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Recombinant human fusion collagen as well as efficient hydroxylation method and application thereof

A collagen and hydroxylation technology, applied in the field of genetic engineering, can solve the problems of reporting and achieve high biological activity and good stability

Pending Publication Date: 2022-08-05
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] CN111087464A discloses a recombinant human type III collagen prepared by hydroxylation through the pACYCDute-1 plasmid induced by IPTG and simultaneously expressing the giant virus proline hydroxylase Hy726 in Escherichia coli; Amino acid hydroxylase and human collagen were constructed into a single plasmid Pkk223-3-TPH-COL3A1, and the expression of collagen and hydroxylase was induced at the same time when IPTG was added to E. coli, and the plasmids pPICZB-TPH and plasmids were constructed respectively pPIC9k-COL1A1 was integrated into the genome of Pichia pastoris GS115 for methanol induction and simultaneous co-expression to obtain hydroxylated recombinant collagen. The content of hydroxyproline in the above two methods was not reported, but the content of hydroxyproline in collagen and its structure Stability is closely related to cell receptor binding

Method used

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  • Recombinant human fusion collagen as well as efficient hydroxylation method and application thereof
  • Recombinant human fusion collagen as well as efficient hydroxylation method and application thereof
  • Recombinant human fusion collagen as well as efficient hydroxylation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] According to the amino acid sequence SEQ ID No. 1 of the recombinant human fusion collagen, according to the codon preference expressed by the host E. coli, the NdeI and BamHI restriction sites were avoided during the design process, and the gene sequence was optimized. The optimized gene sequence is SEQ ID As shown in No. 2, in the same way, the amino acid sequence of Bacillus anthracis hydroxylase (BaP4H) was reversely designed to obtain its gene sequence, and the two gene sequences were handed over to Shanghai Sangon Biological Co., Ltd. for full gene synthesis. The recombinant collagen gene sequence is directly connected between the restriction sites NdeI and BamHI of the expression vector pET28a to obtain the recombinant vector pET28a-rhCOL(I-III); the Bacillus anthracis hydroxylase (BaP4H) gene sequence is connected to the vector On pUC57, the recombinant vector pUC57-BaP4H was obtained.

[0074] Construction of recombinant expression vector pGro7-BaP4H, after ins...

Embodiment 2

[0096] Example 2: Co-expression and purification of recombinant human fusion collagen and proline hydroxylase in E. coli host

[0097] Co-expression conditions were optimized, including medium composition, induction temperature, inducer concentration, IPTG addition time, etc., and a large amount of expression was carried out under optimal conditions. The optimal expression conditions were as follows:

[0098] (1) Seed liquid preparation: pick a single colony on the transformation plate in Example 1 on the ultra-clean workbench and inoculate it in 10 mL of LB liquid medium (Cm + and Kan + ), cultured at 37°C, 220rpm in a constant temperature shaker for 10-12h as a seed solution.

[0099] (2) Co-expression: The above-mentioned seed solution was transferred to 3 shake flasks (Cm + and Kan + ), where no inducer was added to the No. 1 shake flask, and the No. 3 shake flask was added with an arabinose solution with a final concentration of 2 mg / mL before inoculation to induce BaP...

Embodiment 3

[0107] Example 3: Structure Detection of Recombinant Human Fusion Collagen

[0108] (1) Analysis of amino acid composition

[0109] The BCA method was used to measure the protein concentration after ultrafiltration and concentration using a protein concentration assay kit (Thermo Fisher). Take a 10 mg protein sample, add 5 mL of 6M hydrochloric acid into a hydrolysis tube, vacuumize and fill with nitrogen, and place at 110 °C The oven was hydrolyzed for 24h. After hydrolysis, the hydrolysis tube was cooled to room temperature, and ddH was used. 2 O dilute to 25mL, take 2mL nitrogen to dry (add a small amount of ddH 2 O was dried twice), and finally 1 mL of pH2.2, 0.02M hydrochloric acid buffer was added to resuspend, filtered with a 0.45 μm membrane filter, and detected by Hitachi L8900 amino acid automatic analyzer (Hitachi Co., Ltd., Japan).

[0110] Its amino acid analysis map such as Figure 5 As shown, the hydroxylation of recombinant collagen was successfully achieved...

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Abstract

The invention discloses recombinant human fusion collagen as well as an efficient hydroxylation method and application thereof. The acid sequence of the recombinant human fusion collagen is shown as SEQ ID No.1, and the cell activity of the recombinant human fusion collagen is higher than that of a single protein. The method comprises the following steps: constructing double-plasmid co-expression by hydroxylation of proline from bacillus anthracis and a human collagen gene, firstly inducing expression of proline hydroxylase from bacillus anthracis, and after a certain amount of intracellular hydroxylase is accumulated, inducing rapid expression of collagen under the control of a strong promoter, so as to obtain the proline hydroxylase. By regulating and controlling the expression time points of the enzyme and the collagen, the expression and synchronous efficient hydroxylation of the large-sub-quantity fusion collagen are realized, the hydroxylation rate reaches 63%, and the collagen with higher hydroxylation rate is better in stability and higher in biological property.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant human fusion collagen and an efficient hydroxylation method and application thereof. Background technique [0002] Collagen is the most abundant protein in the human body and other mammals, accounting for about 30% of the total protein in the human body. There are as many as 30 known collagen families. Collagen consists of its triple helix structure and unique repeats of Gly-Xaa-Yaa, where Xaa is usually proline and Yaa is hydroxyproline (Hyp). Hydroxyproline at the Yaa position stabilizes the triple helix structure of collagen and is extremely important in collagen structure. Type I, II and III collagens are the main components of collagen fibers, among which type I collagen is widely present in the dermis, bones, tendons and ligaments, and is assembled into a highly ordered fibrous tissue that supports the skin; type II collagen Protein us...

Claims

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Application Information

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IPC IPC(8): C07K14/78C12N15/12C12N15/70C12N15/53C12N1/21C12P21/02C07K1/22C07K1/18C07K1/34C07K1/36C12R1/19
CPCC07K14/78C12N15/70C12N9/0071C12P21/02C12Y114/11002Y02A50/30
Inventor 王菊芳刘苏马毅傅宏鑫王蒙
Owner SOUTH CHINA UNIV OF TECH
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