A recombinant human type I collagen fragment COLI-23H and its application

Abstract

The present invention provides a recombinant human type I collagen fragment COLI-23H, the amino acid sequence of which is shown in SEQ ID NO.3. The present invention also provides a coding gene for the above-mentioned protein COLI-23H, a Pichia pastoris engineered bacterium comprising the above-mentioned gene, and a fermentation process of the engineered bacterium. The present invention screened a sequence fragment in the original human type I collagen sequence, optimized the nucleotide sequence of the fragment to make it more suitable for the Pichia pastoris expression system, obtained a high-yield strain of recombinant type I collagen, and optimized its fermentation process. The recombinant human type I collagen fragment COLI-23H of the present invention has excellent biological activity, can be recombinantly expressed, and is high-yielding. At the same time, the present invention also provides the application of the above-mentioned recombinant human type I collagen COLI-23H in wound repair, medical cosmetology and plastic surgery, and cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant human type I collagen fragment COLI-23H and an application thereof. Background Art

[0002] Type I collagen is the most abundant component in dermal connective tissue, accounting for more than 80% of the skin. Type I collagen molecules are long and tough right-handed triple helical structures, and can spontaneously assemble into collagen fibers, which are widely distributed in the extracellular matrix. In addition to structural characteristics, there are also multiple functional binding sites in human type I collagen, such as the GER site where positive and negative charges are adjacently arranged for recognition and binding with extracellular integrins. The GER site is a specific sequence in collagen, and its full name is glycine-glutamic acid-arginine. This sequence plays an important role in collagen, especially in promoting cell proliferation and adhesion. In addition, the GER site is also characterized by high biocompatibility and low immunogenicity. These characteristics make collagen containing GER sites have a wide range of application value in the fields of biomedicine and cosmetics.

[0003] At present, the production of type I collagen is mainly obtained through animal tissue extraction, such as extracting type I collagen from the cartilage of pigs, cattle, sheep and other animals. However, collagen from animals has strong antigenicity, serious rejection reactions in the body, and potential safety hazards such as viruses and infectious diseases. Recombinant collagen is a protein obtained by cloning human collagen genes into selected expression vectors and transforming them into expression cells, and finally through purification technology. Due to its single molecule, clear structure and easy control, recombinant collagen has become the best substitute for animal collagen in biomedicine and tissue engineering.

[0004] Yang Jing and others used Escherichia coli to produce recombinant human type I collagen, with a yield of 0.52 g / L, and it can effectively promote the proliferation of mouse fibroblast 3T3 cells. However, collagen produced by Escherichia coli as an engineered bacterium lacks hydroxylation. The yeast expression system is a popular eukaryotic system in recent years, with high safety, low fermentation cost and high yield. Northwest University fused the human collagen fragment hCOL1A1 with the insulin gene and achieved a yield of 0.30 g / L using Pichia pastoris shake flask fermentation, but for the huge market demand, the output of recombinant collagen needs to be further improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant human type I collagen fragment COLI-23H which has biological activity, can be recombinantly expressed and has high yield, and its application.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A recombinant human type I collagen fragment COLI-23H, whose amino acid sequence is shown in SEQ ID NO.3.

[0008] As one of the preferred embodiments of the present invention, the recombinant human type I collagen fragment COLI-23H contains two "GER integrin recognition sites".

[0009] A gene encoding the above-mentioned recombinant human type I collagen fragment COLI-23H, the gene is COLI-23H, and the nucleotide sequence is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the protein is obtained by codon optimization and Pichia pastoris expression system adaptability optimization of the original fragment of human type I collagen shown in SEQ ID NO.1.

[0011] A recombinant Pichia pastoris engineered bacterium contains the coding gene of the recombinant human type I collagen fragment COLI-23H, and the sequence is shown in SEQ ID NO.2.

[0012] As one of the preferred embodiments of the present invention, the construction method is as follows: first construct a recombinant expression vector pPIC9K-COLI-23H containing the target gene, then linearize the vector, and transfer it into Pichia pastoris GS115.

