CN112941058A

CN112941058A - Recombinant Clostridium histolyticum type-II collagenase as well as preparation method and application thereof

Info

Publication number: CN112941058A
Application number: CN202110358892.2A
Authority: CN
Inventors: 李树刚; 张伟; 龚会英; 但国平; 邓永康; 刘涛; 程丹凝; 辛渝; 杨丹丹; 于廷和
Original assignee: Chongqing Kerun Biomedical R & D Co ltd
Current assignee: Chongqing Kerun Biomedical R & D Co ltd
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-06-11
Anticipated expiration: 2041-04-02
Also published as: CN112941058B

Abstract

The invention discloses a recombinant Clostridium histolyticum type II collagenase, which comprises Clostridium histolyticum type II collagenase and a tag protein connected to the N end of the Clostridium histolyticum type II collagenase; the tag protein comprises hepcidium calcium binding protein (Fh 8); preferably, the amino acid sequence of the Fasciola hepatica calcium binding protein is SEQ ID NO. 2. The invention also provides an expression vector and recombinant engineering bacteria for expressing the recombinant Clostridium histolyticum type II collagenase, and also provides a preparation method of the Clostridium histolyticum type II collagenase. The recombinant Clostridium histolyticum type II collagenase can be efficiently expressed, does not form inclusion bodies, is easy to separate and purify, and is beneficial to the industrial production of the Clostridium histolyticum type II collagenase.

Description

Recombinant Clostridium histolyticum type-II collagenase as well as preparation method and application thereof

Technical Field

The invention relates to the field of bioengineering, and particularly relates to high-purity recombinant Clostridium histolyticum type II collagenase and a preparation method and application thereof.

Background

Clostridium Histolyticum Collagenase (Collagenase Clostridium Histolyticum) is a drug originally developed by the group of Biospecificics Technologies and was approved by the United states Food and Drug Administration (FDA) at 2/2010 and 2/2011 and European drug administration (EMA) at 28/2011 and Japanese pharmaceutical and medical device integration (PMDA) at 2015 and 7/3/2015. The name of the commodity is

The product of (a) is marketed by Auxilium pharmaceutical (now Yuancao pharmaceutical, Endo International) in the United states and by Asahi Kasei (Aasahi Kasei) in Japan. The name of the commodity is

Is marketed by Swedish Orange Biovitrum (SOBI) in Europe. Further, the trade name is

And

the product of (2) is sold by the Biospecifics Technologies group.

Approved indications are Dupuytren's contracture, pelonie's disease and orange peel tissue (Cellulite).

Is the first non-surgical treatment option approved by the FDA for use in male patients with peyronie's disease.

Is prepared by mixing Clostridium collagenase type I (AUX-I) and Clostridium collagenase type II (AUX-II) according to the mass ratio of 1: 1. AUX-I and AUX-II are both single peptide chains encoded by ColG and ColH, respectively, of Clostridium histolyticum, both of which are zinc ion and calcium ion-binding metalloenzymes.

The role of collagenase under physiological conditions is to hydrolyse collagen in its native triple helix conformation, allowing the collagen deposit to be solubilised. Injection of drugs

To the palpable connective tissue of Dupugile's palm contracture patients or to the vagina of patients with Pelonias diseaseThe stem is destroyed by the induration (the main component of both is the deposited collagen). In vitro studies indicate that AUX-I and AUX-II act synergistically based on their different substrate affinities, hydrolysis efficiencies, and selective cleavage sites.

Biospecifics of original research corporation purified AUX-I and AUX-II from the fermentation supernatant by culturing Clostridium histolyticum. But the clostridium histolyticum is a pathogenic bacterium of gas gangrene, various toxins are often mixed in fermentation supernatant liquid of the clostridium histolyticum, the components are complex, and if the strain and the production and purification process of the original research company are not available, the refined product reaching the medicinal standard is difficult to ensure. The production of Clostridium histolyticum collagenase type II by genetic recombination is a possible approach to solve this problem, but conventional E.coli expression is difficult to achieve due to the large molecular weight of AUX-II (113 kD). Even if the expression is mostly accumulated in the cell, inclusion bodies are easily formed. The inclusion body renaturation process is complex, the technical difficulty is high, the yield is low, the production cost is high, and the inclusion body renaturation process cannot be applied to industrial production at present.

