Establishment And Application Of Detection Assays Of Mink IL-4,IFN-?,and TNF-? Genes By Real Time RT-PCR And Indirect ELISA Based On The IFN-?

Mink(Neovison vison)is a kind of small fur animal,it is Mammalia,Carnivo,Mustela.For fur-manufacture industry developed rapidly in current,health of mink affect the social and economic benefits greatly.It has been increasing that mink infected by canine distemper,parvovirus and aleutian virus in recent years.Immunization was less than satisfactory because of virus variation,as well as cacoethic immune status of the animals.To evaluate the immune status can provide the basis for the choice of optimal immunization program and better immunization time.Therefore,it is important to found a new detection and evaluation of vaccine immune effect tecnique system,for prevention and control of special animal disease.The main contents of the research was as follows:1.Clone and sequence analysis of mink interleukin 4,interferon-gamma and tumor necrosis factor-alpha genes.Mink lymphocyte was in vitro cultured for 24 h and collected by centrifuge,after isolation and induction by PHA,and extracted the total RNA.The specific primers were designed and synthesized according to the other animals interleukin 4,interferon-gamma,tumor necrosis factor-alpha genes sequence in GenBank,to clone the whole sequence of the three genes of mink by RT-PCR,the length of the genes were 399 bp,501 bp and 702 bp,respectively,and all sequences were compared and analysed.The results gave a foundation for further study of mink interleukin 4,interferon-gamma,tumor necrosis factor-alpha genes.2.Establishment of real time quantitative RT-PCR detection assay for target genes.In order to detect the effect on related cell ranscription factors mRNA of mink,after infected by canine distemper virus virulent strain and attenuated strain,respectively,the standard plasmids were constructed according the MiIL-4,MiIFN-? and MiTNF-? genes sequences amplified by RT-PCR,and also the mink glyceraldehyde-3-phosphate dehydrogenase gene sequences as an internal control in GenBank.The methods of SYBR Green ?real-time quantitative RT-PCR assay for detection of three target genes mRNA was established.The peripheral blood monocytes were collected as clinical samples,which have been in vitro cultured after infected by phytohemagglutinin,canine distemper virus virulent strain and canine distemper virus attenuated strain,respectively,at different time points.Test results showed that IL-4 and IFN-? have efficiently expressed in mink peripheral blood monocytes induced by PHA,while canine distemper virus has inhibited the production of IL-4 and promoted production of IFN-?.This study provided an effective method for quantitative analysis of mink cytokines mRNA.3.Preparation of monoclonal antibody of mink IFN-? and the foundation of indirect ELISA detection assay.The prokaryotic expression vector pCold-MiIFN-? of mink IFN-? gene was constructed,and expressed at a soluble condition.BALB/C mice have been immunized by mature MiIFN-? protein for four times.The spleen cells from the BALB/ C mice were fused with SP2/0 myeloma cells,the recombinant soluble protein pCold-MiIFN-? was used as the detection antigen for indirect ELISA detection,a total of 24 cell strains have been screened that could steadily secrete antibodies.Five strains among them have been selected showed specificity by Western-blot analysis,because of good sensitivity,were named 31 A,31B,31 G,E44 and G46,respectively.Vero cells were transfected by recombinant plasmid pcDNA3.1-MiIFN-?,monoclonal antibodies secreted steadily were used as the first antibody for indirect immunofluorescence assay.The results showed that two of them were monoclonal antibodies secreted by the cells,which were specific antibodies against IFN-?.The two strains were identified as IgG2 a and IgG2 b,respectively,and the light chains were both ? chains.In order to detect the IFN-?prepare expression level after canine distemper virus virulent strain and canine distemper virus attenuated strain infection,and the purified monoclonal antibody has been used as primary antibody,and HRP-labeled rabbit anti-mouse IgG as secondary antibody for ELISA assay.The results demonstrated that the expression of IFN-? were both highest at 48 h after attenuated strain CDV-Hebei and CDV3 infection,and was inhibited at 72 h,while the expression level of IFN-? was influenced weakly by CDV3 strain infection compared to that of CDV-Hebei strain.