Expression Vectors Construction Of Three Cytokines From Grass Carp And Establishment Of Double Antibody Sandwich ELISA For Detecting Interleukin-10

Like mammals,fish cytokines are important immune molecules that regulate fish innate immunity and acquired immunity.At present,there are about 24 kinds of fish cytokines.Among them,IL-6 can promot proliferation of macrophages and lymphocytes,and antibody synthesis;TNF-? is a pro-inflammatory cytokine that resists pathogen infection by mediating inflammatory responses and activating immune responses;In addition to inhibiting the inflammatory response and promoting the recovery of damaged tissue cells,IL-10 also can promot B cells to produce antibodies.Therefore,the level of these three cytokines in the serum or cells of immunized animals can be used as important indicators for evaluating the vaccine efficacy.Among the reported assays for cytokine,ELISA method has the advantages of being fast,conveniently,specific,and sensitive,and is the most commonly used detection method.At present,ELISA assay kits for mammalian varied cytokines have been developed at home and abroad,but commercial assay kits for fish cytokines have not yet been found.Therefore,it is necessary and urgent to establish a double antibody sandwich ELISA method for detecting grass carp cytokines.In this paper,based on the construction of cloning and expression vector of grass carp cytokines,preparation of recombinant gc IL-10 protein and its antibody,a double antibody sandwich ELISA method for detecting gc IL-10 was established.The main research work and results completed are summarized as follows:1)Construction and expression of cloning and expression vectors for three cytokines from grass carp.Three pairs of specific primers were designed based on the gc IL-6,gc IL-10 and gc TNF-? gene sequences.The c DNA of grass carp was used as a template,and the target gene was amplified by RT-PCR and ligated to p MD18-T vector by TA cloning method.The PCR results showed that three gene bands with a size of approximately 645 bp(gc IL-6),558 bp(gc IL-10),and 738 bp(gc TNF-?)were amplified;the sequencing results showed that gc IL-6 and gc TNF-? There was only one unintentional mutation in the gene.There was no mutation or deletion in the IL-10 gene,indicating that the cloning vectors of the three grass carp cytokines were successfully constructed.The method of using the enzyme-linked enzyme gc IL-6,gc IL-10 and gc TNF-? were subcloned into the prokaryotic expression vector p ET32 a and in p Creat-SII.The p ET32a-gc IL-6,p ET32a-gc IL-10 and p Creat-SII-gc TNF-? were successfully constructed by PCR and double enzyme digestion.After the expression of the recombinant inclusionsp ET-32a-IL10/BL21 induced inclusion body products were denatured,renatured and purified by Ni-Charged Resin affinity chromatography,rgc IL-10 protein with a purity of about 97% was obtained,Western-blot analysis rgc IL-10 has good immunoreactivity.These results indicate that the prepared rgc IL-10 can be used as a preparation and detection antigen for subsequent immunogens.2)Preparation of polyclonal and enzyme-labeled antibodies for detection.An immunogen was prepared from the purified rgc IL-10 protein and immunized three times with New Zealand white rabbits and BALB/c mice to prepare rabbit anti-gc IL-10 antibody and mouse anti-gc IL-10 antibody.Rabbit anti-gc IL-10 immune serum was purified by saturated ammonium sulfate fractionation and Protein G affinity chromatography to obtain rabbit anti-gc IL-10 antibody with a purity of 95%,a concentration of 1.7 mg/m L and an ELISA titer of 1:838860800.The rabbit anti-gc IL-10 purified antibody was labeled with horseradish peroxidase(HRP)using sodium periodate method.After being precipitated with saturated ammonium sulfate,HRP-labeled rabbit anti-gc IL-10 antibody with a purity of more than 97% was obtained.Used as a detection antibody in the subsequent method establishment.Mouse anti-gc IL-10 immune serum was purified using Protein G affinity chromatography to obtain a purified antibody with a purity of 95%,a concentration of 1.2mg/m L,and an ELISA titer of 1:6553600,which can be used as a capture in the subsequent method establishment.antibody.3)Establishment of a sandwich ELISA assay for detection of gc IL-10.The mouse anti-gc IL-10 purified antibody was used as the capture antibody,the rgc IL-10 purified protein was used as the detection antigen,and the enzyme-labeled rabbit anti-gc IL-10 antibody was used as the detection antibody.The optimal concentration of the antigen and antibody was optimized,and the blocking solution type and closed condition were optimized.A gc IL-10 double antibody sandwich ELISA assay was established.The sensitivity of this method was 23.54 ng/m L,and the coefficient of variation between plate and intraplate assays was less than 10%,and rgc IL-6,rgc TNF-?,m IL-10,m IFN-?,and m MCP-1 and other cytokines were not detected,only the detected rgc IL-10.In summary,this study successfully established a double-antibody sandwich ELISA for gc IL-10 detection.This method has good specificity,sensitivity and stability.