Disclosure of Invention

The invention aims to provide a tag protein which is easy to express efficiently and used for preparing recombinant Clostridium histolyticum type II collagenase, the tag protein comprises calcium binding protein (Fh8) derived from Fasciola hepatica, the amino acid sequence of the tag protein is shown as SEQ ID NO.2, and the nucleotide sequence of the tag protein is shown as SEQ ID NO. 3. The tag protein can promote the soluble and high-efficiency expression of the recombinant Clostridium histolyticum type II collagenase, and does not influence the biological activity of the recombinant Clostridium histolyticum type II collagenase.

A recombinant clostridium histolyticum type II collagenase comprising clostridium histolyticum type II collagenase and a tag protein attached to the N-terminus of the clostridium histolyticum type II collagenase; the tag protein comprises hepcidium calcium binding protein (Fh 8); preferably, the amino acid sequence of the hepcidin calcium binding protein is SEQ ID NO: 2.

in one embodiment according to the invention, the tag protein further comprises one of a transcription termination anti-termination factor (NusA), escherichia coli thioredoxin a (trxa), disulfide oxidoreductase (DsbA, DsbC) or glutathione S-transferase (GST).

In one embodiment according to the invention, the tag protein is linked to the N-terminus of clostridium histolyticum collagenase type II by a flexible linker peptide; preferably, the amino acid sequence of the flexible connecting peptide is SEQ ID NO: 7.

in one embodiment according to the invention, the amino acid sequence is SEQ ID NO: 4.

the invention also provides a recombinant gene for encoding the recombinant Clostridium histolyticum collagenase type II, which comprises a Clostridium histolyticum collagenase type II encoding gene SEQ ID NO:1 and a signature protein encoding gene located upstream of the clostridium histolyticum collagenase II encoding gene, the signature protein encoding gene comprising a gene encoding a fasciola hepatica calcium binding protein (Fh 8).

In one embodiment according to the present invention, the gene encoding the hepcidin calcium binding protein (Fh8) is SEQ ID NO: 3.

in one embodiment according to the invention, the nucleotide sequence of the recombinant gene is SEQ ID NO: 5.

the present invention further provides a recombinant vector for expressing recombinant Clostridium histolyticum collagenase type II, comprising a recombinant gene according to the above claims;

preferably, the backbone plasmid of the recombinant vector is selected from any one of pET-28a-c (+), pET29a, pET-30a-c (+), pET39b (+), pET-40b (+), pET-41a (+) or pET-43.1a (+).

The invention also provides a recombinant engineering bacterium for expressing the recombinant Clostridium histolyticum type II collagenase, which comprises the recombinant vector; preferably, the host bacterium of the recombinant engineering bacterium is a bacterium or a fungus; preferably, the host bacterium is escherichia coli, and more preferably any one of BL21(DE3), BL21(DE3) P1ysS, or TB 1.

The invention further provides a preparation method of the recombinant Clostridium histolyticum type II collagenase, which comprises the following steps:

1) connecting the coding gene of the tag protein to the upstream of the Clostridium histolyticum type II collagenase coding gene to obtain a recombinant gene for coding recombinant Clostridium histolyticum type II collagenase;

2) connecting the recombinant gene to a skeleton plasmid to obtain a recombinant vector;

3) transforming the recombinant vector into host bacteria to obtain recombinant engineering bacteria;

4) inoculating the recombinant engineering bacteria into a fermentation tank, and performing induced expression;

5) separating and purifying the culture to obtain recombinant Clostridium histolyticum type II collagenase after the concentration of the bacteria and the protein reaches a preset threshold value;

6) enzymolyzing the recombinant Clostridium histolyticum collagenase II;

7) and sequentially carrying out QFF chromatography, Butyl-4FF chromatography, Phenyl 6FF chromatography and QFF flow-through chromatography on the sample subjected to enzymolysis to obtain the pure Clostridium histolyticum collagenase II product.

