A modified recombinant human type III collagen mature peptide containing hydroxyproline, its preparation method and application

A technology of collagen and hydroxyproline, applied in biochemical equipment and methods, chemical instruments and methods, animal/human protein, etc., can solve the problems of natural collagen gap, lack of post-modification of proline, etc., and achieve high molecular weight , good biological activity, and the effect of expanding the scope of application

Active Publication Date: 2021-11-23
肽源(广州)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent CN103725623A claims to have obtained the mature peptide of type III collagen containing 1069 amino acids, and achieved secreted expression, but did not provide any evidence, not even the basic electrophoresis. In addition, proline, etc. also lack post-modification, There is a certain gap with natural collagen

Method used

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  • A modified recombinant human type III collagen mature peptide containing hydroxyproline, its preparation method and application
  • A modified recombinant human type III collagen mature peptide containing hydroxyproline, its preparation method and application
  • A modified recombinant human type III collagen mature peptide containing hydroxyproline, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of Pichia pastoris expression system containing proline hydroxylase gene

[0055] Using pPICZaA (purchased from Invitrogen) as the backbone, Pichia pastoris anthranilate synthase (Anthrailate synthase) gene was introduced to obtain the pPZSaA vector; then using the pPZSaA vector as the backbone, the P4HA2 and P4HB2 genes were respectively introduced into the multiple cloning sites, Obtain pPZSaA-P4HA2 and pPZSaA-P4HB2, and then connect the reading frames of the two through the BamHI / BglII restriction site to obtain the proline hydroxylase expression vector pPZAStA-P4HA2-P4HB2, and finally transform it into Pichia pastoris SMD1168 In this way, Pichia pastoris containing the proline hydroxylase gene was obtained. The detailed steps are as follows:

[0056] 1. Preparation of pPZSaA vector

[0057] (1) Utilize the Yeast Genome Purification Kit (purchased from TAKARA Company, product number 9082) to extract the Pichia pastoris genome, the steps are a...

Embodiment 2

[0089] Example 2 Expression of human type III collagen of different lengths in Pichia pastoris with proline hydroxylase gene

[0090] (1) The Pichia pastoris expression system containing the proline hydroxylase gene prepared in Example 1 is prepared into competent cells (the method is the same as in Example 1), and Pichia pastoris SMD1168 / pPZSaA-P4HA2-P4HB2 competent ;

[0091] (2) According to the protein resource database UniProt (URL https: / / www.uniprot.org / ) released the human type III collagen mature peptide sequence (P02461-1), combined with the codon preference of Pichia pastoris to optimize the design, and entrusted Shanghai Jierui Biotechnology Co., Ltd. to synthesize the Col3A1 gene, the corresponding amino acid sequence and nucleotide The sequences are as follows:

[0092] COL3A1 amino acid sequence:

[0093]QQEAVEGGCSHLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPNGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGP...

Embodiment 3

[0113] Example 3 Production Process of Recombinant Human Type III Collagen

[0114] (1) Take the strains SMD1168 / pPZSaA-P4HA2-P4HB2, pPIC9k-COL3a1(428..1172) preserved at -80°C for activation, dip the bacterial liquid with an inoculation loop and streak on the YPD solid plate; culture at 30°C For 2 days, pick a single colony in YPD liquid medium, culture at 30°C, 200rpm for about 24h, until OD 600 Reaching the range of 5-40, this is the first-class seed liquid, and the seed liquid can be adjusted according to the size of the culture volume.

[0115] (2) Inoculate the above-mentioned prepared seed solution into a 50L fermenter according to the aseptic inoculation method (the initial fermentation BSM medium is 30L, the conditions are: the temperature is 28°C, the dissolved oxygen is not less than 20%, and the fermenter is added by feeding 25% ammonia water control pH is 5.5 ± 0.5.After the glycerin is exhausted, continue to add the sterilized glycerin that mass volume ratio is ...

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Abstract

The invention relates to the technical field of biological genetic engineering recombination, in particular to a recombinant human type III collagen mature peptide modified with hydroxyproline and its preparation method and application. The invention provides a gene encoding a recombinant human type III collagen mature peptide, and combines the proline hydroxylase expression vector pPZAStA-P4HA2-P4HB2 with the nucleotides of the gene encoding a recombinant human type III collagen mature peptide The recombinant vector obtained by sequence ligation is transferred into the yeast expression system to obtain a yeast that can express the mature peptide of recombinant human type III collagen modified with hydroxyproline, and use the yeast to express the recombinant human type III modified with hydroxyproline Collagen mature peptide, the recombinant human type III collagen mature peptide has a large molecular weight, a high expression level, is closer to natural collagen, and exhibits good biological activity.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering recombination, in particular to a recombinant human type III collagen mature peptide modified with hydroxyproline and its preparation method and application. Background technique [0002] Collagen is the main structural protein of vertebrates, accounting for about 30% of the total protein. Collagen is composed of more than half of the extracellular matrix proteins in human tendon, cartilage, and skin. Collagen plays a role in protection, nutrition, transportation and so on for cells. The collagen in human skin is mainly type I and type III, and the content of type III collagen will gradually decrease with age, which is closely related to skin aging. [0003] The molecular weight of the collagen subunit is generally about 120kDa, which is much larger than the subunit of the general biologically active protein. The amino acid sequence contains a large number of Gly-X-Y (X, Y...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/81C12N1/19C07K14/78A61L26/00C12R1/84
CPCA61L26/0033A61L26/0085C07K14/78C12N15/815C08L89/00
Inventor 黄亚东张齐肖巧学杨艳周帅印赵文谢伟权
Owner 肽源(广州)生物科技有限公司
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