| Along with the market demand, the mink breeding industry has developed rapidly in recent years. Shandong became the important province of the mink breeding in China.However, as the continuous expansion of the mink breeding scale, some epidemic diseases become the key factor restricting mink breeding development. Aleutian Mink Disease(AD),as one of the three epidemic diseases(Aleutian Mink Disease 、 virus enteritis 、 canine distemper)of fur-animals, will cause the deterioration of fur quality, which will bring about the great economic loss of the mink breeding. However, there are not specific drugs or vaccines to treat this disease. The only way we could use is to eliminate these infected animals. Therefore, it is important to research the molecular genetic features of ADV in Shandong province and establish a specific diagnostic method.In 2014-2015, we grind mink visceral muscle that infected ADV and then inoculate the lapping liquid to CRFK. When there is stable cytopathy in CRFK,we collected genome DNA for PCR test. The result is ADV positive test. Comparing reference sequence ADV-G and determination of DNA sequences using DNASTAR software, the nucleotide homology is 92.6%. But, with increasing serial passages of ADV in the CRFK, the cytopathy effect gradually disappeared. We collected genome DNA for PCR test. The result is ADV positive test. It is indicated that the ADV can’t steadily passage in CRFK.Meanwhile, we collected 134 mink parenchymal organ samples from ZhuCheng.it is discovered that 37 mink samples were positive. The positive rate is 27.6%.Amplifing and determining the sequence of VP2, we found that the 37 nucleotide sequences showed88%-99.8% identity with each other and 88.0%-100% identity with reference sequences.There are large amounts of mutational sites in the 37 VP2 genes. The 37 sequences and reference sequences is divided into 5 branches(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ), analyzed with phylogenetic tree. 89% domestic strains is pointed in the group of ⅠandⅤ, in which is not included the foreign strains. 78.4% domestic strains is pointed in the group of Ⅰ. The close genetic relationship exists in the rest of the 11% domestic strains and foreign strains. It showed that the Aleutian mink virus in domestic is formed the individual branch, which isdifferent from the foreign strains. Owing to the most of the isolates lie in the groupⅠ, it is tentatively considered that the isolate strains have the evolutionary trend to group Ⅰ. This research will help us to find the standard strains about Shandong province, which will provide a scientific basis for detecting this virus.In order to investigate more precisely the characteristics of the molecular biology in VP2 gene of 37 isolate strains, we using DNASTAR software analyzed and aligned the deduced amino acid sequences of each protein and analyzed relatively with reference strains.It is discovered that there had 89.2%-99.7% similarity with isolate strains and 87.7%-100%similarity with reference sequences at amino acid level. More than 100 amino acid mutations lied in the VP2 gene. Most of the mutations existed in the three areas, including VP2 immune response sequences: 429-524. The 14 mutation sites existed merely in the Chinese strains, including 204:V-I、305:K-T、308:T-S、419:L-I、436:I-M, which consisted in more than 10 domestic strains. Two mutation sites(419 : L-I 、 436 : N-D)lie in VP2:428-446. This region was likely to affect the scope of host selection about Aleutian mink virus. There is no exact research to show the meaning of its mutations for the other mutation sites.In the next place, we induced prokaryotic expression for the immune response sequence of VP2 region(430-525) and established the indirect ELISA detection method.Connecting the amplification sequence with prokaryotic expression vector and constructing recombinant plasmids, we import the recombinant plasmids into Escherichia coli BL21. The expression protein reached a maximum deduced at 37℃with 4h and 1mmol/L IPTG and had a high reactogenicity after detected with Western Blot. Coating with purified protein into plates and optimizing the condition, we established the indirect ELISA detection method. Detecting of 84 serum samples, it is discovered that the positive rate of ADV is26.2%, which is consistent with the result of PCR detection method. |