[0013] A fermentation process of the above-mentioned recombinant Pichia pastoris engineering bacteria comprises the following steps:

[0014] (1) Seed culture

[0015] Pick a single colony of the recombinant Pichia pastoris, inoculate it into a centrifuge tube containing BMGY medium, and culture it at 30°C, 240 rpm for 18 to 24 hours, preferably 22 hours; transfer it to a shake flask containing BMGY seed medium at a 2% inoculum amount, and culture it at 30°C, 240 rpm until OD 600 =5~6, preferably OD 600 =5; inoculate the seed culture solution into a fermentation tank containing the optimized BSM basal salt medium at an inoculation rate of 10%;

[0016] The optimized BSM basal salt medium has the following formula: CaSO4 0.651 g / L, K2SO4 12.74 g / L, MgSO4·7H2O 10.43 g / L, KOH 2.891 g / L, glycerol 40 g / L, defoamer 1 g / L; after sterilization, 85% phosphoric acid 18.69 mL / L is added;

[0017] (2) Fermentation culture

[0018] During the glycerol culture stage, the temperature is maintained at 30°C and the pH is 5.0, and the rotation speed is adjusted to 400-800 rpm, preferably 600 rpm, to maintain DO>20%; when the glycerol in the optimized BSM culture medium is exhausted, DO=70-80, preferably DO=80, the glycerol addition stage is entered, and 50% glycerol is started to be added; when the wet weight of the bacteria is 180-220 g / L, preferably 200 g / L, the starvation period is entered, and after starvation for 1 hour, the methanol addition period is entered, the induction temperature is adjusted to 28°C, and methanol is started to be added to maintain DO at 18-22%, preferably 20%; the pH of the entire fermentation process is maintained by ammonia water and phosphoric acid; the tank is released after 108 hours of induction, and the supernatant is collected by centrifugation.

[0019] An application of the above-mentioned recombinant human type I collagen fragment COLI-23H in the preparation of wound repair, skin care, medical cosmetology and plastic surgery, and cosmetic products.

[0020] The advantages of the present invention compared to the prior art are:

[0021] (1) The present invention screened a sequence fragment containing two "GER integrin recognition sites" in the original human type I collagen sequence; then, according to the expression characteristics of Pichia pastoris, the nucleotide sequence of the fragment was optimized to make the nucleotide sequence encoding recombinant human type I collagen more suitable for the expression system of Pichia pastoris, and a strain of recombinant type I collagen high-yield strain was quickly screened;

[0022] (2) According to the characteristics of the constructed high-yield strain of recombinant type I collagen, the present invention has established a new fermentation process based on optimized culture medium, that is, based on the Pichia pastoris fermentation guide provided by Invitrogen, the protein yield is increased by reducing the inorganic salt content in the basic BSM culture medium; based on the new fermentation process of the present invention, the final yield of recombinant type I collagen COLI-23H is as high as 3.432 g / L;

[0023] (3) The recombinant human type I collagen fragment COLI-23H of the present invention has excellent biological activity and has a significant effect on promoting the proliferation of mouse embryonic fibroblasts. It can be widely used in wound repair, skin care, medical cosmetic surgery, cosmetic materials and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the active site map in the amino acid sequence of the recombinant human type I collagen fragment COLI-23H in Example 1 (GER in the map is the integrin recognition site);

[0025] Figure 2This is the protein electrophoresis diagram obtained by separation and purification of the recombinant human type I collagen fragment COLI-23H in Example 4 (in the figure, the first lane is the molecular weight marker, and the second lane is the purified COLI-23H protein; the molecular weight of the COLI-23 protein detected by electrophoresis is about 23 kDa);

[0026] Figure 3 This is a graph showing the effect of recombinant human type I collagen COLI-23H on the proliferation of mouse embryonic fibroblasts in Example 5 (in the graph, "**" indicates P<0.01, and "*" indicates P<0.05);

