CN111363029B

CN111363029B - Recombinant human III-type collagen, expression strain and construction method thereof

Info

Publication number: CN111363029B
Application number: CN201811590564.XA
Authority: CN
Inventors: 赵健烽; 高力虎; 冯丽萍; 黄建民
Original assignee: Jiangsu Jland Biotech Co ltd
Current assignee: Jiangsu Jland Biotech Co ltd
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2022-07-12
Anticipated expiration: 2038-12-25
Also published as: CN111363029A

Abstract

The invention discloses a recombinant human III-type collagen, an expression strain and a construction method thereof. The recombinant human type III collagen contains 498 amino acids and has theoretical molecular weight of about 54.5kd, and the constructed recombinant human type III collagen expression strain can express the recombinant human type III collagen effectively, stably and massively. The recombinant human collagen of the invention has good hydrophilicity and stability, the structure of the recombinant human collagen is 100 percent identical to the corresponding part of a natural collagen gene sequence, the recombinant human collagen can not cause immunological rejection when being applied to a human body, and the recombinant human collagen can be widely applied to the fields of biomedical materials, cosmetics and the like.

Description

Recombinant human III-type collagen, expression strain and construction method thereof

Technical Field

The invention belongs to the technical field of bioengineering, and relates to recombinant humanized type III collagen, a pichia pastoris engineering bacterium for expressing the recombinant humanized type III collagen and a construction method thereof.

Background

Collagen (Collagen) is the most important structural protein in the bodies of vertebrates, and accounts for about 25-30% of the total amount of the protein. Collagen, the most abundant protein in the extracellular matrix, is the most prominent structural protein in connective tissue and in the interstitial tissue of almost all parenchymal organs, and serves to stabilize tissues and organs and maintain their structural integrity. Because the natural collagen is not easy to dissolve in water, and the used collagen is generally derived from cattle or pigs, the molecular weight is different, the property is very complex, the further processing is difficult, and the hidden danger of animal viruses exists, so that the application of the natural collagen serving as a biological material in the fields of medical appliances and the like is limited. Therefore, studies have been made to solve these problems by gene construction techniques using bioreactors for expression of recombinant collagen, and good progress has been made.

The Pichia pastoris (Pichia pastoris) expression system is an exogenous protein expression system, has the advantages of simple operation, easy culture, high growth speed, high expression level, low cost and the like of a prokaryotic expression system, and has the characteristics of exogenous protein modification such as glycosylation, protein phosphorylation and the like which are not possessed by the prokaryotic expression system. Over 200 different proteins have been successfully expressed in pichia over the last 20 years, many of which have been widely used in clinical diagnostic and therapeutic work and scientific research. In a pichia pastoris expression system, an exogenous gene is not present in an autonomously replicating expression vector, but is integrated on a yeast chromosome together with the expression vector through homologous recombination, and is replicated and inherited along with the chromosome, so that the phenomenon of loss of the exogenous gene cannot occur; the pichia pastoris has strong aerobic growth preference, can realize high-density cell culture and is beneficial to large-scale industrial production; the pichia pastoris can secrete and express exogenous protein at high level, the accumulation of fermentation products can not generate toxic or side effect on the pichia pastoris, and the protein secreted into a culture medium by the pichia pastoris is little, so that the purification is convenient.

Disclosure of Invention

The invention aims to provide a recombinant human type-III collagen with excellent hydrophilicity, a pichia pastoris engineering bacterium for secretory expression of the recombinant human type-III collagen and a construction method thereof, and extracellular secretory expression of the recombinant human type-III collagen can be efficiently and safely carried out.

The technical scheme for realizing the purpose of the invention is as follows:

the amino acid sequence of the recombinant human type III collagen is shown in SEQ No. 2.

The coding gene III alpha 498aa of the recombinant humanized III-type collagen has a nucleotide sequence shown in SEQ No. 3.

The nucleotide sequence of the recombinant human type III collagen plasmid ppic 9K-IIIalpha 498aa constructed by the invention is shown in SEQ No.4, and the recombinant human type III collagen plasmid is constructed by double-enzyme digestion of a coding gene IIIalpha 498aa of the recombinant human type III collagen to be connected between EcoR I and Not I of a ppic9K vector.

