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Peparation of human recombined blood-vessel generation element and skin whitening product

An angiogenin, skin whitening technology, applied in the direction of angiogenin, angiogenesis factors, animal/human protein, etc., can solve the problem that there are no cosmetics containing human angiogenin

Inactive Publication Date: 2003-06-11
上海福缘生化药学研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In short, so far, due to various technical and cost factors, there is no cosmetic product containing human angiopoietin.

Method used

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  • Peparation of human recombined blood-vessel generation element and skin whitening product
  • Peparation of human recombined blood-vessel generation element and skin whitening product
  • Peparation of human recombined blood-vessel generation element and skin whitening product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Gene expression of human angiopoietin

[0051] In this example, according to the amino acid sequence of human angiogenin, its coding nucleotide sequence was synthesized by chemical synthesis. Considering the specificity of strains used in genetic engineering, codons favored by Escherichia coli B21 cells were used in the process of designing coding nucleotides. The obtained human angiopoietin coding sequence is shown in SEQ ID NO: 1 and Figure 1.

[0052] Figure 1 lists the nucleotide sequences encoding human angiogenin used in the present invention. The nucleotide sequence used in the present invention is different from the human natural gene sequence encoding angiogenin (SEQ ID NO: 3 and Fig. 1). However, the amino acid sequences of their encoded proteins (SEQ ID NO: 2 and Fig. 1) are identical. Using the nucleotide sequence of angiogenin designed in the present invention, the highly expressed angiogenin can be obtained in bacteria by means of genetic engineering. ...

Embodiment 2

[0063] Biochemical Purification of Human Angiopoietin

[0064] In the embodiment, the angiogenin expressed by the genetic engineering technique in the embodiment 1 was purified by a biochemical method. This purification method, with proper modification, is also suitable for extracting angiogenin from other materials including human plasma, milk and tumor cell secretion.

[0065] (1) The inclusion body containing angiopoietin prepared by the above expression was dissolved with 7 equivalents of guanidine hydrochloride, and after further sonication, 2-mercaptoethanol (0.7% final concentration) was added and incubated at 37°C for 3 hours to make the disulfide bond completely Open.

[0066](2) Slowly add the guanidine hydrochloride solution of angiopoietin with the above-mentioned peptide chain fully extended, dilute it and add it to 100 times the volume of the peptide chain refolding solution (50mM Tris-HCl, 100mM NaCl, pH8.5), statically at 4°C Leave to insulate for 8 hours, th...

Embodiment 3

[0077] Angiogenin activity assay

[0078] In this example, the inhibitory effect of the angiogenin obtained in Example 2 on melanin secretion by normal human melanocytes was tested.

[0079] (1) Normal human melanocytes were purchased from Clonetics Company in the United States and cultured in a special medium provided by the company. The culture conditions are constant temperature and humidity at 37°C and 5% carbon dioxide gas phase. The medium was changed every two days, and the culture medium was subcultured at a ratio of 1:3.

[0080] (2) The adherent melanocytes were digested with trypsin-EDTA, the cells were collected by centrifugation, suspended in complete medium, and seeded in a 96-well cell culture plate at a density of 30,000 cells per well.

[0081] (3) Add different concentrations of samples to be tested (human angiogenin or bovine angiogenin at different concentrations) to each well, and incubate at 37° C. under the above culture conditions.

[0082] (4) Measu...

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Abstract

The present invention provides a skin-whitening product which contains human angiogenin with effective concentration to inhibit the production of melanose of skin, said effective concentration is 0.000001% to 0.1% of total weight. Said product can be used for removing freckle, black spot, aged-mark, melanotic nevus and dermal abnormal change due to melanosis, and / or can be used for eliminating wrinkles, raising skin elasticity and preventing dermal senility.

Description

technical field [0001] The present invention relates to the field of cosmetics. More particularly, the present invention relates to a skin whitening product containing human angiogenin to suppress the production of skin melanin. The invention also relates to novel coding sequences and recombinant expression of human angiogenin. Background technique [0002] Human angiogenin (Angiogenin) is a protein consisting of 123 amino acids [Fett JW, Strydom DJ, Lobb RR, Alderman EM, Bethune JL, Riordan JF, Vallee BL (1985): Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 24:5480-6]. Angiopoietin exists in the blood of normal people, and its content is generally 250-360ng / ml [Blaser J, Triebel S, Kopp C, Tschesche H (1993): A highly sensitive immunoenzymometric assay for the determination of angiogenin. European Journal of C1inical Chemistry & Clinical Biochemistry 31:513-6]. [0003] The main physiological function of ang...

Claims

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Application Information

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IPC IPC(8): C07K14/515C12N15/12
Inventor 胡国富
Owner 上海福缘生化药学研发有限公司
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