The invention also provides application of the recombinant Clostridium histolyticum type II collagenase to preparation of a medicament for treating Dupuytren's contracture, Pelonie's disease or orange peel tissue (Cellulite).

The invention has the beneficial effects that:

the tag protein in the recombinant Clostridium histolyticum type II collagenase provided by the invention can promote the soluble and high-efficiency expression of the recombinant Clostridium histolyticum type II collagenase, and does not influence the biological activity of the recombinant Clostridium histolyticum type II collagenase;

the tag protein is fused to the N end of the Clostridium histolyticum type II collagenase through a joint to form a fusion protein for fusion expression, and the product is completely separated by a purification means, so that the soluble and high-efficiency expression of the Clostridium histolyticum type II collagenase is realized.

Meanwhile, the flexible connecting peptide provided by the invention ensures that the fusion protein label does not influence the structure of the target protein, and the product has biological activity; contains enterokinase site-DDDDK-to effectively remove the tag protein, the finally obtained N-terminal sequence of the recombinant Clostridium histolyticum type II collagenase is completely consistent with the natural sequence, and a histidine tag can be further added to facilitate purification and improve the yield.

The nucleotide sequence of the Clostridium histolyticum type II collagenase is optimized, and simultaneously, Fh8 is selected as a tag protein, so that the recombinant Clostridium histolyticum type II collagenase can realize efficient and soluble expression in escherichia coli.

The recombinant Clostridium histolyticum type II collagenase fusion protein prepared by the method is soluble expression, and the expression amount is more than 20% of the total protein of the thallus. Finally, the yield of the recombinant Clostridium histolyticum type II collagenase is more than 300mg pure product/100 g thallus, the purity is more than 96%, the biological activity reaches 170000U/mg, and the endotoxin is less than 5 EU/mg.

The invention utilizes the fasciola hepatica calcium binding protein Fh8 as the label protein, can efficiently promote the soluble expression of the Clostridium histolyticum type II collagenase (ColH), avoids the renaturation of the inclusion body, reduces the production cost and can meet the requirement of industrial production.

Drawings

FIG. 1 is a diagram showing the structure of expression plasmid pET30-a (+) -ColH constructed in the present invention;

FIG. 2 shows the shake flask induction screening result of pET30-a (+) -ColH/BL21(DE3) engineering bacteria constructed by the present invention;

FIG. 3 shows the RP-HPLC detection result, the purity is more than 96%;

FIG. 4 shows the result of detecting the N-terminal amino acid sequence of recombinant ColH;

FIG. 5a is a mass spectrometric total ion flux (TIC) profile of recombinant Clostridium histolyticum collagenase type II according to the invention;

FIG. 5b is a first mass spectrum of recombinant Clostridium histolyticum collagenase type II according to the invention;

FIG. 5c is a deconvolution of recombinant Clostridium histolyticum collagenase type II according to the invention;

FIG. 5d is an enlarged view of the deconvolution of recombinant Clostridium histolyticum collagenase type II according to the invention;

FIG. 6a is a graph of a leucine standard curve for determining enzyme activity;

FIG. 6b is a statistical chart of the results of the enzyme activity assay for recombinant Clostridium histolyticum collagenase type II according to the invention.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of recombinant Clostridium histolyticum type II collagenase fusion protein engineering bacteria

1. Construction of pET30-a (+) -ColH/BL21(DE3) engineering bacteria

The Clostridium histolyticum collagenase II mature protein contains 991 amino acids, and the Clostridium histolyticum collagenase II gene sequence is shown in SEQ ID NO. 1.

The tag protein is selected from Fasciola hepatica calcium binding protein Fh8(GenBank accession number: AAF 31420.1); the amino acid sequence of the tag protein is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 3.