[0027] Figure 4 It is a curve diagram of the growth of recombinant human type I collagen COLI-23H in a fermenter under the fermentation process of the present invention in Example 6, the change of wet weight of the bacteria and the expression amount of the target protein;

[0028] Figure 5 This is the protein electrophoresis diagram obtained by separation and purification of the recombinant human type I collagen fragment COLI-14H in Comparative Example 1 (in the figure, the first lane is the molecular weight marker, and the fifth lane is the purified protein; the molecular weight of the target protein detected by electrophoresis is about 14 kDa);

[0029] Figure 6 This is a graph showing the effect of the recombinant human type I collagen fragment COLI-14H on the proliferation of mouse embryonic fibroblasts in Comparative Example 1;

[0030] Figure 7 This is a curve chart of the growth of recombinant human type I collagen COLI-23H in a fermenter under the traditional fermentation process in Example 2, the change in wet weight of the bacteria and the expression level of the target protein. DETAILED DESCRIPTION

[0031] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0032] The Pichia pastoris GS115 strain used in the following examples was from Shanghai Angyu Company, and the expression vector pPIC9K was from Shanghai Shenggong Company, both of which are directly commercially available strains and vectors. The medium formula used is as follows:

[0033] 1) YPD complete medium (1 L): Add 10 g / L yeast extract and 20 g / L peptone to 900 mL water, sterilize by high pressure, and then add 100 mL sterile 10× glucose solution. The solid medium contains 1.5% agar.

[0034] 2) MD medium (1 L): First, sterilize 800 mL of water by high pressure, cool to 60°C, then add 100 mL of 10XYNB, 2 mL of 500X biotin, and 100 mL of 10X glucose. The solid medium contains 2% agar.

[0035] 3) BMGY medium (1 L): add 10 g / L yeast extract and 20 g / L peptone to 700 mL water, sterilize by autoclaving, cool to room temperature, and then add 100 mL 1 M potassium phosphate buffer (pH 6.0), 100 mL 10XYNB, 2 mL 500X biotin, and 100 mL 10X glycerol.

[0036] 4) BMMY medium (1 L): Add 10 g / L yeast extract and 20 g / L peptone to 700 mL of water, sterilize by autoclave, cool to room temperature, and then add 100 mL 1 M potassium phosphate buffer (pH 6.0), 100 mL 10XYNB, 2 mL 500X biotin, and 100 mL 10X methanol.

[0037] 5) BSM culture medium: CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, defoaming agent 1 g / L; after sterilization, add 85% phosphoric acid 26.7 mL / L.

[0038] 6) Optimized BSM medium: CaSO4 0.651 g / L, K2SO4 12.74 g / L, MgSO4·7H2O10.43 g / L, KOH 2.891 g / L, glycerol 40 g / L, defoamer 1 g / L; after sterilization, add 85% phosphoric acid 18.69 mL / L;

[0039] 7) PTM1 trace elements: CuSO4·5H2O 6g / L, NaI 0.08g / L, MnSO4·H2O 3g / L, NaMoO4·2H2O 0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20g / L, FeSO4·7H2O65g / L, biotin 0.2g / L, H2SO4 5mL / L.

[0040] Meanwhile, the DMEM basal culture medium, PBS, fetal bovine serum (FBS), and trypsin used in the following examples were from Vivacell, and the calf serum (BCS), and mouse embryonic fibroblast cell line NIH / 3T3 were from Oricell.

[0041] Example 1

[0042] The encoding gene of recombinant human type I collagen fragment COLI-23H:

[0043] The recombinant human type I collagen fragment COLI-23H encoding gene screened and optimized by the present invention is named COLI-23H, and its nucleotide sequence is shown in SEQ ID NO.2, and the corresponding amino acid sequence is shown in SEQ ID NO.3 (the GER active site in the amino acid sequence of the recombinant human type I collagen fragment COLI-23H is shown in Figure 1 ).

