Preparation method for biosynthesis of human body structural material
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Solution Overview
Problem
Current methods for obtaining type VII collagen, such as enzyme digestion and lentiviral transfection, face challenges including low content, complex extraction processes, animal-derived immune responses, and difficulties in achieving high-purity and precise control, limiting its application as a structural material.
Innovation Solution
A biosynthetic method involving functional region screening, large-scale biological fermentation, inducible expression, and purification of recombinant humanized type VII collagen, using a PET-28a-Trx-His expression vector in E. coli BL21 cells, followed by Ni affinity chromatography and ion exchange column purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If extraction from animal tissues is used, then type VII collagen can be obtained, but the extraction process is complicated and animal-derived immune responses occur
Solution Approach 1:
The patent uses recombinant DNA technology to copy the human type VII collagen gene into host cells (such as Chinese hamster ovary cells or human embryonic kidney cells), enabling the cells to produce humanized collagen proteins. This molecular copying approach replaces the complex animal tissue extraction process, eliminating immune responses while simplifying the production workflow.
Solution Approach 2:
The patent replaces the mechanical extraction process (enzymatic digestion, tissue homogenization, filtration) with a biological production system. By using gene expression vectors and cellular fermentation, the production mechanism shifts from mechanical/chemical extraction to biological synthesis, thereby simplifying the overall process and eliminating animal-derived immune issues.
2Object-affected harmful factors
If lentiviral transfection is used, then type VII collagen can be produced with lower immunogenicity, but the operation is difficult and target gene capacity is limited
Solution Approach 1:
The patent segments the collagen production system into distinct functional components: the type VII collagen gene (encoding the structural protein), a eukaryotic expression vector (providing transcriptional control), and host cells (providing translational machinery). This segmentation allows each component to be optimized independently, simplifying the overall operation while maintaining low immunogenicity through humanized protein production.
Solution Approach 2:
The patent changes the production parameters by using eukaryotic expression systems instead of viral transfection. By adjusting parameters such as host cell type (CHO cells, HEK293 cells), vector type (plasmid, adenovirus, or lentivirus with optimized promoters), and culture conditions, the system achieves both ease of operation and low immunogenicity simultaneously.
3Quantity of substance
If enzyme digestion is used, then type VII collagen derivatives can be extracted, but the collagen loses original biological activity
Solution Approach 1:
The patent performs preliminary action by expressing the complete, functional type VII collagen gene in host cells before production. The gene includes all necessary coding sequences and regulatory elements, ensuring that the synthesized protein maintains its native biological activity from the outset, eliminating the need for subsequent enzyme digestion that would compromise functionality.
Solution Approach 2:
The patent copies the complete functional sequence of human type VII collagen into the host cell's genetic material. This molecular copying ensures that the produced protein is identical to the native human collagen, preserving all biological activities such as cell adhesion, proliferation, and tissue repair functions, without requiring enzymatic processing that would degrade these activities.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method allows for the large-scale production of recombinant type VII humanized collagen with high activity in promoting cell adhesion and proliferation, without immune responses, and is applicable in various medical and cosmetic applications.
Implementation Method 1
large-scale biological fermentation
Implementation Method 2
inducible expression
Implementation Method 3
Ni affinity chromatography
Implementation Method 4
ion exchange column purification
Data Source
AI summary
The present application provides a preparation method for biosynthesis of a human body structural material. A polypeptide has the amino acid sequence of SEQ ID NO. 4, 5 or 6. Recombinant type-VII humanized collagen prepared in the present application has high activity of promoting cell proliferation and does not produce an immune response when applied to a human body, and the preparation method therefor is novel and can obtain the recombinant type-VII humanized collagen on a large scale, and is widely applied in the preparation of human body structural materials.