Design linker sequence is SEQ ID NO: 7-GSGSGHMHHHHHHSSGPDLDDDDK-connecting the C end of the Fh8 tag protein with the N end of the mature protein sequence of Clostridium histolyticum collagenase II through a joint, wherein the amino acid sequence of the obtained fusion protein is shown as SEQ ID NO.4, and the nucleic acid sequence of the fusion protein is shown as SEQ ID NO. 5.

The designed fusion protein nucleic acid sequence (SEQ ID NO.5) was committed to the general biological System (Anhui) Co., Ltd for whole gene synthesis and subcloned into an expression vector via NdeI/NotI site, wherein the vector was selected from the group consisting of commercial vector pET-28a-c (+), pET29a, pET-30a-c (+), pET39b (+), pET-40b (+), pET-41a (+), and pET-43.1a (+), and the host cell of the vector was a bacterium or a fungus, and the host cell was Escherichia coli selected from commercial strain BL21(DE3), BL21(DE3) PlySS, and TB 1. In this example, an expression vector pET30-a (+) -ColH was constructed using an E.coli expression vector pET30-a (+), and the construction scheme is shown in FIG. 1.

The constructed expression vector is transferred into an escherichia coli expression host bacterium BL21(DE3) by a chemical transfection method, and a recombinant expression engineering bacterium pET30-a (+) -ColH/BL21(DE3) is obtained by screening LB + kan solid plates.

2. Induced screening of engineering bacteria

The above-mentioned pET30-a (+) -ColH/BL21(DE3) engineered bacteria were randomly picked up and inoculated into 10ml of LB medium (100ug/ml kan), cultured in 100ml Erlenmeyer flask, and shake-cultured overnight at 37 ℃ and 220 rpm. The next day, the overnight cultured mother solution was transferred to 40ml LB liquid medium (100ug/ml kan) at a ratio of 1%, and cultured in a 250ml Erlenmeyer flask at 37 ℃ and 220rpm for 2.5 hours to OD₆₀₀The value is about 0.6-1.0. A final concentration of 1mM IPTG was added and induction was carried out at 30 ℃ for 4 h.

SDS-PAGE, the results are shown in FIG. 2, lanes 1-4: pET30-a (+) -ColH/BL21(DE3) No. 1-4 induces total protein for 4 h; comparison: total protein was not induced; lane M: protein molecular weight Marker. Compared with a control, the specific expression is realized at the position higher than 97kD, and the expression amount accounts for more than 20 percent of the total protein of the thallus. The bacteria breaking analysis is carried out on the induced expression bacteria, which shows that all soluble expression is realized.

Example 2 fermentation of recombinant Clostridium histolyticum type II collagenase engineering bacteria

Activating seed liquid: 60ul of the pET30-a (+) -ColH/BL21(DE3) glycerotube strain was transferred to 100ml of 2XYT medium, kan +100 ug/ml; the culture temperature is 33 ℃, 220r/min, and the culture time is about 12h (OD value is 4.5-5.5).

And (3) fermentation process: fermenting with 5L fermentation tank, setting temperature at 37 deg.C, ventilating for 3vvm, inoculating 4ml microelement, kan +0.06g sterile filtering, inoculating into fermentation tank, and adjusting pH to 7.0; inoculating 5% (100ml) of seed prepared in LB medium stored at 4 deg.C into 2L fermentation medium (M9 modified medium); starting the rotation speed of 300rpm, starting the rotation speed dissolved oxygen linkage, setting the dissolved oxygen value to be 30%, when the rotation speed is increased to 600r/min, setting the dissolved oxygen value to be 15%, and increasing to 800r/min, adjusting the ventilation volume to be 4vvm, changing the rotation speed linkage into manual operation, rebounding the dissolved oxygen (about OD value of 31) for material supplement, and setting the material supplement speed to be 100ml/h-130ml/h according to the dissolved oxygen not less than 30%; when the culture OD value is 36-42 (about 3-4h), setting the temperature to be 25 ℃, when the temperature reaches below 26 ℃, inducing for 4h, taking samples every 1h after induction, measuring the OD value, and observing the growth of the thalli and the protein expression condition, wherein the inducer is isopropyl-beta-D-thiogalactoside (IPTG).

