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Peptide hormone prolactin (prolactin, PRL) is secreted by the pituitary prolactin cells, the major physiological role in human and animal reproductive To promote the development of the mammary gland, the start of lactation and maintain lactation. In livestock, especially sheep breeding production, postpartum physiological high prolactin levels suppress ovarian function, manifested as ovarian follicular development and ovulation of the ewes for feeding not breeding during pregnancy, so that the sheep the lambing interval prolongation. In sheep production, is one of the most effective ways to improve breeding efficiency as much as possible to shorten the interval of ewes. And the most effective measures to shorten the lambing interval is based on measures of early weaning and improve ewe nutritional status, as soon as possible to reduce the ewes prolactin levels, restore ovarian activity. The researchers already using bromocriptine to reduce prolactin levels, to promote the recovery of the ewes ovarian function and estrus. Rapidly reduce the ewes in vivo to promote the secretion of prolactin, another way is to use the prolactin polyclonal or monoclonal antibodies. With sheep to promote prolactin pure and Freund's adjuvant as immunogen routine immunization program, indirect ELISA test results show that, one day, after 15 days and 29 days of immunization three serum antibody titers 1:8 000 to 1:10 000, indicating that the immunogenicity of the antigen good fourth strengthen the immune spleen cells for cell fusion. In order to screen the positive hybridoma cells secreting antibody in order to promote the prolactin immune serum of BALB / C mice as the positive control, to the non-immunized BALB / C mouse serum as negative control, through the optimization of the ELISA test conditions, the establishment of detection sheep prolactin antibody indirect ELISA detection method, the main criteria are as follows: pH9.6, 0.05 mol / L carbonate buffer solution (CBS) for the package is liquid, 0.1μg/mL antigen as coating antigen , 1% BSA blocking solution, and 1:200 dilution of serum, 1:10 000 for the optimum working concentrations of the enzyme labeled secondary antibodies, 90 min, the HRP-anti-action time, 30 min for the role of a substrate time. Test proved that the method is specific and repeatability. Of the immunized mice prolactin serum antibody and hybridoma monoclonal antibody supernatant were detected by this method. 1 mL 40% polyethylene glycol as a fusion agent 4 000 (PEG 4 000), the choice of SP2 for the logarithmic growth phase / 0 myeloma cells and the immune spleen cells was conducted at room temperature in accordance with the ratio of 1:5 to 1:10 fusion, the entire process of the integration of control at 1 min, try to maintain a constant speed, and then within 3 min with 8 mL of basal medium diluted PEG speed to slow after the fast, it lost the role of promoting financial centrifugation, washed, integration cells with HAT selective medium culture medium was changed every 3 d ~ 4 d half the amount of time, three days after the fusion of small clone fusion, seven days hybridoma covered 50% of the bottom of the hole, using the established indirect ELISA assay positive wells, the clone was 69%, was 23% positive clones. Positive cultures hole screening after limited dilution positive clones were screened for monoclonal antibody induced ascites legal system, and preliminary identification of chromosome karyotype analysis of hybridoma. By cell fusion, get the five can promote prolactin specific monoclonal antibody hybridoma cell lines secreting anti named AA5B1 AB6C8, BB3C1 CA6E4, CE2H6. The cell supernatant titer of 1:100 to 1:500, ascites titer of 1:10000 to 1:40000 map of karyotype analysis shows five hybridoma cell chromosome number between 92 to 103, proved SP / 20 and the spleen cell fusion to produce a hybridoma cell. Cryopreserved 5 months after the recovery of the hybridoma cell lines cultured continuously passaged for 16 generations, and still have the ability to stably secrete antibodies.
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