in the overlapping area of VP5 and VP2 where the sequence is highly homologous according to the seq
uence of genome segment A of classic strains "STC" from Genebank and 267bp intent nucleotide fragment were amplified .The same size of intent gene fragment were got from the test of specificity, sensitivity and replicate detecting IBDV antigen and its sensitivity got to 1.995ng/ml.This method was most sensible one in whole method of detecting IBDV antigen. Our test also verified PCR method have the excellence of celerity, nicety, easy to performing, high sensitivity, easy to reading, high specificity and good repeating and this was a most nicety and most available method for detecting IBDV, which can be used for detecting a majority of IBDV specimens.
引文
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