CN117903287B

CN117903287B - Recombinant type IV collagen with antioxidant activity and application thereof

Info

Publication number: CN117903287B
Application number: CN202410077995.5A
Authority: CN
Inventors: 范代娣; 张疆睿; 宇文伟刚; 畅迎雪; 刘琳
Original assignee: Xi'an Giant Biogene Technology Co ltd
Current assignee: Xi'an Giant Biogene Technology Co ltd
Priority date: 2024-01-18
Filing date: 2024-01-18
Publication date: 2025-01-28
Anticipated expiration: 2044-01-18
Also published as: CN117903287A

Abstract

本申请公开了一种具有抗氧化活性的重组人IV型胶原蛋白及其应用。所述具有抗氧化活性的重组人IV型胶原蛋白是人IV型胶原蛋白的截短蛋白，其氨基酸序列如SEQ ID NO：1所示。所述具有抗氧化活性的重组IV型胶原蛋白可在毕赤酵母表达系统中表达且表达产量高，而且其生物学活性显著优于天然IV型胶原蛋白。The present application discloses a recombinant human type IV collagen with antioxidant activity and its application. The recombinant human type IV collagen with antioxidant activity is a truncated protein of human type IV collagen, and its amino acid sequence is shown in SEQ ID NO: 1. The recombinant type IV collagen with antioxidant activity can be expressed in a Pichia pastoris expression system with high expression yield, and its biological activity is significantly better than that of natural type IV collagen.

Description

Recombinant type IV collagen with antioxidant activity and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and relates to recombinant IV type collagen with antioxidant activity, a preparation method and application thereof.

Background

The natural antioxidant is a bioactive protein with effects of scavenging free radicals, inhibiting lipid peroxidation and chelating metal ions, and has antioxidant and antiaging effects, and can be used for preventing and treating diseases, delaying aging and prolonging food shelf life. The natural antioxidant has wide application prospect in medicine, cosmetics, health care products, food and feed additives and the like, and has high safety and effect compared with the additive which is industrially synthesized in food, medicines and cosmetics and feed on the market, so that the natural antioxidant becomes a current international hot research topic, and the screening of natural resources with strong antioxidant activity becomes a new trend of food science, biology, medicine and other researches.

Type IV collagen is an important collagen family member, which is widely present in tissue matrix and plays an important role in the formation of extracellular matrix and the regulation of cellular functions. Type IV collagen (IV-C) is the major collagen found in the extracellular basement membrane separating various epithelial and endothelial cells, and is the major component of the dermis-epidermis junction, mainly present in the dense layer.

Conventionally, type IV collagen is isolated from human placenta and purified using a multi-step process, and the collagen extraction method has certain limitations such as low extraction efficiency and low purity. On the other hand, many methods have been tried by those skilled in the art to recombinantly express type IV collagen in prokaryotic or eukaryotic expression systems, but unfortunately suffer from the problems that (1) recombinant expression of the full length of type IV collagen has not been successful and (2) some truncated proteins of type IV collagen are recombinantly expressed but have low yields and/or have much poorer biological activity than the full length of type IV collagen and are therefore not suitable for industrial scale production.

Disclosure of Invention

In view of the technical problems in the prior art, the invention aims to provide recombinant type IV collagen which has high recombinant expression yield in Pichia pastoris and biological activity remarkably superior to that of natural type IV collagen, and a preparation method and application thereof.

Namely, the present invention includes:

1. a recombinant human IV type collagen with antioxidant activity is a truncated protein of human IV type collagen, and the amino acid sequence of the truncated protein is shown as SEQ ID NO. 1.

2. Nucleic acid encoding the recombinant human type I collagen of item 1.

3. An expression vector comprising the nucleic acid of item 2.

4. A host cell into which the expression vector according to item 3 has been introduced. The host cell may be E.coli, pichia pastoris, bacillus subtilis, preferably Pichia pastoris.

5. A method for producing the recombinant human type I collagen according to claim 1, comprising the steps of culturing the host cell according to claim 4 and collecting the cell culture.

6. The method according to item 5, further comprising the step of subjecting the cell culture to separation and purification using one or a combination of several of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, membrane separation, preferably a combination of ion exchange chromatography and hydrophobic chromatography.

7. A product comprising the recombinant human type IV collagen according to claim 1, which is a cosmetic, health product or medical device.

8. The use of the recombinant human type IV collagen according to item 1 in the preparation of cosmetics, health products or medical devices.

The recombinant type IV collagen can be expressed in a Pichia pastoris expression system, the expression yield is high, and the biological activity (antioxidant activity and cell adhesion promoting activity) of the recombinant type IV collagen which is tested is obviously superior to that of the natural type IV collagen.

Drawings

FIG. 1 is a SDS-PAGE analysis of recombinant type IV collagen prepared in the example after expression purification in X-33.