[0044] Gene screening optimization process:

[0045] (1) Based on the human type I collagen gene sequence (NM_000088.4) in Genebank, an original sequence fragment containing two "GER integrin recognition sites" (GER sites can be recognized by integrin recognition sites on the cell membrane, and then transmitted to cells through signal transduction to promote cell proliferation and adhesion) was found, as shown in SEQ ID NO.1.

[0046] (2) Sequence optimization was performed based on the codon preference of Pichia pastoris and the GC content of the DNA sequence, and the optimized gene sequence is shown in SEQ ID NO. 2. The optimized gene sequence was artificially synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0047] Example 2

[0048] Construction of recombinant expression vector pPIC9K-COLI-23H:

[0049] An EcoRI restriction site sequence was added to the 5' end of the coding gene sequence (SEQ ID NO.2), and a stop codon, a 6XHis tag and a NotI restriction site sequence were added to the 3' end; subsequently, the sequence fragment was inserted into the pPIC9K expression vector through the EcoRI and NotI restriction sites to obtain the corresponding recombinant expression vector pPIC9K-COLI-23H.

[0050] Example 3

[0051] Construction of recombinant Pichia pastoris engineering bacteria:

[0052] (1) The recombinant expression vector pPIC9K-COLI-23H was linearized and then transformed into the competent cell of Pichia pastoris GS115.

[0053] (2) Single clones were picked from the transformed MD plate and placed in a 48-well culture plate containing 500 μL YPD (1 mg / mL G418) medium. The plates were cultured at 30°C and 240 rpm for 18–24 h. The plates were then transferred to YPD liquid medium with increasing G418 content.

[0054] (3) Fermenting the strain with good growth, the steps are as follows:

[0055] 2% inoculum: inoculate the bacterial solution into 10mL / 50mL BMGY and culture at 30℃, 240rpm for 18-24h;

[0056] After centrifugation, the supernatant was discarded and 10 mL of BMMY was added for resuspending. The cells were cultured at 28°C, 240 rpm for 48 h, and 100% methanol was added every 24 h to a final concentration of 1%;

[0057] The mixture was centrifuged and the supernatant was taken for SDS-PAGE analysis to obtain the recombinant Pichia pastoris with high expression level.

[0058] Example 4

[0059] Production, preparation and purification of recombinant human type I collagen fragment COLI-23H:

[0060] (1) The successfully constructed recombinant Pichia pastoris (glycerol strain) was activated in YPD (G418 containing corresponding resistance) medium at 30°C, 240 rpm, for 18 h.

[0061] (2) 2% inoculum was transferred to 200 mL / 1 L of BMGY and cultured at 30°C, 240 rpm for 26-28 h to completely consume the glycerol in the culture medium.

[0062] (3) Take 200 mL of BMMY to resuspend the cells and culture at 25°C, 240 rpm for 48 h; add 100% methanol every 24 h to a final concentration of 1%.

[0063] (4) After fermentation, the supernatant was filtered through a 0.22 μm water filter membrane to obtain a clarified fermentation supernatant, and the recombinant human type I collagen fragment COLI-23H was separated and purified by nickel column affinity chromatography.

[0064] Figure 2 This is the protein electrophoresis diagram of the recombinant human type I collagen fragment COLI-23H separated and purified. The COLI-23H protein solution after separation and purification was analyzed by gel filtration chromatography and non-reducing-SDS-PAGE, and the purity was >92% and the recovery rate was >85%.

[0065] Example 5

[0066] Effect of recombinant human type I collagen fragment COLI-23H on the proliferation of mouse embryonic fibroblasts NIH / 3T3:

[0067] 1. MTT method to measure the effect of recombinant human type I collagen on the proliferation of mouse embryonic fibroblasts:

[0068] (1) Mouse embryonic fibroblasts NIH / 3T3 were digested, centrifuged, resuspended, and diluted to 2×10 4 / mL, 100 μL was inoculated into each well of a 96-well cell culture plate; at the same time, culture medium without cells was inoculated as a blank control, and cultured at 37°C, 5% CO2, 95% humidity for 24 h.

