| Aleutian Disease (AD), which caused by Aleutian mink disease virus belonged to Parvovirinae Amdovirus, is a major chronic infectious disease of mink. AD would cause immunologic system patho-disharmony. And it was wide spread all over the world in the mink with both vertical and horizontal transmission. The disease caused acute interstitial pneumonia even death of the new birth young mink without antibody and the descent of reproductive capacity, fur quality and health status of the adult minks. AD resulted in increase of death rate and enormous economic loss of mink cultivation. For the past few years, the popular information was reported in Chinese mink cultivation. High level AD infection is one of the all-important effect factors for developing Chinese mink cultivation healthy. Now, regular detection and condemn infected minks to clean the population was used widely. So the clinical diagnosis of AD, prevention and cure methods and more scientific information would be provided by identifying the AD strain, studying the virus molecular characters and founding the fit sero-diagnosis methods.In the study, some Hei Long Jiang and Liao Ning province mink nursery were detected by CIEP and ADV PCR method founded by our laboratory. The PCR productions of positive strains in both methods were sequenced. And four conservative strains were draw-off to sequence compare and homology analyze with the standard contrast strain. The result showed that the nucleate homology with the corresponding ADV-G region was 92.8%, 93.9%, 93.2% and 93.5% respectively. And the homology with the collate strain ADV-Utah was 94.5%, 98%, 94.6% and 95.5% respectively. So the virus strain was determined ADV virus specific nuclear fragment. According to the result, the four viruses were named as MS-1, DL-1, DL-2 and DL-3 respectively.The total DNA of MS-1, DL-1, DL-2 and DL-3 were extracted from infective organizations. Four pairs of primers based on the standard virus strain ADV-G were designed. The recombinant plasmids were constructed with PCR productions and sequenced. The four proper complete DNA sequences were obtained by splicing the sequenced results with the software SeqMan of the DNA Star. The homology compare of the corresponding nuclear sequences and the cladogram analyze between the obtained virus strain and abroad virus collate strain were measured by Jotun Hein method in software DNA Star. The results suggested that there is high homology, from 92.9% to 93.4%, between the ADV-Utah and the experimental virus strain. And ADV-G and ADV-SL3 had the low homology with the experimental virus strain. The four experimental virus strains and the three collate virus strains were divided into two groups, and belonged two evolutionary branches. Although ADV-DL1, DL2 and DL3 were all belonged to the Da Lian separated strains, they had the ulterior genetic relationship. The nuclear sequences of the obtained experimental ADV virus strains, ADV virus collate virus strains and typify virus strains of other four Parvovirus generic were cladogram analyzed with the Clustal W method of software DNA Star. The results show that fourteen virus were divided into five independent evolutionary branches. ADV and Bovine parvovirus, as the new genus of the parvovirus subfamily, had the different evolutionary branches with the other genus viruses. The result validated the rationality of the new classify system reported by ICTV8.ADV MS-1 strain was selected with a relative conserved main antigenic region of VP2 protein. According to the two pairs of primers designed, two main antigen domains of VP2 gene of ADV strain were amplified by PCR. Then the amplified DNA products were cloned into the multiple cloning sites of prokaryotic expression vector pMAL-c2, respectively, through which recombinant vectors pMAL-VP2a and pMAL-VP2b were constructed. The insert position, the size and the reading frame were affirmed all right by restriction digestion, PCR and the sequence analysis which showed that the prokaryotic expression vectors pMAL-VPa and pMAL-VPb were constructed successfully. Then the positive recombinant vectors were transformed into recipient germs TB1 for expression by IPTG inducing. Through SDS-PAGE and Western blot, the two proteins were detected to be expressed successfully in the form of cytoryctes. The molecular weights of the expressed protein were 63KDa and 65 KD respectively which was identical with the theoretically presumed. Induced at a concentration of 1 mmol/L IPTG, it took 4 hours for the quantity of expressed protein to amount to peak value. Western blot indicated that the expressed antigen proteins could be recognized by the rabbit anti-MBP antiserum. To detect the immunoreactivity, the protein was isolated and purified as the antigen and CIEP was performed on the positive serum. The result was showed to be 94.3% similar to the CIEP with control antigen. It was believed that the expressed recombinant protein in this study can be used as the antigen in the clinical diagnosis with better specificity, reproducibility and sensitivity.In a word, this study provided lots of foundational information for the separating, identifying and complete sequencing of the ADV separated strains in China. And the results would be useful for the evolutionary characters and regularities of the ADV virus strains. VP2 major antigens epitope were expressed by the prokaryotic expression vector first time in China. Using the expression vector as the antigens, the CIEP detection was set up initially. All of these would be useful in AD sero-survey of the mink cultivation. |
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