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CN-122011206-A - Heat-resistant stable recombinant humanized V-type collagen and preparation method thereof

CN122011206ACN 122011206 ACN122011206 ACN 122011206ACN-122011206-A

Abstract

The embodiment of the application discloses a heat-resistant stable recombinant humanized V-type collagen and a preparation method thereof, relates to the technical field of recombinant proteins, and aims to solve the technical problem that the existing collagen is difficult to meet application requirements. The preparation method of the heat-resistant stable recombinant humanized V-type collagen comprises the following steps of respectively obtaining recombinant collagen with gene sequences shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6, respectively inserting the recombinant collagen into pPIC9K plasmids to obtain corresponding recombinant plasmids, wherein the recombinant plasmids comprise recombinant plasmids pPIC9K-ColV α1-1, recombinant plasmids pPIC9K-ColV α1-2 and recombinant plasmids pPIC9K-ColV α1-4, and sequentially performing electric transformation treatment, screening treatment, identification treatment and purification treatment on the recombinant plasmids to obtain recombinant V-type collagen α1, and the recombinant V-type collagen α1 comprises recombinant V-type collagen α1-1, recombinant V-type collagen α1-2 and recombinant V-type collagen α1-4.

Inventors

  • ZHAO JUN
  • XIA BINGBING
  • XU GAOTAO
  • XU ZHIYONG
  • Xiang Zongjing
  • ZHOU WEI
  • WU BO
  • ZHANG YONG

Assignees

  • 英特菲尔(成都)生物制品有限责任公司

Dates

Publication Date
20260512
Application Date
20251231

Claims (10)

