Preparation Of Polyclonal Antibody Of VP2 Gene Of Mink Enteritis Virus And Establishment Of IPMA

Mink Enteritis Virus(MEV)is also known as mink parvovirus,the MEV caused by which exerts a great impact on the growth and fur quality of mink.Since the disease was firstly discovered in China in1981,MEV has been detected in succession in mink farms in multiple provinces in China.The objectives of this study are as follows:firstly,to get recombinant protein by constructing pET-32a-VP2 recombinant plasmid and transforming it into E.coli prokaryotic expression system.The recombinant protein will be used to immunize New Zealand white rabbits to obtain polyclonal antibody VP2,so as to lay the foundation for the laboratory subsequent study of the relevance structure of MEV VP2;secondly,to carry out the MEV immunoperoxidase monolayer assay,so as to provide a reliable and convenient method for the detection and control of MEV in mink farms.The results obtained are as follows:According to the MEV VP2 gene sequence in GenBank database,through multiple sequence alignment and the introduction of Bam H I and Xho I restriction enzyme cutting sites to the upstream and downstream regions of the gene,primers VP2 F and VP2 R were designed and MEV whole-genome recombinant plasmid pB-MEV-L was used as the template to amplify VP2 fragment by using PCR technique,then the fragment was connected to pET-32a vector,which was used to transform DH5?competent cells.Through enzyme digestion and sequencing,it was proved that the VP2 gene prokaryotic expression plasmid pET-32a-VP2 was successfully constructed.Then the plasmid was used to transform E.coli BL21(DE3)competent cell,which was induced by IPTG to have high-efficiency expression of pET-32a-VP2 recombinant protein.The results of Western blot showed that the obtained recombinant protein has favorable reactionogenicity.In this study,protein mass spectrometry was used to analyze the amino acid composition of low molecular weight proteins.The purified protein was used as immunogen to immunize New Zealand rabbits and prepare antiserum.After four times of immunizations,the titer of the antiserum was determined by means of ELISA,and the titer reached 1:1024.The IFA showed that the obtained antibody has favorable binding activity with MEV.IPMA method for the detection of MEV antigen in serum was established,and experimental verification was carried out on the specificity,sensitivity,repeatability and detection time of virus infection of the method.The results showed that cell fixation and detection 48 hrs after virus inoculation achieved the optimal effect;the optimal dilution of primary antibodies was 1:100 and the optimum action time was60 min;the highest dilution of secondary antibodies was 1:2000 and the optimal incubation time was 90min;IPMA method has no cross reactions with CDV,CAV or ADV;10~-55 diluted MEV virus could still be detected by this method,and the sensitivity was one dilution degree higher than PCR technique;this method has good repeatability;MEV virus could be detected in the blood of rats 4 days after virus inoculation;the 90 collected mink blood samples were detected and the results showed that 19 samples had MEV positive infection,and the infection rate was 21%.