mrgd immune antigen fragment, mrgd antibody and preparation method and application thereof
A technology for immunizing antigens and fragments, which is applied in immunoglobulins, chemical instruments and methods, and anti-animal/human immunoglobulins to achieve good specificity.
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Embodiment 1
[0024] 1. Polypeptide antigen synthesis
[0025] The following fragment was selected and synthesized from the rat MrgD sequence, SSPAPGLTISPTMDC, as the immune antigen.
[0026] 2. Animal immunization
[0027] Several New Zealand rabbits of 2-3 kg were immunized with the synthesized polypeptide antigen.
[0028] Initial immunization: Antigen 100ug / rat, plus complete Freund's adjuvant (antigen and adjuvant are mixed in equal volumes, ground into a water-in-oil chylus) and subcutaneously injected at multiple points on the back, with a volume of 1.5ml.
[0029] The second immunization: the second immunization was performed at an interval of 2 weeks after the initial immunization, the dosage route was the same as above, and incomplete Freund's adjuvant was added.
[0030] The third immunization: after the second immunization, the third immunization was carried out at an interval of 2 weeks. The dose was the same as above, without adjuvant, intraperitoneally injected, and blood w...
Embodiment 2
[0044] Rat primary cardiomyocytes were extracted, cultured in the medium DMEM+10% FBS for 48 hours, the medium was discarded, washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, blocked with 5% BSA for 30 minutes, and added to the example The MrgD antibody obtained in step 1 was spent overnight at 4, and then washed 3 times with PBS, added fluorescent secondary antibody (cy3) and incubated at room temperature for 2 hours, eluted the secondary antibody, added anti-fluorescent quencher, mounted the slide and took pictures. Such as figure 1 As shown, it can be seen that the content of MrgD in normal cardiomyocytes is low.
Embodiment 3
[0046] Rat primary cardiomyocytes were extracted and added with angiotensin II (concentration 10 -6mol / L) culture medium DMEM+10% FBS for 24 hours, discard the medium, wash twice with PBS, fix with 4% paraformaldehyde for 30 minutes, block with 5% BSA for 30 minutes, add MrgD antibody for 4 nights, Then wash 3 times with PBS, add secondary antibody and incubate at 37°C for 1 hour, wash 3 times with PBS, mount the slides and take pictures. Such as figure 2 As shown, it can be seen that the expression level of MrgD (highlighted part) is higher in hypertrophic cardiomyocytes induced by angiotensin II.
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