| In most mammals, during pregnancy, tolerance of the fetal allo graft without immune rejection appears to involve regulating the expression of MHC antigens on maternal-fetal interface. Expression of MHC-I molecules on the’window period’ of the embryo endometrial implantation plays a vital role in the regulation of immune tolerance and the establishment of pregnancy. MHC-I heavy chain is a kind of classical MHC-I molecules, and it is the common structure for the class I molecules, its expression can completely reflect the overall expression of class I molecules. Dairy cattle MHC-I has been named bovine leukocyte antigen I (BoLA-I). IFN-Υ is expressed in the ruminant trophoblast cells around peri-implantation period only, and acts on the maternal endometrium, is considered to be earliest pregnancy identification signal sent to the pregnant body of a ruminant embryo. Accordingly, we hypothesized that IFN-Υ has a regulatory role in BoLA-class I molecule expression on uterine epithelial cells. Since BoLA-I related to pregnancy, this study examined its expression in uterus, ovaries, liver, and spleen in order to compare the expression characteristics in reproductive organs. Therefore, this study was aimed at proving the basis for exploring the immune regulatory pathways of IFN-Υ-BoLA-I heavy chain and the uterine epithelial cells, and establishing the scientific and theoretical basis of further clarifying the bovine embryo implantation environment.(1) Cloning, expressing and preparing polyclonal antibody of BoLA-I heavy chain. Total RNA was extracted from the Holstein calf liver tissue and reverse-transcribed into cDNA using RT-PCR technique. According to the mRNA sequence of BoLA-I heavy chain registered in GenBank, designed a pair of primers, then amplified cDNA with enzymes to get target fragment. Constructed pGEX-4T expression vectors, transformed into Rosetta (DE3) for inducing prokaryotic expression by ITPG. Polyclonal antibody was prepared by immunization of rabbits and its specificity and potency were identified by Wester Blot and indirect ELISA. The results revealed that the experiment successfully cloned BoLA-I heavy chain CDS sequence, the fragment size is717bp, which is100%identical with the BoLA-I heavy chain isoform1precursor BLAST in NCBI. The weight of the encoded protein is approximately53kD. Indirect ELISA and Western Blot showed that polyclonal antibody has good specificity and reactivity of immune response.(2) Analyzing BoLA-I heavy chain expression on uterine epithelium cell regulated by IFN-Υ with RT-qPCR and Western Blot. Cultivated uterine epithelium cell, set a cell density of5x105/ml as the model of heavy chain expression and stimulated it with exogenous IFN-Υ. After which set time gradient (3h,6h,12h) and concentration gradient of IFN-Υ (50ng/ml,100ng/ml,200ng/ml). In mRNA levels, using RT-qPCR detected the regulation of heavy chain expression, analyzing data results with SPSS software. In protein levels, selected two groups of cells with the max diversity of RQ value. After detected heavy chain expression by Western Blot, valuing OD by alphaEaseFC software then analyzed data with SPSS. At the IFN-Υ intervene time of3h,the experimental groups of100ng/ml and200ng/ml showed no significant difference in BoLA-I heavy chain expression whereas the50ng/ml group made a significant inhibition(P<0.05). When the time up to6h, the BoLA-1heavy chain expression of groups100ng/ml and200ng/ml has increased significantly (P<0.01). By the time of12h, significantly increase in protein expression (P<0.01) were made in all3groups. Wester Blot showed that group3h,50ng/mL was significantly lower than the control group while the6h,200ng/mL group was significantly higher than control group (P<0.01). These results were consistent with the result of RT-qPCR detection.(3) Detecting the distribution of BoLA-I heavy chain in main tissues used immunohistochemistry assay. Polyclonal antibody was used to detect the expression differences on main organs. The results displayed that the BoLA-I heavy chain were mainly distributed in the cytomembrane of liver, spleen, ovary and uterine tissues. It is obvious that the positive signal distribution in immune organs were significantly higher than reproductive organs. Respectively, in ovarian and uterine, positive signal were highly detected in epithelial cells and lymphocytes, positive signal were distributed evenly in term of liver cytomembrane, and the expression of positive products is strongest in spleen red pulp.Conclusion:1) This study successfully cloned BoLA-I heavy chain, completed the preparation of polyclonal antibody and proved the specificity of the antibodies;2) With the increase of time and IFN-Υ concentration, the expression of BoLA-I heavy chain significantly rised on cow uterine epithelial cells. The regulation played a key role in escaping from the maternal placenta pregnancy immune in the process of embryo implantation;3) Expression of BoLA-I heavy chain on cell membrane in distict organs and tissues were widely different. |
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