Example 3 separation and purification of recombinant Clostridium histolyticum collagenase II

1. Crushing and clarifying thallus

And (3) freezing and centrifuging the thallus at a high speed to collect the thallus, and performing centrifugation according to the following steps of 1: 10 (1 g: 10ml buffer solution: 20mM Tris-HCl pH7.6), suspending the thalli, breaking the thalli for 3 times under the ice bath temperature of 10-15 ℃ controlled by 700-800 Bar, then freezing and centrifuging at high speed, collecting supernatant, and discarding the precipitate; and (4) performing ultrafiltration and clarification on the bacteria-breaking liquid by using a hollow fiber column with the diameter of 0.65 mu m.

2、Ni²⁺Chelate chromatography

Loading the disrupted solution on an equilibrium chromatographic column by using an equilibrium buffer solution of 25mM Tris-HCl pH8.0, and then eluting the hybrid protein by using an elution buffer solution of 25mM Tris-HCl pH8.010mM imidazole; the fusion protein of interest was eluted with elution buffer 25mM Tris-HCl pH 8.050mM imidazole.

3. Enterokinase enzymolysis

Adding 0.8U of recombinant enterokinase into the fusion protein solution according to each milligram of protein, carrying out enzymolysis on the fusion protein for 12 hours at the enzymolysis temperature of 4-8 ℃.

4. QFF preliminary purification

Filtering the sample after enzymolysis with 0.45 μ M cellulose membrane, adjusting pH to 7.6 with 4M hydrochloric acid, diluting with ultrapure water to conductivity less than 2800 μ s/cm, equilibrating the chromatographic column with equilibration buffer (20mM Tris-HCl pH7.6), introducing sample with the sample volume of 10-15 times the column volume, eluting with elution buffer 50mM NaCl, 20mM Tris-HCl pH7.6 to elute impurities; the desired protein was eluted with 100mM NaCl, 20mM Tris-HCl pH7.6 buffer.

5. Butyl-4FF chromatography

The column was equilibrated with equilibration buffer 20mM Tris-HCl pH7.6, 2M NaCl, loaded with the sample obtained from the previous QFF chromatography, then eluted with elution buffer 1.6M NaCl, 20mM Tris-HCl pH7.6 for impurities, eluted with 1.4M NaCl, 20mM Tris-HCl pH7.6 for the protein of interest, and washed with 20mM Tris-HCl pH 7.6.

6. Phenyl 6FF chromatography

Equilibration buffer 20mM Tris-HCl pH7.6, 1M NaCl equilibration chromatography column, loading the sample obtained from the previous step of F chromatography, eluting impurities with elution buffer 0.4M NaCl, 20mM Tris-HCl pH7.6, eluting the target protein with 0.1M NaCl, 20mM Tris-HCl pH7.6 buffer,

7. QFF flow-through chromatography

The Phenyl 6FF chromatography was concentrated by ultrafiltration membrane to 5-fold, the conductivity was controlled at 4800-. Collecting flow through (target component), ultrafiltering to concentrate flow through component, concentrating by about 5 times, and protein concentration reaching 1.0-1.5 mg/ml. Purity by HPLC was greater than 96%, see fig. 3.

Example 4 determination of the physico-chemical Properties of the collagenase type II of recombinant Clostridium histolyticum

1. Determination of N-terminal amino acid sequence

The recombinant Clostridium histolyticum collagenase (rColH) prepared by the method is subjected to N-terminal amino acid sequence determination by the limited Biotech of Beijing Baitai park, and the determination result is consistent with the N-terminal sequence of the mature ColH protein, which is shown in figure 4.

