Preparation method and application of a non-human mammalian model of premature ovarian failure
A technology for non-human mammals and premature ovarian failure, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., and can solve problems such as hereditary premature ovarian failure
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[0047] Preparation method of model animals
[0048] According to the bioinformatics analysis on the molecular structure of FSHR, the invention clones the extracellular region fragments, constructs prokaryotic expression plasmids for expression and purification, and synthesizes three polypeptides that may be combined with the ligand FSH.
[0049] After actively immunizing female non-human mammals with the obtained recombinant protein rFSHR and artificially synthesized polypeptides, high-titer levels of antibodies were produced in the blood of the animals, and then it was detected that the animal's estrous cycle was disturbed, the estrus period was shortened, the blood FSH level increased, And blood estrogen levels decreased. The FSHR antibody in the blood of the animal can specifically bind to the FSHR on the ovarian granulosa cells, resulting in a decrease in their reactivity to gonadotropins, thereby triggering the appearance of symptoms of premature ovarian failure, thereby ...
Embodiment 1
[0101] Example 1. Preparation of immunogens directed against FSHR extracellular region
[0102] We cloned the corresponding cDNA sequence of the extracellular region of rat FSHR, constructed bacterial recombinant expression plasmids, and expressed the recombinant proteins GST-rFSHR and his-rFSHR of the extracellular region of rat FSH receptor (rFSHR). The recombinant proteins GST-rFSHR and his-rFSHR of the rat FSH receptor (rFSHR) in DE3 expressing bacteria were mainly expressed in the form of inclusion bodies. SDS / PAGE and Coomassie Brilliant Blue staining verified that a sufficient amount of protein with high purity was obtained ( figure 2 ). At the same time, the purified protein was verified by western blot with tag antibodies (rabbit anti-GST polyclonal antibody and mouse anti-His monoclonal antibody) and specific rabbit anti-FSHR polyclonal antibody ( image 3 ). At the same time, through the prediction of antigen reactivity, the three peptide sequences were obtained...
Embodiment 2
[0103] Embodiment 2.ELISA detects the FSHR antibody titer in immune rat antiserum
[0104] Seven days after rats were immunized for three times, about 0.1 ml of blood was collected from the sublingual vein, and the antiserum was separated. The microtiter plate was coated with purified protein GST-rFSHR, and the antigen content per well was 1 μg. The antibody titer of the sera of active immunized rats was detected. The synthetic peptide group and the adjuvant control group started with a dilution ratio of 1:200, and the recombinant protein group started with a dilution ratio of 1:2000. The antibody titer of the corresponding serum was calculated by measuring the A450 value of the immune detection reaction. The serum antibody titer was continuously detected for six weeks, and it was found that the antiserum titer level remained at a fairly high level. It can be seen from Figure-5 that after the last immunization at the age of 52 days, the serum antibody titer of the recombinant ...
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