Recombinant plasmid of GSDMD-N (gasdmermin D-N) gene, expression method in mammary gland and application
A technology of GSDMD-N and gene recombination, applied in the field of genetic engineering, can solve the problems of antibacterial and anti-inflammatory functions that have not been reported, achieve good application prospects, and reduce the effect of mastitis
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Embodiment 1
[0040] The construction of the GSDMD-N recombinant expression plasmid includes the following process steps and results:
[0041] Design of GSDMD-N gene domain CDS full-length fragment primers:
[0042] According to the full-length sequence of GSDMD protein, the GSDMD-N gene sequence was determined by GenBank, and the GSDMD-N gene domain CDS full-length fragment primers were designed, and the GSDMD-N gene primers (GSDMD-N-F, GSDMD-N-R) contained BamH I and EcoRI enzymes respectively cutting site, used to amplify the 840 bp GSDMD-N fragment.
[0043] According to the upstream regulatory sequence of whey acid protein (WAP) gene and the plasmid containing WAP gene sequence, primers (WAP-F, WAP-R) containing XhoI and EcoRI restriction sites were designed to amplify a 1050 pb WAP fragment, According to the EGFP sequence in the pEGFP-1 plasmid, the primers EGFP+HiS-F and EGFP+HiS-R contained BamHI and NotⅠ restriction sites, respectively, and were used to amplify the 752 bp EGFP fragm...
Embodiment 2
[0111] GSDMD-N breast cell expression, the specific process steps are as follows:
[0112] 1. Electrotransfer of pEGFP-GSDMD-N-WAP-His and pEGFP-WAP-His plasmids into attenuated Salmonella
[0113] 1) Preparation of SL7207 Competent State
[0114] ①The attenuated Salmonella SL7207 taken out from -80°C was streaked on the LB plate, and placed in a 37°C incubator for about 16 h-18 h;
[0115] ②Pick two single colonies and inoculate them into 3 mL LB for overnight expansion culture, and culture overnight on a shaking table;
[0116] ③The next day, add 1 mL of the activated bacterial solution to 100 mL of LB liquid medium at a volume of 1:100, culture on a shaker at 37°C and 200 r / min until OD 600 =0.4;
[0117] ④ Divide 100 mL of the shaken bacterial solution into two 50 mL centrifuge tubes and place in ice bath for 1 h. Pre-cool the centrifuge, 3500 r / min, 10 min, discard the supernatant at 4°C;
[0118] ⑤ Sterilize the bacteria at the bottom of the tube with 5 mL, resuspen...
test example 1
[0158] GSDMD-N mammary gland expression and evaluation of mastitis treatment effect, the specific process steps are as follows:
[0159] 1. Establishment of mouse Staphylococcus aureus mastitis model
[0160] 1) Inoculate Staphylococcus aureus frozen in a -80°C refrigerator into BHI medium at a ratio of 1:100, and culture it in a shaker at 37°C at 200r / min until the logarithmic phase (OD 600 ≈0.9), centrifuged at 7000 r / min for 3 min, washed three times with sterile PBS, and adjusted the concentration to 3.3×10 7 CFU / mL;
[0161] 2) Randomly select female mice that were born for 3-7 days, and anesthetize them with 200 uL pentobarbital sodium intraperitoneally, and then fix them on the cardboard with paper tape;
[0162] 3) Disinfect the fourth pair of teats of the mouse with 75% alcohol, gently clamp the teats with tweezers under a stereomicroscope, and cut off the tip of the teats with scissors to expose the opening of the teat ducts, and then hold the teats for 30 Insert...
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