The constructed strain for secreting and expressing the recombinant human-derived type III collagen is Pichia pastoris JY0301, the preservation number is CGMCC No.16462, the strain is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms in 2018, 9 and 12 months, and the preservation address is No.3 of the national institute of sciences, No.3 of West Lu No.1 of the Beijing city and the rising district, China. The Pichia pastoris JY0301 is constructed by inducing a linearized recombinant human type I collagen plasmid ppic 9K-III alpha 498aa obtained by Sac I enzyme digestion into Pichia pastoris.

The invention selects a nucleotide sequence with strong water solubility and stability from a helical region of human collagen gene type III with a known sequence, inserts an optimized gene segment into a pichia pastoris expression plasmid, converts pichia pastoris and screens to obtain a high-expression pichia pastoris gene engineering bacterium, and obtains high-purity recombinant human-like collagen through preliminary fermentation and purification steps. The recombinant human type III collagen has good hydrophilicity and stability, has the same structure with 100 percent of the corresponding part of a natural collagen gene sequence, can not cause immunological rejection when being applied to a human body, and has potential application prospect in the fields of biomedical materials, cosmetics and the like.

Drawings

FIG. 1 is a diagram showing the hydrophobicity analysis of amino acids in human type III alpha-chain collagen.

FIG. 2 is a graph showing the hydrophobicity analysis of amino acids in recombinant human type III collagen.

FIG. 3 is a schematic diagram of the construction of recombinant human type III collagen plasmid ppic 9K-IIIalpha 498 aa.

FIG. 4 shows the Sac I agarose gel electrophoresis of recombinant human type III collagen plasmid ppic 9K-IIIa 498 aa.

FIG. 5 is a photograph of a Pichia pastoris GS115 after electroporation.

FIG. 6 is a gel electrophoresis of the positive clone strains.

FIG. 7 is a PCR gel electrophoresis of the positive clone strains.

FIG. 8 is a graph showing protein expression analysis of samples cultured for 48 and 72 hours from #3, #4, #5, #10 positive clone strains.

FIG. 9 is an SDS-PAGE electrophoresis of protein expression of #10 positive clone.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

1. Protein sequence selection

The amino acid sequence (SEQ No.1) of human type III alpha-chain collagen was subjected to hydrophobicity analysis, and the results are shown in FIG. 1. The lower the hydrophobicity evaluation score, the better the hydrophilicity. Low scoring amino acid fragments were selected and integrated into a new protein, recombinant human type iii collagen of the invention (SEQ No. 2). The amino acid hydrophobicity analysis of the recombinant human type III collagen is carried out, the result is shown in figure 2, all the amino acid hydrophobicity evaluation in the protein is lower than zero, and the protein is proved to have good hydrophilicity.

2. Plasmid construction and linearization

The recombinant human type III collagen is translated into a base sequence (SEQ No.3), a PAS (PCR-based Accurate Synthesis) method is adopted to synthesize a gene III alpha 498aa, double enzyme digestion is carried out between EcoR I and Not I connected to ppic9K vector, and FIG. 3 is a construction schematic diagram of a recombinant human type III collagen plasmid ppic 9K-III alpha 498 aa. The obtained recombinant plasmid ppic 9K-IIIalpha 498aa is transferred into a TOP10 clone strain, positive clones are picked for sequencing, and the sequencing result is shown as SEQ No. 4. The sites of the 100-105 bases and 1603-1610 bases of SEQ No.4 are the restriction sites.

Extracting 20 mu g of plasmid, using Sac I to perform enzyme digestion linearization, freeze-drying and concentrating for later use. Digest for 3h at 37 ℃. Mu.l of the sample was subjected to 1% agarose gel electrophoresis, and the results of the electrophoresis are shown in FIG. 4. Wherein M is a DNA standard substance, and is 1000, 2000, 3000, 4000, 5000, 6000, 8000 and 10000bp from bottom to top; 1 is Sac I enzyme digestion; 2 for recovery of the desired fragment.

