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Sulfide quinone oxidoreductase intestinal tract directed expression vector and cell line thereof

A technology for expressing vectors and enzymatic intestines, applied to cells modified by introducing foreign genetic materials, using vectors to introduce foreign genetic materials, microorganisms, etc., can solve problems such as impossibility, harmlessness, and secondary pollution, and achieve The effect of reducing pollutant emissions

Inactive Publication Date: 2012-08-22
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, pig farms can only control these waste gases to a certain extent, and cannot truly make them harmless.
The main control methods are adsorption method, incineration method, chemical and biological deodorant method, washing method and biological filtration, etc. These methods have a certain control effect on harmful gases such as H2S and NH3, but due to high cost or secondary secondary pollution problems and cannot be used on a large scale

Method used

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  • Sulfide quinone oxidoreductase intestinal tract directed expression vector and cell line thereof
  • Sulfide quinone oxidoreductase intestinal tract directed expression vector and cell line thereof
  • Sulfide quinone oxidoreductase intestinal tract directed expression vector and cell line thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of SQR eukaryotic expression vector pcDNA3.1(-)-SQR-Myc

[0057] 1. Experimental Materials

[0058] Plasmids: pcDNA3.1(-) plasmid was purchased from Shanghai Invitrogen Life Technology Co., Ltd.; pMD18T plasmid was purchased from Dalian Bao Biological Co., Ltd.; pRSETA-SQR plasmid was constructed and donated by Professor Li Jiaqi, School of Animal Science, South China Agricultural University (Yu Fengxiang et al., Jiangsu Agricultural Journal, 2011, 27(5): 1043-1046).

[0059] Strains: Escherichia coli DH5α strains were purchased from Dalian Bao Biological Co., Ltd.

[0060] Main enzymes and reagent kits: restriction endonucleases XhoⅠ, KpnⅠ, EcoRI, HindⅢ, Prime STAR high-fidelity DNA polymerase, Taq DNA polymerase, T4 DNA ligase were purchased from Dalian Takara Company; medium plasmid extraction kit, The gel recovery kit was purchased from Promega; other common chemical reagents were domestic, imported or imported.

[0061] 2. Solution prep...

Embodiment 2

[0092] Example 2 Construction of intestinal-specific expression vector pcDNA3.1-IFABP-SQR-GFP and preparation of lentivirus

[0093] 1. Main Reagents

[0094] Restriction endonucleases NheI, XbaI, XhoI, KpnI, Prime STAR high-fidelity DNA polymerase, Taq DNA polymerase, and T4 DNA ligase were purchased from Dalian Takara Company; kits, quick link kits (Liga Fast TM Rapid DNA Ligation System) was purchased from Promega, transfection reagent Lipofectamine TM LTX, plasmid pLenti4, pC-GP, pR-Rev, and pC-VSVG were purchased from Invitrogen; trypsin, penicillin, and streptomycin were purchased from SIGMA; DMEM (high glucose) and fetal bovine serum (FBS) were purchased from GIBCO .

[0095] 2. Solution

[0096] HEK293 cell complete medium (1L): DMEM (high glucose) 13.4g, FBS (fetal bovine serum) 100ml, penicillin 0.06g, streptomycin 0.1g, ultrapure water to 1L, adjust the pH value to 7.2, pump Filter out bacteria and store at 4°C for later use.

[0097] Ca 2+ / Mg 2+ free ...

Embodiment 3

[0135] Example 3 Construction and identification of porcine fibroblast cell lines expressing SQR

[0136] 1. Reagents

[0137] LipofectamineTM LTX (GEⅡ), DMEM (high glucose), DMEM-F12, and fetal bovine serum (FBS) were purchased from Invitrogen; pancreatin, EGF, insulin, penicillin, and streptomycin were purchased from SIGMA. Anti-Myc mouse primary antibody, horseradish peroxidase-conjugated goat anti-mouse secondary antibody, and Pro-light HRP chemiluminescent detection reagent were all purchased from Tiangen Biochemical Company. Reagents related to SDS-PAGE and Western Blot were purchased from Shanghai Sangong, and developer and fixer were purchased from Nanjing Zhengran Technology Company.

[0138] 2. Solution preparation

[0139] HEK293 cell culture medium (1L): same as Example 2

[0140] Fibroblast complete medium (1L): DMEM (high sugar) 13.4g; FBS (fetal bovine serum) 100ml; penicillin 0.06g; streptomycin 0.1g;

[0141] IEC (intestinal epithelial cells) complete me...

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Abstract

The invention relates to a sulfide quinone oxidoreductase intestinal tract directed expression vector and a cell line thereof, belonging to the field of biotechnology. The SQR (sulfur quinine oxidoreductase) gene is inserted into a eukaryotic expression vector pcDNA (deoxyribonucleic acid) 3.1 (1), so that a slow virus recombinant vector Lanti4-blockit-IFABP (intestinal fatty acid binding protein)-SQR-GFP (green fluorescent protein) can be built, and the primarily cultured pig intestinal epithelial cell is infected by the slow virus recombinant vector, so that the eukaryotic cell line of specific expression SQR-GFP protein can be built. In order to further prepare the somatic cell nuclear transfer clone trans-SQR gene, necessary transgenic biological materials and key technical system support can be provided for cultivating new transgenic pig species reducing the pollutant discharge.

Description

1. Technical field [0001] The invention relates to an intestine-directed expression vector of sulfide quinone oxidoreductase and a cell line thereof, and belongs to the field of biotechnology. 2. Background technology [0002] Environmentally friendly breeding is a new direction for the sustainable and healthy development of animal husbandry. Since the reform and opening up, with the comprehensive and rapid development of my country's national economy and the continuous improvement of people's living needs, the scale of my country's animal husbandry industry has achieved unprecedented development. However, with the improvement of the scale, intensification and mechanization of livestock farms, livestock manure has become a pollution source that cannot be ignored. Livestock and poultry manure not only pollute water and soil, but also cause serious air pollution. According to tests, a pig farm that produces 100,000 pigs a year can discharge nearly 148 kg of ammonia (NH3), 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/867C12N15/66C12N5/10C12R1/91
Inventor 王公金于建宁徐小波于峰祥陈哲
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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