| Aleutian Mink Disease virus (ADV) belongs to the family Parvoviridae, the subfamily Parvovirinae within the genus Amdovirus, and ADV is the pathogen of Aleutian disease of mink (AD). Aleutian mink disease is a chronic progressive disease of mink, characterized by decreased reproductive capacity, body weight loss, autoimmune disease, high immunoglobulin acidosis, the increase in susceptibility to bacterial infections and kidney failure death. Aleutian mink disease been discovered in1940, has been prevalent in the farmed mink populations of the world, caused very serious economic losses in the mink industry. Heilongjiang, Jilin, Liaoning, Shandong, Xinjiang, Inner Mongolia mink farm has the disease, antibody-positive rate from20%to30%, the individual up to71%. In this study, ADV strains of three different regions were isolated and identified, the coding region gene Cloning and sequence analysis, the molecular characteristics of structural protein genes were analyzed. And the TaqMan real time PCR and indirect ELISA were established for ADV detection by means of molecular biology. Finally, ADV structural proteins VP2gene was cloned and expressed in baculovirus, and their immunogenicity were determined in a mouse model.The contents of the paper contain six parts as following:1. Isolation and identification of aleutian mink disease virus in ChinaMink from mink breeding areas (Shandong, Liaoning, Heilongjiang provinces) suspected infection ADV symptoms were tested with CIEP method. The mink with antibody to ADV were selected and culled. And the liver, spleen, kidney and mesenteric lymph node samples sterile were taken for pathological examination and the viruses were observed under electron microscope. The grinded tissue fluid filter was added penicillin and treptomycin and inoculated into cells and passaged by6times for virus isolation. And three cells cultures were identified as ADV by PCR. And then they were inoculated into healthy mink. Three days later, the mink showed clinical signs, which including the loss of appetite, anemia, hair dull, antifeedant and binge drinking. Some minks showed neurological symptoms, manifested symptoms of convulsions, cramps, staggering gait, ataxia, or hind limb paralysis and died. The three virus strains isolated and identified were named as the ADV-LN1, ADV-the LN2, and ADV-LN3.2. Cloning and sequence analysis of the gene of Chinese Aleutian Mink Disease Virus strainsThe coding region gene of three Chinese ADV isolates ADV-LN1, ADV-LN2and ADV-LN3were cloned and sequenced by means of PCR fragment amplification technology. The results showed that the coding region of gene were4543bp,4566bp and4566bp, respectively. And the Chinese strains were compared with reported European and American strains, in terms of sequences of structural proteins and non-structural proteins. The results indicated that the highest nucleotide homology of structural protein VP1between the Chinese ADV-LN1, ADV-LN2and ADV-LN3strains and European and American ADV-G, ADV-Utahl and ADV-SL3strains, was97.4%. And the highest nucleotide homology of non-structural protein NS1, NS2and NS3was92.6%,93.3%and93.9%, respectively. The highest deduced amino acid sequence homology of VP1was97.2%, and the highest deduced amino acid sequence homology of NS1, NS2and NS3was89.2%,91.2%and92.0%, respectively. Phylogenetic tree analysis of VP1showed that the Chinese ADV isolates were probably evolved from new strains distantly related to European strains or American ADV-Utahl strain. The bio-informatics results showed that the gene encodes a small virus VP1protein had the closest relationship with Parvovirus. The protein is a conserved outer membrane protein without signal peptide, with seven potential N-glycosylation sites and34phosphorylation sites. Secondary structure analysis showed that the highest content of random coil, up68.75%, a helix, β fold, respectively16.42%and14.83%; Homology modeling compared to experiment, build a reasonable and high reliability of the three-dimensional structure,predicted epitope mainly located in the peptide chain254-265,96-BF112,317-348,514-523,629-645sections.3. Development of real-time PCR for detection of Aleutian Mink Disease VirusOne set of primers and probe were designed based on the nucleotide sequence within VP2of Mink Aleutian Virus (ADV), and a quantitative TaqMan real-time PCR was developed firstly in China for detecting ADV which characterized with highly specificity, reproducibility and more sensitivity than a conventional PCR. Standard dilutions allowed absolute quantitation of the amount of viral DNA to47.7copy/μL, which is106times as sensitive as that by a conventional PCR. The result of detecting ADV from tissues of mink with ADV also confirms the highly sensitivity and specificity of real-time PCR. The method can be used for the inspection and quarantine of imported mink and domestic mink farms for ADV instant diagnosis.4. Prokaryotic expression of the major functional areas of VP2gene and establishment of indirect ELISAThe VP2gene fragment (950bp) of ADV was amplified using PCR technology and cloned into the pET-28a (+) prokaryotic expression vector, and the recombinant pET-28a (+)-VP2prokaryotic expression vector was obtained. Enzyme digestion, PCR amplification and sequencing analysis confirmed the correct insert into the expression vector and the correct reading frame. The recombinant plasmid was transformed into host strain BL21(DE3), and the protein expression was induced by Isopropyl-β-D-galactoside (IPTG), and confirmed by using SDS-PAGE and Western-blotting. The expression product molecular weight was42kD, which being consistent with the theoretical speculation, and it could be reacted with ADV-specific antibody. And then the recombinant protein was purified and used as diagnostic antigen. On this basis, an indirect ELISA was established with the purified VP2protein. The results showed that the method has the advantage of easily purification of antigen, high specificity and sensitivity and good repeatability.5. Construction and immunogenicity of recombinant VP2protein of ADV expressed in recombinant baculovirsesVP2gene was amplified from ADV-LN2isolate and cloned into vecter pFastBacTM ual. The transfer vector was transformed into E.coli DH10Bac cells and the VP2gene was integrated into the Bacmid shuttle plasmid. Then a BacmidVP2of recombinant shuttle plasmid was obtained and then transfected into the Sf9insect cells by lipofectin to produce a recombinant baculovirus (rBacVP2). Expression of ADV VP2in Sf9cells infected with the recombinant virus was detected by Western blot and indirect immunofluorescence assay (IFA).The results showed that the molecular weight of the recombinant VP2protein is35kD. Moreover, BABL/c mice were inoculated with the recombinant VP2protein in oil adjuvant. Meanwhile, some mice were inoculated with inactived ADV-LN2in the same oil adjuvant, and wild type bacuvirus (wtBac) in the same oil adjuvant was used as negative control group and no vaccinantion as blank control group. Serum of experimental animals were detected by ELISA at21and42days post vaccination, and the results showed that the recombinant protein prompted mice to produce specific antibodies of the ADV. Spleen lymphocyte proliferation experiments showed that the lymphocyte proliferation was significantly higher that those in the wtBac negative control group and blank control group. It indicated that the recombinant protein should be useful for developing new vaccine against this disease. |
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