TABLE 1 preparation table of enzyme digestion linearization system

3. Pichia electrotransformation cell GS115

The electric rotor was ice-cooled, 10. mu.L of linearized plasmid was added to a 1.5mL EP tube containing 80. mu.L of Pichia competent cells, and the mixture was transferred to an electric rotor having a diameter of 0.2cm, followed by ice-cooling the electric rotor for 5 min. The electric shock conditions are as follows: the voltage is 1.5 kV; a capacitance of 25 μ F; the resistance is 200 omega, and the electric shock time is 4-10 msec. After the shock was complete, 650. mu.L of 1M sorbitol solution pre-chilled on ice was added to the shock conversion cup and the solution was gently swirled uniformly with a pipette tip. The whole liquid in the cuvette was transferred to a new 2ml EP tube and incubated at 30 ℃ for 2 hours. And (4) centrifugally collecting the thalli at a low speed, completely coating the thalli on an MD (MD) plate, and culturing at a constant temperature of 30 ℃ for 3-4 days. FIG. 5 is a photograph of a Pichia pastoris GS115 after electroporation.

PCR identification of Positive clones

When bacterial colony grows on the plate, the single bacterium growing on the plate is picked by using an inoculating loop and is inoculated into a centrifugal tube filled with 500 mu L of YPD liquid culture medium for overnight culture at 30 ℃ and 180 r/min. 10 clones were selected and genomic DNA was extracted, respectively, as shown in FIG. 6. M is a DNA standard substance, and is 1000, 2000, 3000, 4000, 5000, 6000, 8000 and 10000bp from bottom to top; 1-10: genome extracted from each cloned strain. The results of PCR identification using primers on the vector are shown in FIG. 7, where M: DNA standard, from bottom to top 100, 200, 500, 750, 1000, 2000 bp; 1-10: PCR amplified fragments of each cloned strain.

5. Small test expression identification

Inducing expression: inoculating 50 μ l of the identified positive strain (No. 3, No.4, No. 5, No. 10) into a conical flask containing 10ml of BMGY, culturing overnight at 30 deg.C and 220r/min, and shaking to OD₆₀₀2-6 (logarithmic growth, about 16-18 h); centrifuging at room temperature for 5min at 5000r/min, collecting cells, removing supernatant, resuspending the cells with 10ml BMMY, and performing induced expression; sampling 1ml of the culture medium every 24h, and adding methanol to a final concentration of 0.5% to continue induction; centrifuging the samples at 10000r/min for 2min at the following time points of 0, 24, 48, 72 and 96h, collecting the supernatant, and placing the supernatant at the temperature of minus 20 ℃ for detection.

And (3) concentrating the expression product by a trichloroacetic acid precipitation method:

(1) adding 500. mu.l of culture supernatant and 1/9 volume of 100% TCA into a centrifuge tube, shaking and mixing, and precipitating at 4 ℃ overnight;

(2) centrifuging at 12000r/min for 10min to obtain viscous yellowish brown jelly, removing supernatant, collecting precipitate, placing the EP tube on absorbent paper, and standing in a 37 ℃ oven for 10-20 min to ensure that no obvious liquid remains on the tube wall;

(3) adding 200 mul of cold acetone, oscillating and uniformly mixing, standing the sample at room temperature for 10min, and washing off residual TCA on the tube wall and the tube bottom;

(4) centrifuging at 12000r/min for 10min, discarding the supernatant, repeating the steps (2) and (3), and repeating for 2-3 times;

(5) adding 30 mu l of loading buffer solution, incubating for 1h at 37 ℃, and dissolving the precipitate; if the precipitate does not dissolve, the precipitate can be blown up with a 100. mu.l lance tip until the precipitate dissolves.

6. Identification assay

Western Blotting detection:

(1) sampling 10 μ l of sample;

(2) after the sample loading is finished, the polyacrylamide gel runs out of laminated gel at 90V, and then the voltage is increased to 200V until the electrophoresis is finished;

(3) after electrophoresis is finished, taking down the gel for membrane conversion, performing membrane conversion at constant voltage of 100V for about 1.5h and performing constant current of 250 mA;

(4) after the electrotransformation is finished, taking off the membrane, and washing with PBST for 4 times, 5min each time;

(5) placing the membrane in 5% skimmed milk powder sealing solution, sealing at 37 deg.C for 1 h;

(6) diluting primary antibody (Rabbit anti-His) with a blocking solution, and allowing the membrane to stay overnight at 4 ℃ in the primary antibody dilution (dilution ratio 1: 1000);

(7) taking out the membrane the next day, washing the membrane with PBST for 5min for 4 times;

(8) diluting the secondary antibody (goat anti-rabbit) with blocking solution containing 5% milk, and reacting the membrane in the secondary antibody diluent (dilution ratio 1:5000) at 37 deg.C for 1 h;

(9) after the reaction is finished, taking out the membrane, and then placing the membrane in a clean box to wash the membrane for 4 times, 5min each time;

(10) and ECL developing and exposing.

