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Xylanase intestinal directional expression vector and cell line thereof

A technology of xylanase and expression vector, which is applied in the direction of using a vector to introduce foreign genetic material, cells and microorganisms modified by introducing foreign genetic material, etc., and can solve the compensatory increase, pollution and destruction of digestive organs. Intestinal normal structure and micro-ecological balance, etc., to achieve the effect of high feed utilization rate and low pollutant discharge

Inactive Publication Date: 2015-05-06
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xylan can prevent the release of intracellular nutrients, increase the viscosity of chyme, hinder the contact between feed nutrients and digestive juice, increase the thickness of the immobile water layer in the intestinal tract, increase the compensatory increase of digestive organs, and destroy the normal structure and microstructure of the intestinal tract. Ecological balance affects the digestion and absorption of nutrients. Therefore, xylan is a typical anti-nutritional factor. Due to the lack of corresponding endogenous digestive enzymes in the digestive tract of pigs to decompose such anti-nutritional factors in feed, corn used as pig feed 1. The xylan in soybean meal is difficult to be effectively utilized and is directly excreted from the body, resulting in waste of feed resources and serious environmental pollution

Method used

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  • Xylanase intestinal directional expression vector and cell line thereof
  • Xylanase intestinal directional expression vector and cell line thereof
  • Xylanase intestinal directional expression vector and cell line thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Construction of XynB eukaryotic expression vector pcDNA3.1-XynB-Myc

[0074] 1. Experimental Materials

[0075] Plasmid: pcDNA3.1(-) plasmid was purchased from Shanghai Invitrogen Life Technology Co., Ltd.; pMD18T plasmid was purchased from Dalian Baobio Co., Ltd.; pRSETA-XynB plasmid was constructed and donated by Professor Wu Zhenfang’s research group from the School of Animal Science, South China Agricultural University (Zhang Xianwei et al., Chinese Agricultural Sciences, 2013, 46(22): 4774-4783).

[0076]Strains: Escherichia coli DH5α strains were purchased from Dalian Bao Biological Co., Ltd.

[0077] Main enzymes and reagent kits: Restriction endonucleases xho I. Kpn Ⅰ. Prime STAR high-fidelity DNA polymerase, Taq DNA polymerase, and T4 DNA ligase were purchased from Dalian Takara Company; medium-volume endotoxin-free plasmid extraction kit and gel recovery kit were purchased from Promega Company; other common chemical reagents were Domestic, i...

Embodiment 2

[0109] Example 2 Porcine RELMβ gene promoter amplification and promoter activity detection

[0110] 1. Main Reagents

[0111] Premix Taq (La Taq version 2.0) enzyme, T4 DNA ligase, DNA Marker, endonuclease, agarose recovery and purification kit, small extraction plasmid extraction kit, medium extraction plasmid extraction kit were purchased from Baobio (Dalian) Co., Ltd. Company; pGL3-basic, pGL3-Enhancer, pRL-Tk vector and dual luciferase detection kit were purchased from Promega; human embryonic kidney cell line 293T, human colon adenocarcinoma cell line HT29 were purchased from KGI Biotech (Nanjing); Lipo LTX, Opti-MEM, DMEM medium, and fetal bovine serum were purchased from Invitrogen; primer synthesis and DNA sequencing were completed by Invitrogen (Shanghai) Trading Co., Ltd.

[0112] 3. Operation steps

[0113] (1) Pig whole genome DNA extraction

[0114] Purchase 40-day-old three-way hybrid sows from the Liuhe base pig farm of Jiangsu Academy of Agricultural Scien...

Embodiment 3

[0126] Example 3 Construction of the intestinal-specific expression vector pcDNA3.1-RELMβ-XynB-myc-GFP and its lentivirus preparation

[0127] 1. Main reagents

[0128] restriction endonuclease Nhe I. xho I. Kpn Ⅰ, Prime STAR high-fidelity DNA polymerase, Taq DNA polymerase, and T4 DNA ligase were all purchased from Dalian Takara Company; endotoxin-free medium plasmid extraction kit, gel recovery kit, and quick ligation kit (Liga Fast TM Rapid DNA Ligation System) was purchased from Promega, transfection reagent Lipofectamine TM LTX, plasmid pLenti4, pC-GP, 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.

[0129] 2. Solution

[0130] 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 b...

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Abstract

The invention relates to a xylanase intestinal directional expression vector and a cell line thereof and belongs to the biotechnical field. An XynB gene is inserted into an eukaryotic expression vector pcDNA3.1(-), a lentivirus recombinant vector Lanti4-blockit-RELMbeta-XynB-myc-GFP is constructed, and primary culture porcine embryonic fibroblasts are transfected to construct an eukaryocyte line of intestinal directional expression XynB zymoprotein. Therefore, a necessary transgenic biomaterial and a key technical system support can be provided for further cloning of trans-XynB gene embryo by somatic cell nuclear transplantation, and breeding of a new transgenic swine variety with high feed utilization and low pollutant discharge as well as the field of associated biological scientific research and production.

Description

1. Technical field [0001] The invention relates to a xylanase gut-directed expression vector 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. At the same time, my country is a large breeding country, and its feed production ranks second in the world. Due to the rapid development of my country's breeding industry in recent years, the contradiction between humans and animals competing fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/867C12N15/66C12N15/56C12N5/10C12Q1/34C12Q1/02
Inventor 陈哲王公金于建宁徐小波
Owner JIANGSU ACAD OF AGRI SCI
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