Active protein peptide gene with excellent repairing effect and encoded protein peptide

A technology of protein peptides and vitality, applied in the field of active peptides, can solve the problems of high cost, difficulty in fully exerting efficacy, difficulty in mass production of EGF, etc., and achieve the effect of high safety

Active Publication Date: 2021-04-09
四川省恩乐生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, EGF has not been widely used in the fields of medicine and cosmetics. The reason is that EGF is difficult to achieve mass production: At present, EGF extraction methods are mainly divided into two types, one is animal sources, such as mouse jaw, bovine brain, etc. The other is to extract from the culture supernatant...

Method used

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  • Active protein peptide gene with excellent repairing effect and encoded protein peptide
  • Active protein peptide gene with excellent repairing effect and encoded protein peptide
  • Active protein peptide gene with excellent repairing effect and encoded protein peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Obtaining the target DNA

[0028] (1) The gene sequence of the target gene is:

[0029] Using the messenger RNA transcribed by 5'-GCAAGCGCATTGCACATGTACGGCTATAGCAGATA-3' (SEQ ID NO: 1) as a template, it is reverse-transcribed into complementary single-stranded DNA, and then synthesized into double-stranded DNA under the action of T4 DNA ligase, which is the target gene.

[0030] (2) PCR amplification

[0031] Denaturation: High temperature dissociates double-stranded DNA into single strands (94°C, 30s);

[0032] Annealing: The primer (pACT2 GAL4 AD pACT2-R) binds to the complementary region of the template DNA at low temperature to form a hybrid molecule (55°C, 30s);

[0033] Extension: moderate temperature extension, in DNA polymerase, dNTP, Mg 2+ In the presence, DNA polymerase catalyzes the extension of the DNA strand starting from the primer from 5' to 3', and synthesizes a DNA sub-strand complementary to the template DNA strand (70-72°C, 30-60s);

[0...

Embodiment 2

[0035] Embodiment 2: Electrophoresis detection and recovery purification

[0036] (1) Electrophoresis

[0037] ① Prepare an appropriate amount of buffer for electrophoresis and gel preparation (1X TAE Buffer)

[0038] ② Accurately weigh the agarose powder (1g) according to the amount of gel to be prepared and the concentration of the gel, and add it to an appropriate Erlenmeyer flask.

[0039] ③ Add a certain amount of electrophoresis buffer (1X TAE Buffer 100ml).

[0040] ④ Melt completely, cool to about 60°C, add EB5-7ul, and mix well.

[0041] ⑤ Pour the solution into the rubber mold, before inserting the comb in the appropriate position.

[0042] ⑥It solidifies at room temperature. If it is not used immediately, wrap the gel with plastic wrap and store it at 4°C.

[0043] Mix an appropriate amount of PCR product with an appropriate amount of bromophenol blue and add it to the gel tank, and add an appropriate amount of DNAMaker to the right tank to start electrophoresis...

Embodiment 3

[0049] Embodiment 3: Recombinant expression vector construction

[0050] The selected recombinant expression vector plasmid vector is pBR322, and the construction of the recombinant plasmid includes the following steps:

[0051] (1) Since one of the parents of the pBR322 plasmid is the pMB1 plasmid, at first based on pMB1, the Tn3 translocation of the Rldrd19 plasmid is introduced to obtain the pMB3 plasmid of 13.3kb;

[0052](2) The molecular weight of pMB3 obviously makes it unsuitable as a carrier, so most of the useless fragments must be lost by digestion under EcoRI active conditions, and the sticky ends of the remaining small fragments of DNA are connected to form pMB8 plasmid (2.6kb);

[0053] (3) At this time, another pSC101 plasmid was digested under EcoRI activity conditions to produce a DNA fragment containing tetr resistance, and this fragment was integrated with pMB8 to form pMB9 plasmid (5.3kb). At this time, pMB9 is ampstetr phenotype;

[0054] (4) pMB9 has p...

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Abstract

The invention provides an active protein peptide gene with an excellent repairing effect. The effective nucleotide sequence of the gene is shown as SEQ ID NO: 1; and the invention further provides an active protein peptide (EL for short) coded by the active protein peptide gene with an excellent repairing function. The amino acid sequence of the active protein peptide is shown as SEQ ID NO: 2. According to the invention, through an in-vitro gene protein recombination technology, a gene segment of the active protein peptide is grafted to a genetic information carrier and transplanted to a genetically engineered bacterium escherichia coli DH5a strain, the active peptide protein is prepared through density fermentation, protein separation and purification and freeze drying, and it can be ensured that the activity of the active peptide protein is not reduced for 3 years in a freeze-dried state. Cell experiments and human body clinical test experiments prove that the protein peptide has excellent effects on pox muscle repair and cosmetic medicine postoperation repair. The active peptide is relatively high in safety, and the safety is effectively authenticated through microorganism detection, heavy metal content detection and cell experiments; and the process is verified to be feasible, and batch production can be carried out.

Description

technical field [0001] The present invention relates to an active peptide suitable for the field of cosmetics, in particular to an active protein peptide gene with excellent repairing effect and its encoded protein peptide and its preparation. Background technique [0002] Well-known epidermal growth factor (Epidermal Growth Factor) is called for short EGF, is a kind of important cell growth factor of human endocrine, has very strong physiological activity. EGF was discovered by American scientist Dr. Stanley Cohen in 1986. EGF is a growth factor that exists in the human body, and its main function is to promote the division of skin cells. Studies have shown that a very small amount of EGF can strongly stimulate cell growth, inhibit the expression of aging genes, prevent skin aging, and keep the various components of the skin in the best physiological state. In addition, it can stimulate the synthesis and secretion of some extracellular macromolecules (such as hyaluronic a...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11C12N15/70A61K8/64A61Q19/00C12R1/19
CPCC07K7/06C12N15/70A61K8/64A61Q19/00A61K2800/85A61K2800/84Y02A50/30
Inventor 邹弼丞宋筱荭杨丹
Owner 四川省恩乐生物工程有限公司
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