Western Blot was performed on 48 and 72 hour samples of selected #3, #4, #5, and #10 strain positive clones. The results are shown in FIG. 8, where M: a protein standard; 1: the GS115 strain was cultured for 72 hours to obtain a supernatant sample; 2: culturing the 3# positive strain for 48 hours to obtain a supernatant sample; 3: culturing the 3# positive strain for 72 hours to obtain a supernatant sample; 4: culturing the 4# positive strain for 48 hours to obtain a supernatant sample; 5: culturing the 4# positive strain for 72 hours to obtain a supernatant sample; 6: culturing the 5# positive strain for 48 hours to obtain a supernatant sample; 7: culturing the 5# positive strain for 72 hours to obtain a supernatant sample; 8: culturing the 10# positive strain for 48 hours to obtain a supernatant sample; 9: the 10# positive strain was cultured for 72 hours and the supernatant sample was obtained.

Expression analysis:

SDS-PAGE electrophoretic detection: the results of expression analysis of 10# positive bacteria are shown in FIG. 9. Wherein, M: a protein standard; 1: culturing a supernatant of GS115 strain for 72 hours; 2: 10# positive strain is cultured for 0 hour, and supernatant is obtained; 3: culturing the 10# positive strain for 24 hours to obtain supernatant; 4: culturing the 10# positive strain for 48 hours to obtain supernatant; 5: culturing the 10# positive strain for 72 hours to obtain supernatant; 6: the 10# positive strain was cultured for 96 hours as supernatant. The 10# positive strain is named as Pichia pastoris JY0301, and the strain is preserved in China general microbiological culture Collection center (the preservation center address: No.3 of Beijing university Hokko-Yang Beichen Xilu No.1, the institute of microbiology of China academy of sciences, postal code: 100101) in 2018, 9, 12 and 4 days, and the preservation number is CGMCC No. 16462.

The invention selects a nucleotide sequence with strong water solubility and stability from a helical region of human collagen gene III type with a known sequence, inserts an optimized gene segment into a pichia pastoris expression plasmid, converts pichia pastoris to screen and obtain high-expression pichia pastoris gene engineering bacteria, and obtains high-purity recombinant human-like collagen through preliminary fermentation and purification steps. Experiments prove that the recombinant human-like collagen produced by the method has good hydrophilicity and stability, and the structure of the recombinant human-like collagen is 100 percent identical to the corresponding part of a natural collagen gene sequence, so that the recombinant human-like collagen can not cause immunological rejection when being applied to a human body, and can be widely applied to the fields of biomedical materials, cosmetics and the like. The secretory expression vector is adopted, the secretory expression of the recombinant human-like collagen is successfully realized, the expression product is secreted in the supernatant, the purification is convenient, the advantages which are not possessed by other recombinant human-like collagen production are possessed, and the large-scale production operation is convenient. After the carrier is electrically transferred into the pichia pastoris, the gene is integrated on a pichia pastoris genome, so the recombinant strain has good stability, the gene is not easy to lose after multiple passages, the character of high-efficiency expression can be kept, stable production can be well realized, the pichia pastoris production method is aerobic fermentation, the thallus density is high, and the expression amount has a great promotion space.