Detailed Description

EXAMPLE 1 construction and expression of recombinant human type IV collagen

(1) Construction of recombinant human type IV collagen bacterium

1) Construction of recombinant plasmids

According to the amino acid sequences shown in SEQ ID No. 1, 2 and 3, codon optimization of a yeast expression system is carried out to obtain a target gene sequence, the obtained target gene sequence is entrusted to the gene synthesis of the engineering, inc. of the department of Prinsepia, and the synthesized gene is connected into pPicZ alpha A plasmid to obtain pPicZ alpha A-IV plasmid.

2) Construction of Yeast expression Strain

And linearizing pPicZ alpha A-IV with Pme I, then converting the competent pichia pastoris X-33, and screening the transformant by taking bleomycin resistance as a screening mark to obtain the yeast expression strain.

(2) Inducible expression of a protein of interest

1) Single colony of the constructed yeast expression strain is selected and added into 5ml YPD liquid culture medium (1% yeast extract, 2% peptone and 2% glucose), and is cultured for 48 hours at 30 ℃ and 200rpm for activation;

2) Transferring 500ml triangular flask (200 ml, YPD culture solution) with 1% inoculum size, culturing at 30deg.C and 200rpm for 24 hr, and taking as seed for upper tank;

3) Preparing 3L of BSM culture medium, adding the BSM culture medium into a 5L fermentation tank, sterilizing for 20min at 121 ℃, cooling to 30 ℃, adjusting the pH to 5.0, adding the upper tank seeds prepared in the step (2) into the fermentation tank in a flame inoculation mode, and performing fermentation culture;

4) Culturing until OD600 is 70, starting to supplement 50% of glycerol until OD600 reaches about 120, stopping supplementing, and starting to add methanol for induction when dissolved oxygen rebounds to 100%;

Controlling dissolved oxygen not lower than 30% in the induction process, controlling pH to be about 5.0, inducing for 40h, ending fermentation, taking culture solution, centrifuging for 2min under 12000rpm, taking supernatant, and detecting protein yield by adopting an SDS-PAGE method.

The measurement results are shown in Table 1 below

TABLE 1 detection results after fermentation of recombinant type IV collagen

The results in Table 1 show that the yield of the collagen of IV-1 reaches 15g/L, the recombinant expression quantity is high, and the requirements of fermentation production can be met.

EXAMPLE 2 purification of recombinant human type IV collagen

And (3) collecting the obtained engineering bacteria fermentation liquor, separating the bacteria liquor from the bacteria by using a Siemens flier table type centrifuge, and collecting supernatant. According to the characteristics of the protein, 20mM/L potassium phosphate buffer (solution A, pH 6.0) was prepared, and 20mM/L potassium phosphate buffer+1 mol/L NaCl (solution B, pH 6.0) was used as the eluent. And (3) regulating the pH value of the collected supernatant, filtering, loading the supernatant onto a hydrophobic cation exchange chromatographic column, balancing the column by using the solution A before loading, washing impurities by using the solution B with 20% after loading, and eluting by using the solution B to obtain the target protein. SDS-PAGE electrophoresis of purified recombinant human IV-type collagen with SEQ ID No. 1, 2 and 3 is shown in FIG. 1.

EXAMPLE 3 determination of antioxidant Activity of recombinant type IV collagen

DPPH radical scavenging Activity assay

DPPH is dissolved in 95% ethanol to prepare DPPH solution with final concentration of 0.1 mmo/L. The antioxidant recombinant type IV collagen, GSH and natural full-length human type IV collagen (commercially available, obtained by an extraction method) prepared in example 2 were prepared into 1mg/mL with PBS solution, 2mL and 2mL DPPH solution were respectively mixed uniformly, left at room temperature in a dark place for 30min, and the absorbance at 517nm was detected and recorded as Ai. The absorbance measured at 517nm after 2mL of 95% ethanol+2 mL DPPH solution was mixed uniformly was used as a control and was designated as Ac. The absorbance at 517nm was measured after mixing 2mL of 95% ethanol and 2mL of antioxidant recombinant type IV collagen (1 mg/mL) uniformly and designated Aj.

The clearance of the recombinant type IV collagen to DPPH radicals was calculated according to the following formula:

DPPH radical clearance (%) = [1- (Ai-Aj)/Ac ] X100%.

Determination of ABTS radical scavenging Activity

Preparing a mixed solution of ABTS (7 mmol/L) and potassium persulfate (2.45 mmol/L) by using ultrapure water, and incubating for 12-16h at room temperature in a dark place to prepare the ABTS free radical cation-based liquid. Diluting the ABTS free radical cation radical working solution with ultrapure water to ensure that the absorbance value measured at 734nm is 0.700+/-0.050, thereby obtaining the ABTS free radical cation working solution. The antioxidant recombinant type IV collagen, GSH and natural full-length human type IV collagen (commercially available, obtained by an extraction method) prepared in example 2 were prepared into 1mg/mL by PBS solution, 0.1mL and 3.9mL of ABTS free radical cation working solution were respectively mixed uniformly, and then placed at room temperature in a dark place for 6min, and the absorbance at 734nm was measured and recorded as Ai. The absorbance measured at 734nm after 0.1mL of ultrapure water+3.9 mL of ABTS radical cationic working solution was mixed uniformly was used as a control and was designated as Ac. The absorbance at 734nm was measured after mixing well 0.1mL of ultrapure water and 3.9mL of antioxidant recombinant type IV collagen (1 mg/mL), and was recorded as Aj.

