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CN-122012616-A - Method for producing active rhBMP9 bone repair material based on double-transgenic silkworm silk gland biosynthesis system, product and application thereof

CN122012616ACN 122012616 ACN122012616 ACN 122012616ACN-122012616-A

Abstract

The invention discloses a method for producing an active rhBMP9 bone repair material based on a double-transgenic silkworm silk gland biosynthesis system, a product and application thereof, realizes the cooperative expression of complex precursor protein Pro-mhBMP and precursor protein invertase hFurin in silkworm rear silk glands, establishes a hBMP9/hFurin double-gene biosynthesis system capable of completing precursor protein shearing, curing and functional silk material synthesis in vivo, further constructs an rhBMP9/CaP/Sericin composite scaffold by extracting silk protein of double-gene transformed silkworm, and provides a firm experiment and theoretical basis for a 'functional protein-material-tissue regeneration' integrated strategy based on a silk gland biosynthesis system.

Inventors

  • XIA QINGYOU
  • TIAN CHI
  • WANG YUANCHENG
  • HUANG GUANGCHENG
  • ZHAO PING

Assignees

  • 西南大学

Dates

Publication Date
20260512
Application Date
20260210

Claims (8)

  1. 1. The method for producing the active rhBMP9 bone repair material based on the double-transgenic silkworm silk gland biosynthesis system is characterized by comprising the following preparation steps: (1) Preparing a Pro-mhBMP transgenic silkworm strain, and integrating a nucleic acid sequence Pro-mhBMP9 encoding a humanized BMP9 precursor into the genome of the transgenic silkworm strain; (2) Preparing hFurin transgenic silkworm strain, and integrating nucleic acid sequence hFurin for coding human Furin protease in its genome, wherein the nucleotide sequence of hFurin is shown as SEQ ID NO. 2; (3) Crossing the Pro-mhBMP9 transgenic silkworm strain and the hFurin transgenic silkworm strain to obtain a double transgenic silkworm strain which can co-express Pro-mhBMP9 and hFurin in the rear silk gland of the silkworm, so that Pro-mhBMP9 is sheared into mature rhBMP9 dimer with biological activity in the silk gland; (4) Extracting fibroin containing active rhBMP9 from silkworm cocoons of double transgenic silkworm strains, and preparing the active rhBMP9 bone repair material.
  2. 2. The method of claim 1, wherein Pro-mhBMP is a silkworm codon optimized sequence as set forth in SEQ ID NO. 1.
  3. 3. The method according to claim 1, wherein the hFurin is a silkworm codon optimized sequence as shown in SEQ ID NO. 2.
  4. 4. The method of claim 1, wherein the Pro-mhBMP or hFurin is expressed under the control of the hr3 CQ enhancer, the FibH promoter and the Ser1pA terminator.
  5. 5. The method of claim 1, wherein in the step (4), the specific preparation method comprises the steps of firstly extracting Sericin containing active rhBMP9 from silkworm cocoons of a double transgenic silkworm strain by using a calcium salt solution, dialyzing by using deionized water, mixing with phosphate to induce and form a mineralized composite rhBMP 9/CaP/Sericin, then extracting the Sericin from the rest silk, and mixing with the composite rhBMP 9/CaP/Sericin after molding and freeze drying to obtain the active rhBMP9 bone repair material.
  6. 6. The method of claim 5, wherein the calcium salt is calcium chloride and the phosphate source is disodium hydrogen phosphate.
  7. 7. An active rhBMP9 bone repair material prepared by the method of any one of claims 1-6.
  8. 8. Use of the active rhBMP9 bone repair material of claim 7 in the manufacture of a medical device or biomaterial for promoting bone tissue regeneration and repairing bone defects.

Description

Method for producing active rhBMP9 bone repair material based on double-transgenic silkworm silk gland biosynthesis system, product and application thereof Technical Field The invention relates to the technical field of biotechnology and genetic engineering, in particular to a method for producing active rhBMP9 bone repair material based on a double-transgenic silkworm silk gland biosynthesis system, and also relates to the active rhBMP9 bone repair material prepared by the method and application of the active rhBMP9 bone repair material in preparation of medical instruments or biological materials for promoting bone tissue regeneration and repairing bone defects. Background Silkworm is an important economic insect in China, and silk gland thereof has extremely high protein synthesis and secretion capacity and is known as a natural protein processing plant. Along with rapid development of genetic engineering and synthetic biology technologies, silkworm silk glands are gradually evolving into a bioreactor with high plasticity and application potential, and the bioreactor is widely used for synthesizing various recombinant proteins and functional substances with biological activities. Meanwhile, the silk protein material has gained a great deal of attention in the fields of tissue repair and biological material engineering by virtue of excellent biocompatibility, mechanical property and processability. However, to meet the needs of diverse applications, continuous exploration and optimization is still needed in terms of functional modification of silk proteins and performance improvement of silk gland expression systems. Not all secreted proteins can directly achieve complete biological activity under high level expression conditions. Many growth factors and cytokines are initially synthesized in larger precursor forms and require hydrolytic cleavage by specific proteases to release the active mature domains. The post-translational processing not only determines the spatial conformation of the protein, but also directly affects its secretion efficiency and functional stability. Among them, human bone morphogenic proteins (hBMPs) are the most representative secreted protein family. In the case of hBMP9, which plays a key role in osteogenic differentiation and tissue regeneration, its precursor form must be cleaved by Furin enzyme to be converted into the biologically active dimeric mature protein. Because the endogenous expression level of Furin homologous genes in the rear silk gland of silkworms is extremely low, the expression of hBMP9 precursor alone is difficult to obtain a sufficient amount of active products, so that the exertion of the biological functions of the active products is limited. Although the silk gland of the family has strong protein synthesis capability, the problems of low expression efficiency of exogenous genes, limited post-translational processing mechanism, difficult stable maintenance of the activity of target functional proteins and the like commonly exist in the existing system, and the deep expansion of the silk protein in high-added-value application is severely restricted. Therefore, a silkworm exogenous protein biosynthesis system with an accurate post-translational processing function is established, and the silkworm exogenous protein biosynthesis system is a key technology break which needs to be overcome in order to realize innovation and leap of the silkworm industry. Disclosure of Invention In view of the above, the invention aims to provide a method for producing active rhBMP9 bone repair material based on double transgenic silk gland biosynthesis system, the second aim of the invention is to provide active rhBMP9 bone repair material prepared by the method, and the third aim of the invention is to provide application of the active rhBMP9 bone repair material in preparing medical equipment or biological material for promoting bone tissue regeneration and repairing bone defect. In order to achieve the above purpose, the present invention provides the following technical solutions: 1. a method for producing active rhBMP9 bone repair material based on double transgenic silk gland biosynthesis system comprises the following preparation steps: (1) Preparing a Pro-mhBMP transgenic silkworm strain, and integrating a nucleic acid sequence Pro-mhBMP9 encoding a humanized BMP9 precursor into the genome of the transgenic silkworm strain; (2) Preparing hFurin transgenic silkworm strain, and integrating nucleic acid sequence hFurin for coding human Furin protease in its genome, wherein the nucleotide sequence of hFurin is shown as SEQ ID NO. 2; (3) Crossing the Pro-mhBMP9 transgenic silkworm strain and the hFurin transgenic silkworm strain to obtain a double transgenic silkworm strain which can co-express Pro-mhBMP9 and hFurin in the rear silk gland of the silkworm, so that Pro-mhBMP9 is sheared into mature rhBMP9 dimer with biological activity in the silk gland; (4) Extracting fibr