Preparation And Identification Of Monoclonal Antibodies Against Stearoyl-coa Desaturase Of Dairy Goat

Stearoyl-Co A desaturase(SCD)is the rate-limiting enzyme of regulating unsaturated fatty acids synthesis in milk,and being as a key enzyme for endogenous synthesis of the conjugated linoleic acid in the meat and milk of ruminants.It has the ability to catalyze desaturation of the saturated fatty acids,goat specific SCD antibody is necessary for the function research of SCD gene fatty acid regulation.The objective of this study was to prepare and identify monoclonal antibodies(MAbs)against Stearoyl-Co A desaturase(SCD)through prokaryotically expression,SCD protein purification and mice immune.The clarified function of the antibody would provide effective tools for the study of SCD functions in fatty acids metabolism.The SCD gene sequence was chosen through bioinformatic analysis mainly using DNAStar software.SCD was amplified by PCR from the c DNA.We sub-cloned SCD gene of goat into the vector p ET32a(+)to construct prokaryotic expression vector p ET32a(+)-SCD,which was transformed into Escherichia coli Rosetta(DE3)competent cells.Recombinant protein was purified by His-tag protein purification kit.Purified recombinant protein emulsified with adjuvant was used as antigen to immune 4~6 weeks of Balb/c mice to prepare monoclonal antibodies.Enzyme-linked immunosorbent assay(ELISA)were used to detect the titer of the immune serum.The preparation of hybridoma cells were using PEG to fuse immune spleen cells and SP2/0 cells.ELISA were used to screen positive fusion cells,and then subcloning for 3 times.Mice inoculated hybridoma cells were used to induce ascites and MAbs were purified from ascites.Purified SCD monoclonal antibodies were used for biological characteristics identification.The main results of this research are as follows:(1)The sequence with a total length of 210 bp at SCD N-terminal was amplified by PCR,and the prokaryotic expression vector p ET32a(+)-SCD was successfully constructed.Recombinant protein was successfully expressed in E.coli Rosetta(DE3).Western blot result showed that the recombinant protein had 6×His tag which could be used for purification.Recombinant protein molecular weight was about 30 k D,consistent with the expected protein molecular weight.(2)We optimized the expression conditions of recombinant protein,the best condition is as follows,25 ?,1 mmol/L of IPTG,induction for 12 h;the target protein was mainly expressed in the fraction of supernatant under these conditions.Then,the recombinant protein from supernatant of lysates was purified using His-tag protein purification beads.Theconcentration of the obtained protein was 2.5 mg/m L determined by BCA Protein Assay Kit.(3)The purified protein was used as antigen to immune Balb/c mice.Using PEG for fusion of immune spleen cells and myeloma cells to prepare hybridoma cells.ELISA were used to screen positive fusion cells.1 positive hybridoma strain was obtained after 3 times of subcloning.(4)Mice were inoculated hybridoma cells to induce ascites and MAbs were purified from ascites.The obtained monoclonal antibodies were with high purity and concentration of2 mg/m L,which satisfied the test requirement.(5)Purified SCD monoclonal antibodies were used for titer,class and specificity etc.biological characteristics identification and preliminary application.ELISA analysis showed that the titer of the obtained antibody was 106.Western blot result revealed that the antibody could specifically identify the specific antigen epitope and specificly combine with the SCD protein expressed in HEK-293 cells.In conclusion,the SCD protein was successfully expressed and purified.The monoclonal antibody with high affinity and specificity was successfully produced,which would provide an important tool for further study of the regulatory mechanism of SCD in the medium and short-chain fatty acid metabolism of goat milk.