Expression Of VP2 Protein Of Aleutian Mink Disease Virus And Application Of Detection Technology

Aleutian Mink Disease virus(ADV), a member of genus ADovirus, belongs to the family Parvoviridae, the subfamily Parvovirinae, and is a parvovirus which can cause severe immune complex-mediated chronic progressive disease in mink, so-called Aleutian mink disease(AD),characterized by violation of the immune system, autoimmune disease, high immunoglobulin acidosis, lifelong viremia, glomerulonephritis, arteritis, hepatitis, the increase in susceptibility to bacterial infections and kidney failure death. Aleutian mink disease has been prevalent in the wild and farmed mink population of the world, and caused very serious economic losses in the mink industry. In our country, Jilin, Shandong, Liaoning, Heilongjiang, Inner Mongolia mink farm has the disease, antibody-positive rate range from 20% to 30%. In this study, a local virulent ADV strain of Liaoning region was isolated, identified and named as ADV-LN; the complete sequence of VP2 structural protein gene of ADV-LN was amplified and analyzed for the first time. And a rapid, cheep, and sensitive enzyme-linked immune-sorbent assay(ELISA)method based on the yeast expressed whole VP2 protein for screening antibodies against ADV has been developed. Furthermore, three rapid, accurate and sensitive testing technology that directly detects the genetic materials of ADV, include Taq Man-MGB fluorescence quantitative PCR, LAMP, and PCR-DHPLC were successfully established and applied. These methods would be laid an important foundation for the disease prevention and control of the ADV in our country. The contents of the paper contain six parts as following:1. solation and identification of local virulent strain ADV-LN and evolutionary analysis of its VP2 gene:Mink from Liaoning provinces suspected infection ADV symptoms were tested with CIEP method. The mink with antibody to ADV were selected and culled. And the spleen, liver,mesenteric lymph node, kidney samples sterile of mink was taken for pathological examination,and observation of the virus under electron microscope. For virus isolation, the grinded tissue fluid filter was added 100 ng/μL of penicillin and 50 ng/μL of streptomycin, inoculated into CRK cells and passaged by six generations. And four cells cultures were identified as ADV by PCR.And then the isolated virus was inoculated into healthy mink. After three days, the mink showed clinical signs, which including apocleisis, hair dull, the loss of appetite, anemia, and binge drinking. Some minks showed neurological symptoms, manifested symptoms of convulsions,cramps, staggering gait, ataxia, or hind limb paralysis and died. The virus strain which isolated and identified was named as ADV-LN. Subsequently, a pair of primers to amplify the full-length VP2 gene of ADV-LN was design, and the full-length VP2 gene was cloned into p MD18-Tvector; resulting vector was named as p MD18-VP2. Sequencing and sequence analysis showed that, the ADV-LN strain was more closely related virulent strain ADVADV-Utah1 and ADVADV-BEIJING than the weak strains ADVADV-G.2. high levelly secretive expression of major antigenic protein vp2 in yeast and its immunogenity assay:A codon-optimized DNA sequence that encodes major antigenic protein VP2 was synthesized by Jinweizhi Technology and Services Co., Ltd. Subsequently, the optimized VP2 gene was subcloned into the plasmid p PICZαA to form p PICZαA-VP2-opt. the recombinant plasmid p PICZαA-VP2-opt was then linearized by Sac I and electric-transfected into Pichia pastoris X-33, an engineering strain X-33/p PICZαA-VP2-opt which with high VP2 secretory expression level was obtained. Small-scale Mut+ clone cultures were subjected to methanol induction for secretory protein production. SDS-PAGE analysis of the culture supernatants revealed that there were major protein bands about 90 k Da, which was about 17 k Da larger than the calculated molecular weight of the VP2 protein. Furthermore,N-glycosylation sites analysis by Net NGlyc 1.0 Serve show that, ADV-VP2 proteins have seven N-linked glycosylation sites Asn-Xaa-Ser/Thr sequons. When the recombinant VP2 protein was denatured and treated with PNGase F, a single band around 73 k Da as expected was appeared in the SDS-PAGE. The results suggested that the VP2 produced in this study was highly glycosylated. Finally, the recombinant protein was tested by western-blot and indirect ELISA,results showed that, The VP2 recombinant protein was able to effectively combine and reacted with ADV-specific antibody, and the immunogenicity was good.3. Development of an ELISA based on yeast produced VP2 antigen for detecting antibodies against ADV:An indirect ELISA method for detection of ADV antibody was established using the purified yeast expressed VP2 proteins as coating antigen, After the reaction conditions(such as antigen concentration, serum dilution, antigen, sealing liquid and sealing time, serum dilution,serum reaction time and enzyme resistance two time, time, substrate color) were optimized,established detection methods had no cross reaction wih common mink infectious virus, with good specificity. Finally, 285 serum samples were detected by established ELISA and CIEP simultaneously; the results showed that the coincidence rate was 95.7%, and ELISA method was proved to be specific and sensitive for detecting ADV antibody.4. Establishment and application of Taq Man-MGB fluorescence quantitative PCR detection method for ADV:According to the sequence of ADV-G strain which published in Gen Bank,probes and a pair of specific primers were designed, and a Taq Man MGB fluorescence quantitative polymerase chain reaction(PCR) detection method with strong specificity, high sensitivity was uccessfully established. The sensitivity of MGB fluorescence quantitative PCR assay, which was determined to be 10 copies/L of the standard plasmid, was 100 fold higher than conventional PCR method. Finally, 40 clinical samples were detected by established method, the positive rate was 52.5%(21/40), increased by 7.5% compared with the common PCR. The development of this method provides an important rapid detection method for ADV.5. Est ablishment and preliminary application of ADV loop-mediated isothermal amplification diagnostic Method:According to the sequence of VP2 of ADV which published in Gen Bank,LAMP primers were designed, and LAMP method which targeting a hithly conserved region of VP2 gene has been developed to diagnose ADV. Sensitivity test, specificity test and replicate test of this method were promoted and then collecting clinical samples to develop preliminary application. Postive reaction tube turns green when the flurescent dye is added. The sensitivity of the LAMP assay, which was determined to be 10 copies of the standard plasmid, was 100 fold higher than common PCR. Furthermore, the method detection of CPV, PPV, CEV, and CDV is negative. Finally, 126 clinical samples were detected by established LAMP method, the positive rate was 48.4%(61/126), increased by 7.5% compared with the conventional PCR.6. Establishment and preliminary application of ADV(PCR-DHPLC) diagnostic Method:To identify the ADV, a PCR-denaturing high performance liquid chromatography(DHPLC) assay was performed. Primers specific for the VP2 gene of ADV were selected to conduct the DHPLC assays. The specific testing was performed with CPV, PPV, CEV, and CDV, without cross reaction, with excellent specification and repeatability. Sensitivity analysis showed that the developed PCR-DHPLC method could detect 10 copy/L reactions. Then the established method was used to detect the clinical samples, the positive rate was 48.4%(61/126), increased by 7.5%compared with the conventional PCR. The method could be used in clinical diagnosis and epidemiological investigation.