The Expression And Distribution Of BoLA-A In Main Tissues And Cells In Dairy Cows

The infertility of dairy cows is a major problem which constraining livestock production. Perimplantation instability or failure is the main cause of animal infertility. Implantation failure related to pregnancy recognition, the uterine receptivity state formation, and the process of the zygotic gene mutation, blastocyst and uterine receptivity. It is natural phenomena of all mammals that pregnancy immune related to pregnancy recognition and uterine receptivity state formation. Pregnancy recognition is mainly mediated by the major histocompatibility complex I （MHC-I）.The dairy cow’s MHC known as bovine leukocyte antigen （BoLA）, BoLA-Ⅰ molecules is divided into BoLA-Ⅰa and BoLA-Ib. BoLA-Ia is highly polymorphic. Histocompatibility complex class I express in bovine trophoblast cells is tightly regulated and biologically relevant which is the key to accept the allograft avoiding from maternal immune. The BoLA-Ⅰa has a heavy chain （a chain） and one light chain β2m constituted by non-covalent heterodimer. BoLA-A can code a chain of Bola-Ia molecules.The expression of BoLA-A is high in Peripheral blood in the third phase of pregnancy. BoLA-A is high expression in ear tissue of newborn calves whereas almost no expression in fetal placental apoptosis. In early peri-implantation, IFN-τ has a regulatory role to BoLA-A in the UEC. The BoLA-A study revealed BoLA-Ⅰ regulate the molecular mechanism of the formation of immune pregnancy from the molecular pathways and functional studies section.（1） Cloning and prokaryotic expression of BoLA-AThe primers were designed based on BoLA-A sequences published in GenBank.We extracted uterine epithelial tissue’s mRNA, got BoLA-A fragments amplified by RT-PCR. Recycled and connected to the PET-28a, provided on the PCR, restriction enzyme digestion and sequencing of the recombinant plasmid. It is99%identical between the cloned sequence and the sequence in GenBank of BoLA-A gene accession which numbered AC₀00180. The cloned sequence BLAST with the published sequence showed that there is one mutation base, but it is synonymous mutations, which means that we successfully cloned the BoLA-A. We put recombinant plasmids transformed into competent cells BL21. The fusion protein His-A apparent expression with a molecular weight of about44kDa in37℃, IPTG0.4mmol/L,4h of induction, and mainly as inclusion bodies forms. （2）Preparation BoLA-A polyclonal antibodyUsing the purified fusion protein His-A as an immunogen, in accordance with the immunization program designed for immunizing rabbits and successfully prepared rabbit anti-bovine BoLA-A hyperimmune serum. The BoLA-A polyclonal antibody titer up to1:256000detected by ELISA. It has a good specificity in Western blot analysis the BoLA-A expression in UEC.（3）Western blot analysis BoLA-A expression regulated by IFN-τ in UECSet UEC density of5×105/mL, IFN-τ concentration of100ng/ml as IFN-τ expressed BoLA-A model of intervention trials according to the BoLA-A gene quantitative results and test design. Select6h,12h and control groups （no IFN-τ） for the trial, Western blot detection, Alpha software processing system analysis optical density values of the target band. The results showed that IFN-τ inhibits UEC’s BoLA-A expression in6h. At12h, compared to the amount with the control group, UEC’s BoLA-A expression was increased. The results were coinsident with IFN-τ regulation BoLA-A gene expression. But the numerical correlation is unknown and need to be further explored.（4） The distribution of BoLA-A in main tissues by SABCStudy the distribution of BoLA-A in cows liver, spleen, epithelial tissue of the uterus and ovaries by SABC. The results showed that the BoLA-A expression in cow liver, spleen, epithelial tissue of the uterus and ovary,spleen and liver have significant positive signal, and immune cells have positive signal.Control group did not detect the positive signal.Conclusions:BoLA-A has a widely distribution in main tissues in dairy cows. The distribution of BoLA-A in tissus were different. IFN-τ at physiological concentrations （100ng/ml） regulated UEC’s BoLA-A expression which coding BoLA-I a class of molecules.