[0069] (2) Prepare COLI-23H protein suspensions at 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL, and 0.078125 mg / mL.

[0070] (3) The culture medium was discarded, and the blank control was added with culture medium, the negative control was added with culture medium, the positive control was added with culture medium containing 4% DMSO, and the experimental group was added with culture medium containing different concentrations of protein solution. The culture was carried out at 37°C, 5% CO2, and 95% humidity for 48 h.

[0071] (4) Add 20 μL MTT to each well and incubate in an incubator for 4 h at 37°C, 5% CO2, and 95% humidity. Add 150 μL DMSO and incubate on a shaker for 10 min to fully dissolve the crystals. Detect the absorbance at 490°C using an ELISA reader.

[0072] Test results such as Figure 3 As shown. Figure 3 It can be seen that the survival rate of the negative control group was 100%, and the survival rate of the NIH / 3T3 experimental group supplemented with COLI-23H was 125%.

[0073] Example 6

[0074] Optimized fermentation process of recombinant Pichia pastoris (expressing recombinant human type I collagen COLI-23H):

[0075] (1) Seed culture

[0076] Pick a fresh single colony on the YPD plate, inoculate it into a 50 mL centrifuge tube containing 5 mL BMGY medium, and culture it at 30°C, 240 rpm for 22 h; transfer it to a 1 L shake flask containing 200 mL BMGY seed medium at a 2% inoculum, and culture it at 30°C, 240 rpm for 14 h until the OD 600 = 5. With an inoculation volume of 10%, 200 mL of seed culture solution was inoculated into a 5 L fermenter (containing 2 L of optimized BSM basal salt medium).

[0077] (2) Fermentation culture

[0078] During the glycerol culture stage, the temperature was maintained at 30°C, pH 5.0, the speed was adjusted to 600rpm, and DO>20%. When the glycerol in the optimized BSM medium was exhausted, DO = 80, and the glycerol addition stage was entered, and 50% glycerol was added to further increase the cell density. When the wet weight of the cells was about 200g / L, the starvation period began. After starvation for 1h, the methanol addition period began. The induction temperature was adjusted to 28°C, and methanol was added to maintain DO at around 20%. The pH of the entire fermentation process was maintained by ammonia and phosphoric acid. After 108h of induction, the tank was released and the supernatant was collected by centrifugation.

[0079] The high-density fermentation of recombinant human type I collagen COLI-23H in a 5L fermenter shows the growth of bacteria, changes in wet weight of bacteria, and expression of the target protein. Figure 4 As shown. Figure 4 It can be seen that induction started from 35 hours of culture and the total culture time was 143 hours. At 72 hours of induction, the highest protein expression level was 3.432 g / L.

[0080] Comparative Example 1

[0081] A recombinant human type I collagen fragment COLI-14H in this comparative example:

[0082] The coding sequence of the recombinant human type I collagen fragment COLI-14H is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0083] The sequence optimization process corresponding to SEQ ID NO.5 is as follows:

[0084] (1) Based on the human type I collagen gene sequence (NM_000088.4) in Genebank, a segment different from the target sequence of the present invention is selected, as shown in SEQ ID NO.4.

[0085] (2) Referring to the method of Example 1 of the present invention, sequence optimization was performed based on the codon preference of Pichia pastoris and the GC content of the DNA sequence. The optimized gene sequence is shown in SEQ ID NO.5.

[0086] The corresponding recombinant expression vector pPIC9K-COLI-14H and recombinant Pichia pastoris were constructed according to the method of the above embodiment, and the biological activity of the recombinant human type I collagen fragment (its effect on the proliferation of mouse embryonic fibroblasts NIH / 3T3) was tested.

[0087] Figure 5 This is the protein electrophoresis diagram obtained by separation and purification of the recombinant human type I collagen fragment COLI-14H in this comparative example. Figure 6This is a graph showing the effect of the recombinant human type I collagen COLI-14H on the proliferation of mouse embryonic fibroblasts. Figure 6 It can be seen that only the recombinant human type I collagen fragment COLI-23H based on the sequence of the present invention has the characteristics of good biological activity, recombinant expression and high yield.

