| Aleutian mink disease,a chronic and progressive disease infected with Aleutian mink disease virus,is one of three diseases which hinder the development of the fur industry,and the morbidity and direct mortality for AMD can be 75% and 30% respectively,which causes huge economical losses to the mink industry.For the special pathogenic mechanism of the disease,there is no effective medicine for AMD prevention,and to screen and kill the AMD test positive minks is the only way to control this disease.Counterimmunoelectrophoresis(CIEP)is recognized as a gold standard for AMD test,whose principle is that antigen and antibody move oppositely under electric conditions and a clear white precipitation line performs by their combination.The result which shows white precipitation line is positive otherwise negative.The quality of antigen which plays a significant role in AMD test decides the accuracy and sensitivity in the experiment.Therefore,the research and preparation for antigen is significant in AMD test.The aim in this study is to find the way to produce antigen at low cost and convenient operation,which may provide some base for AMD test and production of colloidal gold test paper.The results were as followed:VP2 gene sequences from AMDV-G was expressed in prokaryotic system by constructing prokaryotic expressing plasmid PMAL-c4x-VP2 a,with which band of 1944 bp would be obtained.From the SDS-PAGE result,the molecular mass was coincident with that in previous reports;Western blot results indicated that the protein can combine with AMD positive serum distinctively;maltose binding protein was obtained with amylose resin affinity chromatography method to purify the expressed protein,and finally 80 mg recombinant protein was obtained with 1L cultures;The main epitope in VP2 gene was analyzed by bioinformatic software based on whole genetic sequences published on GenBank.A pair of specific primers were designed,with which band of 710 bp would be obtained,and connected with pEASY-Blunt Zero plasmid.PCR,double enzyme digestion and sequencing techniques were used to select positive plasmids.PMAL-p5x-VP2 b prokaryotic expressing plasmid was constructed with target genes and prokaryotic expressing vetor PMAL-p5 x.Results of SDS-PAGE showed the molecular mass was23 kDa;Western blot results indicated that the protein can combine with AMD positive serum distinctively;the expressed protein was separated and purified with Osmotic shock method,and120 mg recombinant protein was obtained with 1 L cultures;The VP2 wholes gene of AMDV-G was expressed in bac-to-bac eukaryotic expressing system.VP2 gene was connected to transfer vector pFast BacHT-B and the combination was transferred into DH10 Bac competent cell,after transposition between shuttle vector and previous combination in DH10 Bac cell,the white colony was picked and grown in large cultures for transposons Bacmid-VP2 extractions.These target transposons Bacmid-VP2 were transfected into sf9 cells with PCR identification,incubated at 27 ?for 72 h.Some cells with pathology were collected against light,meanwhile,the rest cells were used for immunofluorescence analysis.Results showed strong fluorescence signal and that 100 mg recombination proteins were obtained form 1 L cell cultures by purifying virus with plaque assay and expressed protein with nickel column;Ultrafiltration concentration method was used to purify traditional AMDV antigen,with which purity increased,and the antigen results still showed positively when the serum was diluted to 16 times,enhancing sensitivity in the AMD test;From results with biologic software,the target protein expressing ratios for PMAL-c4x-VP2 a and PMAL-p5x-VP2 b were 49.55% and 71.07% in prokaryotic expressing system;three recombination proteins and AMDV antigens were used for CIEP with 240 serum samples which were collected from mink industry,by contrast,purified AMDV antigen had best sensitivity and specificity for test.And among recombination proteins,theses expressed in eukaryotic expressing system showed highest ratios for positive test which were 91.2%,compared with AMDV antigens;for these two proteins expressed in prokaryotic expressing system,target proteins expressed with short sequence had higher sensitivity and expressing amount than that with long sequence between which coincidence ratio was 85.3%;proteins expressed with short sequence indicated 87.2% for coincidence ratios compared with AMDV antigen. |
PDF Full Text Request