  1. 1. The preparation method of the heat-resistant stable recombinant humanized V-type collagen is characterized by comprising the following steps of: recombinant collagen with gene sequences shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6 is respectively obtained, and the recombinant collagen is respectively inserted into pPIC9K plasmids to obtain corresponding recombinant plasmids, wherein the recombinant plasmids comprise recombinant plasmids pPIC9K-ColV alpha 1-1, recombinant plasmids pPIC9K-ColV alpha 1-2 and recombinant plasmids pPIC9K-ColV alpha 1-4; And carrying out electrotransformation treatment, screening treatment, identification treatment and purification treatment on the recombinant plasmid in sequence to obtain recombinant V-type collagen alpha 1, wherein the recombinant V-type collagen alpha 1 comprises recombinant V-type collagen alpha 1-1, recombinant V-type collagen alpha 1-2 and recombinant V-type collagen alpha 1-4.
  2. 2. The method for preparing heat-resistant stable recombinant humanized V-type collagen according to claim 1, wherein the recombinant collagen having the gene sequence shown in SEQ ID No.2 is obtained based on the target amino acid sequence shown in SEQ ID No. 1; The recombinant V-type collagen with the gene sequence shown as SEQ ID No.4 is obtained based on the target amino acid sequence shown as SEQ ID No. 3; the recombinant V-type collagen with the gene sequence shown as SEQ ID No.6 is obtained based on the target amino acid sequence shown as SEQ ID No. 5.
  3. 3. The method for preparing heat-resistant stable recombinant humanized V-type collagen according to claim 2, wherein the target amino acid sequence shown in SEQ ID No.1 is 643 th to 730 th gene sequences in a gene sequence of a recombinant expression ColV alpha 1 chain, and comprises two RGD domains; the target amino acid sequence shown as SEQ ID No.3 is obtained by repeating the target amino acid sequence shown as SEQ ID No.1 for 2 times in series; The target amino acid sequence shown as SEQ ID No.5 is obtained by repeating the target amino acid sequence shown as SEQ ID No.1 for 4 times in series.
  4. 4. A method for the preparation of heat resistant stable recombinant humanized V-type collagen according to any one of claims 1-3, wherein the electrotransformation process comprises the steps of: and (3) carrying out linearization treatment on the recombinant plasmid by using a restriction enzyme SacI, electrically converting the recombinant plasmid into GS115 competent cells, coating the cell on an MD solid plate, standing for 10min at room temperature, and culturing the cell in a 30 ℃ incubator for 2-5 days in an inverted manner until single colonies appear.
  5. 5. The method for preparing heat resistant stabilized recombinant humanized V-type collagen according to claim 4, wherein the screening process comprises the steps of: the single colonies were inoculated onto YPD solid plates containing 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL and 4mg/mL G418, respectively; single colonies capable of growing simultaneously on YPD solid plates containing 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL and 4mg/mL G418 were obtained as high copy recombinant strains by screening.
  6. 6. The method for preparing heat resistant stabilized recombinant humanized V-type collagen according to claim 5, wherein the identifying process comprises the steps of: 4 high-copy recombinant strains are selected and inoculated in a BMGY culture medium, and 24h is cultured at 30 ℃ and 220 rpm until the OD 600 value is 2-6; According to the measured OD 600 value, the volume of BMGY culture bacteria liquid is adjusted, 3000 rpm and 10min are collected for bacteria re-suspension treatment, and the initial OD 600 value is 2.0; continuously culturing at 30 ℃ and 220 rpm, adding 0.5% methanol into the culture medium every 24: 24h, respectively taking bacterial liquid samples after induction of the methanol for 72 hours, and centrifugally collecting expression supernatant; And analyzing the protein expression condition in the supernatant of the recombinant strain by SDS-PAGE electrophoresis to obtain the target protein.
  7. 7. The method for preparing heat resistant stabilized recombinant humanized V-type collagen according to claim 6, wherein the purification process comprises the steps of: using a strong anion exchange medium to balance a chromatographic column with phosphate flushing until the conductivity value and the A280 light absorption value are unchanged, setting the sample loading flow rate to 5mL/min, detecting the ultraviolet A280 light absorption value, and starting sample receiving when the ultraviolet A280 light absorption value rises; after the sample loading is finished, balancing the chromatographic medium by using phosphate solution until the absorbance value and the conductivity value of ultraviolet A280 are reduced to the minimum and are not changed any more, and finishing the balancing; And eluting with phosphate buffer solution containing NaCl and collecting the corresponding protein to obtain the recombinant V-type collagen.
  8. 8. A heat-resistant stable recombinant humanized V-type collagen prepared by the method of any one of claims 1-7, wherein the heat-resistant stable recombinant humanized V-type collagen comprises recombinant V-type collagen α1-1, recombinant V-type collagen α1-2, and recombinant V-type collagen α1-4.
  9. 9. The thermostable recombinant humanized V-type collagen according to claim 8, wherein the Hacat cell adhesion promoting activity of the recombinant V-type collagen α1-1 is 1.10x 4 U/mg; The adhesion activity of the recombinant V-type collagen alpha 1-2 on Hacat cells is 1.27 multiplied by 10 5 U/mg; The Hacat cell adhesion promoting activity of the recombinant V-type collagen alpha 1-4 is 9.58 multiplied by 10 5 U/mg.
  10. 10. The thermostable recombinant humanized V-type collagen according to claim 8, wherein the proliferation promoting activity of the recombinant V-type collagen α1-1 for NIH 3T3 cells is 1.43 x 10 4 U/mg; The proliferation activity of the recombinant V-type collagen alpha 1-2 for promoting NIH 3T3 cells is 2.34 multiplied by 10 4 U/mg; The proliferation activity of the recombinant type V collagen alpha 1-4 for promoting NIH 3T3 cells is 9.3810 4 U/mg.