The clearance of the antioxidant recombinant type IV collagen to ABTS free radicals was calculated according to the following formula:

ABTS radical clearance (%) = [1- (Ai-Aj)/Ac ] X100%.

The measurement results are shown in Table 2 below

TABLE 2 antioxidant Activity of recombinant type IV collagen

The results in Table 1 show that recombinant human collagen IV-1 has good antioxidant activity, and is obviously superior to natural human type IV collagen and other truncated proteins, and even is obviously superior to the accepted strong antioxidant substance GSH.

EXAMPLE 4 determination of the cell adhesion promoting Effect of recombinant type IV collagen

Normal culture of HSF (human skin fibroblast) cells, taking a lyophilized product after purification of recombinant human IV type collagen and a control natural full-length human IV type collagen (commercially available and obtained by an extraction method), preparing into 0.25mg/L, 0.5mg/L and 1mg/L by using PBS solution, and respectively acting on HSF (human skin fibroblast) cells to carry out cell adhesion promotion experiments. The method comprises the specific steps of inoculating human HSF cells into a sterile 96-well plate, adding 200 mu L of culture medium into each well, adding PBS into a blank control group, standing at 37 ℃ for 2 hours, washing each group with PBS for 3 times, washing off non-adhered cells, adding 100 mu L of culture solution and 50 mu L of MTT solution into each well, shaking uniformly, placing into an incubator for incubation for 4 hours, sucking out the mixed solution, adding 150 mu L of DMSO to dissolve purple crystal formazan, shaking for 10 minutes, placing into an enzyme-labeled instrument, detecting the wavelength at 570nm, and measuring the absorbance value of each well.

The cell adhesion data of recombinant human type IV collagen and native full length human type IV collagen (commercially available) for HSF (human skin fibroblasts) are shown in table 3, wherein,

Cell adhesion rate= (experimental OD-blank OD) ×100%/blank.

TABLE 3 adhesion promotion values of adhesion promotion of recombinant collagen at different concentrations to skin fibroblasts

The results in Table 3 show that recombinant human collagen IV-2 can obviously promote the adherence of HSF cells, and the adhesion promotion effect is obviously better than other recombinant type IV collagen, even better than natural type IV collagen.

Sequence information

SEQIDN01:

GPPGPPGIVIGTGPLGEKGERGYPGTPGPRGEPGPKGFPGLPGQPGPPGLPVPGQAGAPG FPGERGEKGDRGFPGTSLPGPSGRDGLPGPPGSPGPPGQP GYTNGIVECQPGPPGDQGPP GIPGQPGFIGEIGEKGQKGESCLICDIDGYRGPPGPQGPPGEIGFPGQPGAKGDRGLPGR DGVAGVPGPQGTPGLIGQPGAKGEPGEFYF DLRLKGDKGDPGFPGQPGMPGRAGSPG RDGHPGLPGPKGSPGSVGLKGERGPPGGVGFPGSRGDTGPPGPP

SEQIDN0.2:

GLPGLPGQQGAPGIPGFPGSKGEMGVMGTPGQPGSPGPVGAPGLPGEKGDHGFPGSSGPR GDPGLKGDKGDVGLPGKPGSMDKVDMGSMKGQKGDQGEKGQIGPIGEKGSRGDPGTPG VPGKDGQAGQPG QPGPKGDPGI SGTPGAPGLPGPKGSVGGMG LPGTPGEKGVPGIPGPQ GSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGEKGEKGSIGIPGMPGSP GLKGSPGSVGY PGSPG

SEQIDN0.3:

GIPGSKGEQG FMGPPGPQGQ PGLPGSPGHATEGPKGDRGP QGQPGLPGLP GPMGPPGLPG IDGVKGDKGN PGWPGAPGVP GPKGDPGFQG MPGIGGSPGI TGSKGDMGPP GVPGFQGPKG LPGLQGIKGD QGDHGVPGAK GLPGPPGPPG PYDIIKGEPG LPGPEGPPGL KGLQGLPGPK GQQGVTGLVG IPGPPGIPGF DGAPGQKGEM GPAGPTGPRG FPGPPGPDGL PGSMGPPGTP SVDHGFLVTR HSQ

Claims

1. A recombinant human type IV collagen with antioxidant activity, which is a truncated protein of human type IV collagen, and its amino acid sequence is shown in SEQ ID NO: 1.

2. A nucleic acid encoding the recombinant human type IV collagen according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced.

5. A method for producing the recombinant human type IV collagen according to claim 1, comprising the steps of culturing the host cell according to claim 4 and collecting the cell culture.

6. The method according to claim 5, further comprising the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation.

7. A product comprising the recombinant human type IV collagen according to claim 1, wherein the product is a cosmetic, a health product or a medical device.

8. Use of the recombinant human type IV collagen according to claim 1 in the preparation of cosmetics, health products or medical devices.