摘要
鸡传染性贫血(chicken infectious anemia,CIA)是由鸡传染性贫血病毒(chicken infectious anemia virus,CIAV)引起雏鸡的以坏疽、淋巴组织损伤和骨髓发育不全为特征的传染病，是继IBD后的一种重要的免疫抑制性疾病。目前，本病已成为世界性疾病，严重地威胁着养禽业。但在现地，由于并发继发的病毒性、细菌性传染病或球虫病升高了CIA的死亡率，而且以后者症状更为突出，导致临床对本病的忽视，并且因为传统CIA血清学方法中的血清中和试验(SN)和间接免疫荧光试验(IFA)存在着费时、费力、对操作者技术要求高和仅适于实验室诊断的问题，因此，急需一种快速、简便、敏感、廉价和能检测大批量样品的诊断方法用于免疫鸡群的抗体监测和流行病学调查。
CIAV的VP1蛋白不仅是病毒的唯一衣壳蛋白，也是主要的免疫原蛋白。本试验用生物软件分析vp1基因及其编码产物，预测了该蛋白的抗原表位，并对预测的其富含抗原表位的C末端基因进行了原核表达。根据GenBank公布的标准毒株Cux-1基因序列，设计合成了一对引物，对CIAV-M9905株的vp1基因进行PCR扩增，产物经琼脂糖凝胶电泳分析，可见一条1350bp的特异带，用Pstl酶切后，将其回收，克隆到pProEXHTa载体，再亚克隆到pGEX-6p-1载体中，得到重组质粒pGEX-vp1，PCR鉴定为阳性者再用BamHI消化阳性质粒，琼脂糖凝胶试剂盒回收大片段，自连后转化感受态细胞，用BamHI鉴定为阳性者命名为pGEX-vp1(C)，得到目的克隆。
将重组刚性质粒pGEX-vp1(C)转入表达宿主菌BL_(21)中，经终浓度为0.6mmol／L IPTG诱导，成功表达了VP1 C端基因，重组蛋白表达量占菌体蛋白的39.4％。经ELISA检测表明，表达的重组蛋白能被CIAV阳性血清识别，具有良好的抗原性。
把表达的重组蛋白经初步纯化后用作包被抗原，建立了检测CIAV血清抗体的间接ELISA方法，并确定了最适封闭液、最适抗原包被浓度、最适血清稀释度、血清最适作用时间、酶标抗体最适稀释度和最适作用时间及底物最适显色时间。包被的重组抗原不与鸡新缄疫(ND)、马立克氏病(MD)、传染性支气管炎(IB)-Ⅰ型和Ⅱ型、禽流感(AI)-H9和H7型、鸡法氏囊病(IBD)、鸡网状内皮组织增生病(RE)、禽白血病(AL)-J亚群、鸡白痢阳性血清发生交叉反应，表明建立的ELISA诊断方法只有良好的特异性。用该方法可检出人工攻毒后110天81％的CIAV血清抗体，表明其具有很高的敏感性。批内重复试验，变异系数小于10％；批间重复试验，变异系数小于15％，说明具有很好的重复性。与全病毒抗原检测结果符合率为92％以上，与进口试剂盒的符合率达97％。
本研究在国内首次成功表达了CIAV VP1蛋白抗原表位优势区，并成功建立了ELISA诊断方法。CIAV重组抗原间接ELISA诊断试剂盒的研制，为免疫鸡群抗体监测和进行CIA流行病学调查提供了一种快速简便的血清学方法。
Chicken infectious anemia (CIA) is caused by chicken infectious anemia virus (ClAV).It characterized by necrosis and lymphoid depletion in lymphatic tissues, as well as aplasia of the bone marrow in young chickens. CIA is an important and immunosuppressive disease treatening poultry industry and has been a cosmopolitan disease. However, in the field, the disease is usually concurrent with other virosis, bacteroidal disease and coccidiosis, and aggravate the mortality of CIA furthermore the latter sign is more outstanding . That is the one reason that CIA is neglected in prevention. Moreover, in the traditional detecting assay of sera, SN and 1FA have undeniable disadvantages, such as expensive time, operated by a professional, limited in lab. So, a rapid, simple, sensitive, cheap and able to detect many samples assay is urgent need to assess vaccination in the field and develop epidemiological survey.
The VPI protein of CIAV is the unique capcid protein and the tmmunogenic protein. In this study, epitopes of VPI protein was analyzed by biological software, then the epitopes located C-terminal was cloned into vector and expressed successfully in E.coli. A pair of primers was cooperated by sequences of Cux-1 published in GenBank. With the primers, vpl of CIAV-M9905 was amplified by polymerase chain reaction (PCR), the product was analyzed by agar gel ,and vpl, a fragment of 1350bp was obtained. The fragment was digested by PstI and reclaimed with agar gel Kit, then was cloned into pProEXHTa vector and sub-cloned into pGEX-6p-l vector, then was detected by PCR, and the recombinant, positive plasmid was named pGEX-vp1. The positive plasmid was digested with BamHI,, and the bigger fragment was reclaimed with agar gel Kit, after self-connection then transformed into BL21 comptent cell of E.coli. The plasmid identified by BamHI and seguencing was positive and named pGEX-vp1(C), so a recombinant plasmid used to e
xpress purposed protein was constructed. The recombinant bacterial was induced by IPTG with a finial concentration of 0.6mmol/L.SDS-PAGE analysis showed that the recombinant protein antigen expressed could reach 39.4 percent of the whole bacterial protein. The expressed protein could be recoganized with polyclonal antibodies against CIAV by indirect ELISA, sharing a good antigencity.
Using the recombinant and purified protein as antigen to coat micro-plate of 96 wells, an indirect ELISA assay was developed to detect anti-CIAV sera. The optimal conditions including blocking solution, concentration of antigen, dilution and reaction time of sera, dilution and reaction time of conjugate, reaction time of substate were determined for the ELISA. The recombinant antigen showed no reaction with the positive sera of other avian diseases, such as Newcastle Disease(ND) Marek's Disease(MD) Infectious Bronchitis(IB)-I, IB-II, Avian Influenza(AI)-H9 AI-H7 Infectious Bursal
Disease(IBD) Reticuloendotheliosis(RE) Avian Leukosis(AL)-J and Fowl Pullorum. The result suggested the ELISA assay was specific. The intro-batch duplicativity test showed that variation coefficient was less 10%, and the inter-batch duplicativity test showed that it was not more than 15%. The positive rate of 81 % could bedetected in the 110th- day-sera from chickens inoculated CIAV. The result showed the ELISA assay had good replicativity and high sensitivity. When compared with cultured virus antigen, above 92% agreement was obtained; in compare with the commercial Kit, 97% agreement was obtained.
In the domestic field of CIA, this study firstly successfully express immunogenic dominant region of VPl protein to use as detecting antigen and develop indirect ELISA assay. The development of the CIAV indirect ELISA Kit basted on recombinant antigen afforded a simple and rapid means of detecting anti-ClAV in monitoring CIAV infection or assessing vaccination in the field.
引文
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