Description

Heat-resistant stable recombinant humanized V-type collagen and preparation method thereof Technical Field The application relates to the technical field of recombinant proteins, in particular to a heat-resistant stable recombinant humanized V-type collagen and a preparation method thereof. Background Collagen, a group of protein families widely distributed in animal connective tissue, plays an important role in maintaining normal physiological functions of cells, tissues and organs and repairing injury. Collagen is composed of polysaccharide protein molecules, mainly present in the skin, bones, teeth, tendons, ligaments, blood vessels, etc. of animals, and is a major protein component of connective tissue, whose function is to maintain the structure and morphology of the skin and various organs. Type I collagen and type III collagen have been widely used in the skin care field, and type V collagen is a key component ‌ for maintaining skin elasticity, and it acts synergistically with type I collagen and type III collagen, and has remarkable effects on skin fineness, firmness and repairability. ‌ the existing extraction method is poor in water solubility and purity of the V-type collagen extracted from animal source tissues, and is difficult to meet the application requirements of the V-type collagen in the biomedical field. In addition, natural extraction of collagen is difficult to avoid infection and rejection risk of infectious viruses of people and livestock, and application of collagen in medicine, biomedical materials or medicine carriers is greatly limited. The recombinant V-type collagen obtained by the existing biosynthesis method generally faces the industrial problem of poor thermal stability, is difficult to store stably for a long time in liquid preparations such as water cream and the like, and limits the application of the recombinant V-type collagen in the fields of skin care products, skin dressings and the like. Disclosure of Invention The application mainly aims to provide a heat-resistant stable recombinant humanized V-type collagen and a preparation method thereof, and aims to solve the technical problem that the existing collagen is difficult to meet application requirements. In order to achieve the above object, the technical scheme adopted by the embodiment of the application is as follows: in a first aspect, an embodiment of the present application provides a method for preparing a heat-resistant stable recombinant humanized V-type collagen, comprising the steps of: recombinant collagen with gene sequences shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6 is respectively obtained, and the recombinant collagen is respectively inserted into pPIC9K plasmids to obtain corresponding recombinant plasmids, wherein the recombinant plasmids comprise recombinant plasmids pPIC9K-ColV alpha 1-1, recombinant plasmids pPIC9K-ColV alpha 1-2 and recombinant plasmids pPIC9K-ColV alpha 1-4; And carrying out electrotransformation treatment, screening treatment, identification treatment and purification treatment on the recombinant plasmid in sequence to obtain recombinant V-type collagen alpha 1, wherein the recombinant V-type collagen alpha 1 comprises recombinant V-type collagen alpha 1-1, recombinant V-type collagen alpha 1-2 and recombinant V-type collagen alpha 1-4. As some alternative embodiments of the application, the recombinant collagen with the gene sequence shown in SEQ ID No.2 is obtained based on the target amino acid sequence shown in SEQ ID No. 1; The recombinant V-type collagen with the gene sequence shown as SEQ ID No.4 is obtained based on the target amino acid sequence shown as SEQ ID No. 3; the recombinant V-type collagen with the gene sequence shown as SEQ ID No.6 is obtained based on the target amino acid sequence shown as SEQ ID No. 5. As some optional embodiments of the application, the target amino acid sequence shown as SEQ ID No.1 is 643 rd to 730 th gene sequence in the gene sequence of the recombinant expression ColV alpha 1 chain, and comprises two RGD domains; the target amino acid sequence shown as SEQ ID No.3 is obtained by repeating the target amino acid sequence shown as SEQ ID No.1 for 2 times in series; The target amino acid sequence shown as SEQ ID No.5 is obtained by repeating the target amino acid sequence shown as SEQ ID No.1 for 4 times in series. As some optional embodiments of the present application, the electrotransformation process comprises the steps of: and (3) carrying out linearization treatment on the recombinant plasmid by using a restriction enzyme SacI, electrically converting the recombinant plasmid into GS115 competent cells, coating the cell on an MD solid plate, standing for 10min at room temperature, and culturing the cell in a 30 ℃ incubator for 2-5 days in an inverted manner until single colonies appear. As some optional embodiments of the present application, the screening process includes the steps of: the single colonies were inoculated