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Preparation method for oral recombinant nutritive polypeptide supplementing human body essential amino acids

A technology of nutritional polypeptides and recombinant strains 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, large specific gravity, and simple and convenient cultivation methods.

Active Publication Date: 2016-02-24
西安自然界生物学研究有限公司
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  • 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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  • Preparation method for oral recombinant nutritive polypeptide supplementing human body essential amino acids
  • Preparation method for oral recombinant nutritive polypeptide supplementing human body essential amino acids
  • Preparation method for oral recombinant nutritive polypeptide supplementing human body essential amino acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Nutritional peptides np Gene cloning, sequencing and identification

[0041] The gene for the nutritional polypeptide NP was amplified from the synthetic DNA fragment (Shanghai Shenggong) purchased by the company by PCR.

[0042] Nutritional peptides np The primers for PCR amplification of gene fragments are:

[0043] Np-F: GG GGTACC CC GACGATGACGATAAG CTGAAGGTGACCATCAAGAC

[0044] Np-R: CG GAATTC CG TCACTTGTCGTCATCGTCTTTGTAG TC CTTGGTCACCAGCAGGATCAC

[0045] PCR reaction system TaKaRaLATaq The instructions for use are strictly carried out, and the 20μL reaction system contains TaKaRaLATaq 0.2μL, 10×LAPCRBufferⅡ(Mg 2+ Plus) 2μL, dNTPMixture (2.5mM each) 3.2μL, template DNA 100ng, upstream and downstream primers (20μM) each 0.4μL, and finally add sterile ultrapure water to make up to 20μL. The PCR conditions were: denaturation at 94°C for 5 min, 94°C for 1 min, 56°C for 40s, 72°C for 40s, 30 cycles. PCR product size is 180bp, including nutritional peptides ...

Embodiment 2

[0046] Example 2: Preparation and application of recombinant Bacillus subtilis spores displaying NP on the surface of CotC

[0047] 1. Construction of recombinant integrated plasmid pJS700-NP

[0048] Plasmid pJS700 (Li Qian. Research on the recombinant spores of Bacillus subtilis displaying WSSV envelope proteins Vp19 and Vp28 on the surface of CotX as a molecular carrier [D]. Zhenjiang, Jiangsu: Jiangsu University, 2010:36-38), by Ningde, School of Environment, Jiangsu University As a gift from Associate Professor Gang, the size of the plasmid is approximately 5.5kb. In the pJS700 plasmid, amyE 5’-end and amyE 3’-end respectively represents the amylase gene amyE (GeneBank sequence number: NP_388186) The 5’ end and 3’ end of the coding sequence are integrated in the Bacillussubtilis 168( trp - ) In the amylase gene of the chromosome. Amp r , Em r Respectively represent the ampicillin resistance gene and erythromycin resistance gene, in E. coli and Bacillussubtilis 168( trp ...

Embodiment 3

[0060] Example 3: Functional analysis of mouse oral administration of recombinant spores to improve exercise efficiency

[0061] In this experiment, 5-week-old healthy ICR mice (20-25g) were used for oral spore experiment. The experimental mice were reared completely in accordance with the requirements and instructions of the Experimental Animal Center of Yangzhou University. The feeding temperature is controlled at 25±1℃, and sufficient drinking water is provided. Acute toxicological experiments of spores were carried out before the formal experiment. Five mice were selected, and the recombinant spores were orally administered to the mice at a dose of 5 mg / gram body weight at one time, and then observed for a week. After all the physiological activities of the mice were found to be normal, proceed to the next experiment.

[0062] Take 46 ICR mice (20-25g), provided by the Animal Center of Yangzhou University. Randomly divided into 5 groups, 6 in the control group and 10 in the...

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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 (Aminoacid) 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, leucine...

Claims

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Application Information

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Patent Type & Authority Applications(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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