The Expression And Distribution Of Mic1in Main Tissues And Cells In Dairy Cows

Reproduction is one of the most important aspects in the dairy cattle industry. Reproduction makes important affect not only on the increase of quantity and quality, but also on the production performance and economic benefits of dairy industry. Various reasons result in the loss of early embryo, especially preimplantation embryo loss which was the main factor for cow reproductive failure.The maternal-fetal interface is the essential site of implantation,and major histocompatibility complex class Ⅰ （MHC-Ⅰ） takes capital contribution on the semiallogenic immune in mammal animals.Major histocompatibility complex （MHC） class Ⅰ α chain-related protein1（MIC1） is unclassical, which is the ligand of NK.G2D on natural killer cell, and its gene locates in the MHC-Ⅰ region of chromosome23.MIC1which is mainly expressed on epithelial cells, transplanted cells and tumor cells, and heat stress can stimulate epithelial cells expressing MIC1. IFN-τ belonged to type Ⅰ interferon, is a glycoprotein, and secretes in ruminants embryonic trophoblastic cells, which plays an important effect on the luteal formation, pregnant establishment and maintenance. With these characters, we presume that IFN-τ might regulate the expression of MIC1on bovine uterine epithelial cells.The specific rabbit anti-cow MIC1polyclonal antibodies were prepared with the technologies of gene clone and express and antibody preparation.With these antibodies, the expression and localization of MIC1protein in main tissues and uterine epithelial cells of dairy cow were adetected by Western blot and immunohistochemistry.The results showed as follows:1. MIC1CloningAccording to the CDS sequence in Genbank,a pair of primers were designed to amplify MIC1from CDS which were reversely transcripted from mRNA in cattle uterus by RT-PCR.The amplified target fragments were896bp confirmed by gel electrophoresis, and then were further sequenced and compared with bovine MIC1in GenBank（registry numbers:NM₀01127317.1）,the nucleotide homology was98%and amino acid homology was100% 2. MIC1expressionMIC1gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4T-1.The recombinant plasmid was transformed into E.coli BL21and successfully expressed.The expressed GST-MIC1fusion protein was about63kDa with the analysis of SDS-PAGE,which was corresponding to the predicted sizes of interest protein.3. The preparation and purification of polyclonal antibodyAfter rabbits were immunized with the purified protein,the polyclonal antibodies of MIC1was successfully harvested and purified by affinity chromatography,and identified by iELISA and its titer was highly to1:64000.4. Differential expression of MIC1MIC1on the expression of dairy cow uterine epithelial cells regulated with IFN-τ was detected by Western blot with rabbit anti-cattle GST-MIC1polyantibodies. The results showed that MIC1expression was highly suppressed by IFN-τ.Immunohistochemical assay discovered that MIC1were lower expressed in liver and kidney,but higher expression in ovary and endometrium.Conclusion:MICl was successfully prepared with gene cloneand express,the polyclone antibodies were harvested and purifid for the uses of MIC1detection. With the results,we suggested that IFN-τ suppressed MIC1expression in uterine epithelial cells in vitro, and MIC1were highly expressed in endometrium and ovary,but lower expressed in liver and kidney.