[0088] Comparative Example 2

[0089] A traditional fermentation process of the recombinant Pichia pastoris of the present invention in this comparative example:

[0090] (1) Seed culture

[0091] Pick a fresh single colony on the YPD plate, inoculate it into a 50 mL centrifuge tube containing 5 mL BMGY medium, and culture it at 30°C, 240 rpm for 22 h; transfer it to a 1 L shake flask containing 200 mL BMGY seed medium at a 2% inoculum, and culture it at 30°C, 240 rpm for 14 h until the OD 600 = 5. With an inoculation volume of 10%, 200 mL of seed culture solution was inoculated into a 5 L fermentation tank (containing 2 L of BSM basal salt medium).

[0092] (2) Fermentation culture

[0093] During the glycerol culture stage, the temperature was maintained at 30°C, pH 5.0, the speed was adjusted to 600rpm, and DO>20%. When the glycerol in the BSM medium was exhausted, DO = 80, and the glycerol addition stage was entered. 50% glycerol was added to further increase the cell density. When the wet weight of the cells was about 200g / L, the starvation period began. After starvation for 1h, the methanol addition period began. The induction temperature was adjusted to 28°C, and methanol was added to maintain DO at around 20%. Ammonia and phosphoric acid were used to maintain pH throughout the fermentation process. After 144h of induction, the tank was released and the supernatant was collected by centrifugation.

[0094] The high-density fermentation of recombinant human type I collagen COLI-23H in a 5L fermenter shows the growth of bacteria, changes in wet weight of bacteria, and expression of the target protein. Figure 7 As shown. Figure 7 It can be seen that the induction started from 49h of culture and the total culture was 193h. At 72h of induction, the highest protein expression level was 1.699g / L, which was significantly lower than the protein yield of the process in Example 6 of the present invention.

[0095] In summary, the recombinant human type I collagen fragment COLI-23H of the present invention has excellent biological activity, has a significant effect of promoting the proliferation of mouse embryonic fibroblasts, and can be widely used in wound repair, skin care, medical cosmetic surgery, cosmetics and other fields. In addition, based on the original fermentation system of the present invention, the COLI-23H protein yield of the recombinant type I collagen high-yield strain of the present invention is as high as 3.432g / L.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant human type I collagen fragment COLI-23H, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. The recombinant human type I collagen fragment COLI-23H according to claim 1, characterized in that: The recombinant human type I collagen fragment COLI-23H contains two "GER integrin recognition sites".

3. A gene encoding the recombinant human type I collagen fragment COLI-23H as claimed in claim 1 or 2, characterized in that: The gene is COLI-23H , the nucleotide sequence is shown in SEQ ID NO.

2.

4. The coding gene according to claim 3, characterized in that The protein was obtained by codon optimization and Pichia pastoris expression system adaptability optimization of the original fragment of human type I collagen shown in SEQ ID NO.

1.

5. A recombinant Pichia pastoris engineered bacterium, characterized in that: The coding gene of the recombinant human type I collagen fragment COLI-23H is shown in SEQ ID NO.

2.

6. The recombinant Pichia pastoris engineered bacterium according to claim 5, characterized in that: The construction method is as follows: first construct a recombinant expression vector pPIC9K- COLI-23H The vector was linearized and then transferred into Pichia pastoris GS115. A strain with high production of recombinant type I collagen was obtained through rapid screening through small-scale expression test.

7. A fermentation process of the recombinant Pichia pastoris engineered bacteria as claimed in claim 6, characterized in that: By reducing the inorganic salt content in the basic BSM medium, protein production can be increased.

8. Use of the recombinant human type I collagen fragment COLI-23H as claimed in claim 1 or 2 in the preparation of wound repair, medical cosmetic surgery and cosmetic products.

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