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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

Inactive Publication Date: 2019-01-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the protein sequence of GSDMD-N has been successfully detected, but its antibacterial and anti-inflammatory functions have not been reported yet

Method used

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  • Recombinant plasmid of GSDMD-N (gasdmermin D-N) gene, expression method in mammary gland and application
  • Recombinant plasmid of GSDMD-N (gasdmermin D-N) gene, expression method in mammary gland and application
  • Recombinant plasmid of GSDMD-N (gasdmermin D-N) gene, expression method in mammary gland and application

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a recombinant plasmid of a GSDMD-N (gasdmermin D-N) gene, an expression method in mammary gland and an application, and belongs to the field of genetic engineering. An expression recombinant plasmid of the GSDMD-N gene is constructed and enabled to be specifically expressed in breast tissue and breast cells, so that mastitis caused by staphylococcus aureus infection is prevented and treated. GSDMD-N protein is a polypeptide compound found in recent years, which can specifically recognize biofilm component cardiolipin of bacteria and can be combined with cardiolipin to form a plurality of honeycomb-shaped pores, so that bacteria are disintegrated and die. According to the technology, the exogenous gene GSDMD-N is efficiently expressed in animal mammary glands througha specific promoter WAP (whey acidic protein) of breast tissue by a mammary bioreactor, staphylococcus aureus causing cow mastitis is killed, and accordingly, the cow mastitis caused by staphylococcus aureus infection is prevented and treated. The recombinant plasmid and the recombinant attenuated salmonella have the advantages of being efficient, safe, low in cost and the like, provide a a new approach for prevention and control of the cow mastitis caused by staphylococcus aureus infection, and have good application prospect.

Description

Technical field: [0001] The invention provides a GSDMD-N gene recombinant plasmid, which is constructed from the mouse GSDMD-N gene; the invention further provides a GSDMD-N gene recombinant plasmid and a method for expressing it in mammary glands; using attenuated Salmonella to make The method for expressing it in bovine mammary gland cells and mouse mammary gland tissues, so as to achieve the purpose of preventing and treating mastitis infected by Staphylococcus aureus by using GSDMD-N protein, belongs to the technical field of genetic engineering. Background technique: [0002] Gasdermin family is a protein family containing DFNA5 domain but whose function is unknown. This family mainly has 12 members. This family protein is mainly expressed in intestinal epithelium and gastrointestinal epithelium. Mutations of this family protein can cause hair follicle growth Abnormal development, and the abnormal expression of proteins in this family is closely related to the occurrenc...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66A61K48/00A61K38/17A61P15/14A61P31/04
CPCA61K38/17A61K48/0025A61K48/0058A61P15/14A61P31/04C12N15/66C12N15/85
Inventor 杨勇军颜世卿马超颖潘志明马轲陈巍
Owner JILIN UNIV
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