Application of reg1a protein in preparation of medicine for treating and/or preventing retinal cell apoptosis
A technology of retinal cells and retinal pigments, applied in drug combinations, peptide/protein components, pharmaceutical formulations, etc., can solve problems such as incomplete gene therapy mechanism, chronic rejection, patient physiological and psychological trauma, etc., to prevent retinal degeneration , the effect of small dosage
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Embodiment 1
[0038] Expression of neurosecretory factor reg1a gene in mouse retina
[0039] Various parts of mice were taken as experimental materials, including pancreas, kidney, heart, skin, small intestine tissue, brain, testis, choroid, liver, spleen, lung, subfrontal gland, cornea, retina and lens. The mRNA of each material was extracted from the above materials using the kit method.
[0040] After the quality of the extracted mRNA was tested, the mRNA with high integrity was tested by semi-quantitative PCR. Specifically, a reverse transcription kit was used to reverse transcribe mRNA into cDNA, perform semi-quantitative PCR reaction on REG1A gene, and use GAPDH as an internal reference gene. The primer sequences of EG1A gene and mGAPDH are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR program is shown in Table 1. table 3.
[0041] Table 1 Primer sequences of REG1A gene and GAPDH
[0042]
[0043] Table 2 PCR amplification system of reg1a gene and GA...
Embodiment 2
[0051] Immunostaining steps:
[0052] Mouse retinas were dissected, fixed in 4% paraformaldehyde for 1 h, dehydrated with 15% and 30% glucose, embedded in OCT embedding medium, and frozen sections were prepared;
[0053] For washing, the retinal slices were put into DPBS, placed on a shaker and slowly rinsed 3 times, 5 minutes each time.
[0054] After rinsing, 500 μl of 0.5% Triton X-100 penetration solution prepared in DPBS was added to each well, the cells were permeated for 10 minutes at room temperature, and the penetration solution was aspirated.
[0055] Repeat step 3, adding 500 μl of 3% HO in DPBS to each well 2 o 2 , 10min at room temperature to remove the influence of peroxidase.
[0056] Absorb H 2 o 2 , repeat step 3. Block with 4% BSA (prepared with DPBS) for 30 min at room temperature.
[0057] After aspirating the BSA, repeat step 3. The REG1A antibody was diluted 1:100, added to the culture dish, and incubated overnight at 4°C.
[0058] Recover the prim...
Embodiment 3
[0063] Expression and Purification of Recombinant REG1A Protein
[0064] The reg1a gene was amplified according to the PCR method in Example 1, and the reg1a gene fragments were subjected to agarose gel electrophoresis, and a large number of reg1a gene fragments were obtained after gel recovery. Using a ligation kit, the recovered reg1a gene fragment was digested and ligated with the pET28a vector to obtain a recombinant vector. The recombinant vector was transformed into Escherichia coli strain BL-1, cultured on a shaker, and the cultured colonies were verified as positive bacteria by colony PCR. Add isopropylthiogalactopyranoside (IPTG) to the positive strains to induce expression, collect the bacteria by centrifugation, add buffer to make a suspension of bacteria, ultrasonically break, and collect protein by centrifugation. Comparing the protein expression changes in bacteria before and after adding IPTG, the extracted protein was subjected to PAGE gel electrophoresis, and...
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