US 12,473,350 B2
Recombinant humanized type III collagen microsphere with innovative spatial structure, and design, preparation process and usethereof
Yu Jin, Nanjing (CN); Lin Xu, Nanjing (CN); Nianqin Xu, Nanjing (CN); Jinbiao Lu, Nanjing (CN); Dongwu Zhang, Nanjing (CN); Songquan Xu, Nanjing (CN); Kaijin Chang, Nanjing (CN); Mouzhi Liu, Nanjing (CN); and Yuan Jiang, Nanjing (CN)
Assigned to Nanjing Tzone Biotechnology Co., Ltd., Nanjing (CN)
Filed by Nanjing Tzone Biotechnology Co., Ltd., Nanjing (CN)
Filed on Dec. 17, 2024, as Appl. No. 18/984,347.
Claims priority of application No. 202311780528.0 (CN), filed on Dec. 22, 2023.
Prior Publication US 2025/0206807 A1, Jun. 26, 2025
Int. Cl. C07K 14/78 (2006.01); C12N 15/70 (2006.01); C12P 21/02 (2006.01)
CPC C07K 14/78 (2013.01) [C12N 15/70 (2013.01); C12P 21/02 (2013.01)] 12 Claims
 
1. A process of preparing a recombinant humanized type III collagen microsphere with innovative spatial structure, comprising:
designing a gene sequence of humanized type III collagen as shown in SEQ NO: 3, and constructing an expression vector pET28a-MBP-His6-ENLYFQ;
inserting the designed gene sequence of humanized type III collagen into the expression vector to form a recombinant plasmid, and then introducing the recombinant plasmid into an engineering bacterium for fermentation expression; and
extracting a target protein expressed by the recombinant plasmid, treating with Tobacco Etch Virus (TEV) cutting enzyme, and cyclizing with Butelase ligase, and obtaining the recombinant humanized type III collagen microsphere with innovative spatial structure;
wherein the collagen microsphere is formed solely by cyclization of two peptide segments having amino acid sequences with 100% coverage with the amino acid sequence of natural human type III collagen; and the collagen microsphere has an amino acid sequence as shown in SEQ NO: 1, and is formed by cyclization of the two peptide segments by head-to-tail connection, each peptide segment having the amino acid sequence as shown in SEQ NO: 2.