Sequence listing

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attggcccac ctggtccagc cggacaacca ggtgacaaag gcgaaggtgg tgctcccggt 1020

gagcctggaa gagatggtaa tccaggatct gatggtttgc caggtcgtga tggttcccct 1080

ggcggaaaag gtgatagagg cgaaaatgga tccccaggcg caccaggcgc tcccggacat 1140

cccggaccac caggtccagt aggtcctgca ggtaaatctg gtgatagggg agaatctggt 1200

cctgccggta gtagaggtgc ccctggtcct cagggtccac gtggtgacaa aggtgagaca 1260

ggtgaaagag gcgctgctgg tattaagggt catagaggtt ttccaggtaa cccaggtgca 1320

cccggaagtc caggaccagc cggtcaacag ggccctcctg gtaaggacgg tacttctggt 1380

catcctggtc ctattggccc tccaggacca aggggtaata gaggtgaaag gggttctgaa 1440

ggttccccag gtcatccagg tcagcccggt ccaccaggac ctccaggtgc tcca 1494

<210> 4

<211> 1610

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acaaataacg ggttattgtt tataaatact actattgcca gcattgctgc taaagaagaa 60

ggggtatctc tcgagaaaag agaggctgaa gcttacgtag aattcggtgc tagaggtaac 120

gatggtgcaa gaggttctga tggtcaacca ggtccacctg gtcctccagg tgctaaaggt 180

gaagttggtc cagctggttc tcctggatct aatggtgctc caggacaaag aggtgaacct 240

ggtccacaag gtcatgctgg tgctcaaggt ccaccaggac caccaggcat taacggttct 300

ccaggtggaa agggtgaaat gggtgctgct ggtgaaagag gtgcaccagg ttttagaggt 360

cctgctggtc caaatggtat tccaggtgaa aaaggtccag ccggtgaacg tggtgctcct 420

ggtccagcag gtccaagagg tgctgcaggc gaaccaggta gagatggtgt accaggtggt 480

ccaggtatga gaggtatgcc aggatctcca ggcggtcctg gttcagatgg taaacccgga 540

cctccaggat cacaaggtga atcaggtaga cccggtcctc ctggaccttc tggtcctaga 600

ggacaacctg gacctaaggg aaatgatggt gccccaggta agaatggtga gcgtggtggt 660

cccggcggtc caggtcctca aggacctcct ggaaaaaatg gtgaaactgg acctcaaggc 720

cctccaggtc ctaccggtcc aggtggtgat aagggtgata ctggcccacc aggacctcaa 780

ggtacaggtg gtccaccagg tgaaaatggt aaaccaggtg aaccaggtcc taagggtgat 840

gccggcgctc ctggtggcaa aggtgacgct ggcgctccag gcgagagagg acctccaggt 900

cctgaaggtg gtaaaggtgc tgccggtcct ccaggattgc aaagaatgcc aggtgagaga 960

ggtggtttgg gttcaccagg tccaaaaggc gacaaaggtg aacccggcgg acccggtgct 1020

gatggtgttc ctggtaaaga tggaccaaga ggtccaactg gaccaattgg cccacctggt 1080

ccagccggac aaccaggtga caaaggcgaa ggtggtgctc ccggtgagcc tggaagagat 1140

ggtaatccag gatctgatgg tttgccaggt cgtgatggtt cccctggcgg aaaaggtgat 1200

agaggcgaaa atggatcccc aggcgcacca ggcgctcccg gacatcccgg accaccaggt 1260

ccagtaggtc ctgcaggtaa atctggtgat aggggagaat ctggtcctgc cggtagtaga 1320

ggtgcccctg gtcctcaggg tccacgtggt gacaaaggtg agacaggtga aagaggcgct 1380

gctggtatta agggtcatag aggttttcca ggtaacccag gtgcacccgg aagtccagga 1440

ccagccggtc aacagggccc tcctggtaag gacggtactt ctggtcatcc tggtcctatt 1500

ggccctccag gaccaagggg taatagaggt gaaaggggtt ctgaaggttc cccaggtcat 1560

ccaggtcagc ccggtccacc aggacctcca ggtgctccat aagcggccgc 1610

Claims

1. The amino acid sequence of the recombinant human III-type collagen is shown in SEQ No. 2.

2. The nucleotide sequence of the gene for coding the recombinant human III-type collagen is shown in SEQ No. 3.

3. A recombinant human type III collagen plasmid containing the gene of claim 2, the nucleotide sequence of which is shown in SEQ No. 4.

4. The method of constructing a recombinant human type III collagen plasmid of claim 3, which is constructed by double-digesting the gene of claim 2 into EcoR I and Not I ligated to ppic9K vector.

5. The recombinant human III-type collagen expression strain is Pichia pastorisPichia pastorisJY0301 with preservation number CGMCC No. 16462.