The Development And Application Of The Elisa For Detecting Antibodies Against Aleutian Mink Disease Virus

Aleutian m ink disease(AMD) is a chronic progressive disease of mink caused by Aleutian mink disease virus(AMDV). This disease is associated with a variety of abnormalities of the immune system, which leads to plasmacytosis and persistent virusemia. AMDV has been found in all mink breeding countries and caused the greatest financial loss to farmers, including decreased production, loss of breeding animals, and low-quality fur. In China, AMDV cannot be diagnosed effectively deal to the lack of effective diagnose method. To control the AMDV spread, a general survey of AMDV in China is urgent. Counter immunoelectrophoresis(CIEP) has been used widely for routine detection of AMDV antibodies. But the CIEP analys is is labor intensive with many manual tasks, requires sophisticated instruments, and not suited for automation. So, it is urgent to develop a more convenient and effective method for the AMD V detection.In this study, five AMDVs’ coding genes were cloned and sequenced. Their nucleotide sequences were alignmented with that of reference strains. The results indicated that these five strains shared the highest nucleotides homology with the AMDVs in China. The VP2 amino acids alignment showed that there were many mutations in VP2, but VP2332-452 was highly conservative.VP2332-452 was then expressed for the establishment of ELISA. VP2994-1356(between aa 332 and 452) was amplif ied and cloned to pMAL-c2 x. The recombinant plasmid was transfected into BL21 E. coli.. The expressed fusion protein was analyzed by SDS-PAGE and Western blotting. The results showed that the fusion protein could react with AMDV positive sera specifically. The VP2332-452 fusion protein was purified using a His PurTM Cobalt Purification Kit. The ELISA for detecting antibodies against AMDV was developed with the purified protein. This ELIS A had a sensitivity and specificity of over 90% with reference to CIEP. 596 mink sera collected from three farms were detected with the established ELIS A and the AMDV positive rate was 65.9%, which indicated that the AMDV was still prevalent in the local mink farms of northeast China.The ELIS A for detecting antibodies against AMDV can be used for mass detection of clinical samples and it gives support to the researchers for the AMDV rapid diagnosis and epidemiological investigation.