| 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 express 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 BursalDisease(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. |
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