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A preparation method of oral recombinant nutritional polypeptide supplementing human essential amino acids

A technology of nutritional polypeptides and recombinant plasmids is applied in the field of preparation of oral recombinant nutritional polypeptides, which can solve the problems of complex separation and purification process of nutritional polypeptides, and achieve the effects of easy industrial amplification and cultivation, strong survivability and broad market value.

Active Publication Date: 2019-04-30
西安自然界生物学研究有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention of this product overcomes the current limitation of BCAA originating from chemical synthesis, and also overcomes the shortcomings of the complex separation and purification process of nutritional polypeptides produced by E. coli fermentation, and has a good application prospect in the field of health care products

Method used

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  • A preparation method of oral recombinant nutritional polypeptide supplementing human essential amino acids
  • A preparation method of oral recombinant nutritional polypeptide supplementing human essential amino acids
  • A preparation method of oral recombinant nutritional polypeptide supplementing human essential amino acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning, sequencing and identification of nutritional polypeptide np gene

[0040] The gene of nutritional polypeptide NP was amplified from the synthetic DNA fragment purchased by the company (Shanghai Sangong) by PCR.

[0041] The PCR amplification primers of the nutritional polypeptide np gene fragment are:

[0042] Np-F: GG GGTACC CC CTGAAGGTGACCATCAAGAC

[0043] Np-R: CG GAATTC CG TCCTTGGTCACCAGCAGGATCAC

[0044] The PCR reaction system was carried out strictly according to the instruction manual of TaKaRa LA Taq. The 20 μL reaction system contained 0.2 μL of TaKaRaLA Taq, 10×LA PCR Buffer Ⅱ (Mg 2+Plus) 2 μL, dNTP Mixture (2.5 mM each) 3.2 μL, template DNA 100 ng, upstream and downstream primers (20 μM) 0.4 μL, and finally add sterilized ultrapure water to make up to 20 μL. The PCR conditions are: denaturation at 94°C for 5 minutes, 1 minute at 94°C, 40s at 56°C, and 40s at 72°C, 30 cycles. The size of the PCR product is 180bp, including the c...

Embodiment 2

[0045] Example 2: Preparation and application of Bacillus subtilis recombinant spores displaying NP on the surface of the carrier using CotC

[0046] 1. Construction of recombinant integrated plasmid pJS700-NP

[0047] Plasmid pJS700 (Li Qian. Study on recombinant spores of Bacillus subtilis displaying WSSV envelope proteins Vp19 and Vp28 on the surface of CotX [D]. Zhenjiang, Jiangsu: Jiangsu University, 2010:36-38) was provided by Ningde, School of Environment, Jiangsu University Donated by Associate Professor Gang, the size of the plasmid is about 5.5kb. In the pJS700 plasmid, amyE 5'-end and amyE 3'-end respectively represent the 5' end and 3' end of the coding sequence of the amylase gene amyE (Gene Bank sequence number: NP_388186), which are integrated into Bacillus subtilis 168 by homologous recombination (trp - ) in the amylase gene of the chromosome. Amps r ,Em r Represent ampicillin resistance gene and erythromycin resistance gene respectively, in Escherichia co...

Embodiment 3

[0059] Example 3: Functional analysis of oral administration of recombinant spores in mice to improve exercise efficiency

[0060] In this experiment, 5-week-old healthy ICR mice (20-25g) were selected for oral spore experiment. Experimental mice were bred in full compliance with the requirements and guidance of the Experimental Animal Center of Yangzhou University. The breeding temperature was controlled at 25±1°C, and sufficient drinking water was provided. Before the formal experiment, the acute toxicology experiment of spores was carried out in advance. Select 5 mice, orally administer recombinant spores to the mice at a dose of 5 mg / g body weight, and then observe for a week. After all the physiological activities of the mice are found to be normal, the next step of the experiment is carried out.

[0061] Take 46 ICR mice (20-25g), provided by the Animal Center of Yangzhou University. They were randomly divided into 5 groups, with 6 rats in the control group and 10 rats ...

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Abstract

The invention relates to a preparation method for oral recombinant nutritive polypeptide supplementing human body essential amino acids, and belongs to the technical field of recombination fusion protein medicines. The preparation method comprises integrating a nutritive polypeptide gene np bringing with an enterokinase label into a pJS700 vector, then using a spore capsid protein gene cotC as a molecular vector and constructing fusion expression integrated type recombinant plasmid, then performing conversion on bacillus subtilis by using the fusion expression integrated type recombinant plasmid, so as to enable the exogenous gene to be integrated into amylase gene in chromosome, and screening out a recombinant bacillus subtilis strain containing fusion expression cotC-NP, and displaying the nutritive polypeptide on the spore surface by inducing the recombinant bacterial strain. The constructed recombinant spore with the enterokinase-labeled nutritive polypeptide displayed on the surface can be directly orally taken by animals or people and be freely digested and absorbed in intestinal tracts, becomes a novel oral sport nutrition health product, and possesses wide application value. The limit that BCAA comes from chemical synthesis extraction is overcome, and also the disadvantage that separation purification is complex when the nutritive polypeptide is produced through recombination fermentation is overcome.

Description

technical field [0001] The invention relates to a preparation method and product of essential amino acid constituting polypeptides and surface display of recombinant spores, in particular to a preparation method of oral recombinant nutritional polypeptide supplementing human essential amino acids, which belongs to the technical field of recombinant fusion protein drugs. Background technique [0002] Amino acid (Amino acid) is the basic unit of protein, which endows protein with a specific molecular structure and shape, and participates in physiological and biochemical processes such as protein catalysis, transportation, movement, defense and protection information transmission. There are mainly 20 kinds of amino acids that make up proteins in nature. Among them, the amino acids that cannot be synthesized by the human body or cannot meet the metabolic needs of the human body are called essential amino acids. There are usually 8 essential amino acids, namely isoleucine, leucin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N1/21A23L33/18C12R1/125
Inventor 陈克平冯凡陈美萍陈亮吕鹏张春霞陈先涛姚勤夏恒传马上上
Owner 西安自然界生物学研究有限公司
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