2. Mass Spectrometry molecular weight

Mass spectrometry of rColH prepared by the above method was carried out, and the molecular weight determined by Nanjing Kingsry Biotech Co., Ltd was 112971.6Da, which is equivalent to the theoretical molecular weight. The corresponding mass spectrum Total Ion Current (TIC) spectrum is shown in fig. 5a, the primary mass spectrum is shown in fig. 5b, the deconvolution diagram is shown in fig. 5c, and the deconvolution magnification is shown in fig. 5 d.

Example 5 recombinant Clostridium histolyticum collagenase type II bioactivity assay

The biological activity of the recombinant collagenase ColH is detected by a GPA-fluorescence amine method, the principle is that a synthetic peptide Carbobenzoxy-glycyl-L-prolyl-L-alanine (hexapeptide) is a substrate of the collagenase ColH, the synthetic peptide can be cut into Carbobenzoxy-glycyl-L-prolyl-glycyl (ZGPG) and glycyl-L-prolyl-L-alanine (GPA), the N end of the GPA has an alpha-amino group, the content of the GPA can be quantitatively detected through a fluorescamine labeling reaction, and the activity of the ColH is reflected by the fact that the released GPA corresponds to the nmol number of leucine per minute.

1. Experimental methods

(1) Heating a water bath to 37 ℃ for preparation, placing the mixture at 4 ℃ for later use except the fluorescamine solution and the substrate hexapeptide, and taking out the rest reagents from a refrigerator and balancing the reagents to room temperature for later use;

(2) mu.L of substrate (20. mu.g) and 37/42/57. mu.L of HEPES buffer were added to each sample tube; 57/62/67 μ L of HEPES buffer was added to the enzyme control tube; putting the above tubes into 37 deg.C water bath for 10 min;

(3) 18/13/8 μ L of diluted enzyme solution was added to the sample tube and enzyme control tube; shaking and mixing the tubes, centrifuging for a short time, and continuing to carry out water bath at 37 ℃ for 30 min;

(4) adding 25 μ L of 20mM phenanthroline into all tubes, vortex mixing, centrifuging for a short time, placing in 37 deg.C water bath for 10min, taking out, and placing at 4 deg.C (for use on the same day) or-20 deg.C (for use on every other day) for fluorescamine reaction;

(5) leucine gradient solution for standard preparation: adding 10 mu L, 20 mu L, 30 mu L, 40 mu L and leucine working solution into an EP tube, supplementing boric acid buffer solution to 100 mu L, and taking 100 mu L boric acid buffer solution as a blank tube;

(6) fluorescamine reaction: respectively adding 50 mu L of 3.3mg/mL acetone-fluorescamine solution into all the test tubes, respectively adding 850 mu L boric acid buffer solution after uniformly mixing the solution by oscillation, uniformly mixing the solution by oscillation and centrifuging the solution for a short time; placing in water bath at 25 deg.C for reaction for 20 min;

(7) and (3) detection: after the reaction time of all the test tubes with the fluorescamine is up to the reaction time, the test tubes are evenly mixed by oscillation and are centrifuged at 12000rpm for 5min, then 200 mu L of solution is respectively added into a black 96-hole enzyme label plate, and the black 96-hole enzyme label plate is excited on an enzyme label instrument at 390nm and emits at 490nm to read the detection value.

2. Calculation of enzyme Activity

(1) Drawing a standard curve according to the quality of the leucine and the fluorescence value of 490nm, and linearly fitting to obtain a standard curve formula;

(2) subtracting the fluorescence value of the enzyme reference tube from the fluorescence value of each sample tube to obtain the net fluorescence value of each sample tube, substituting the net fluorescence value into a leucine marking formula to obtain the corresponding leucine quality;

(3) the unit of activity of the enzyme is defined as: collagenase AUX-II degrades hexapeptide at 37 deg.C, pH7.2, releasing GPA in 1 activity unit corresponding to 1nmol leucine marked by fluorescamine, i.e. U is leucine quality g × 1000 × 5/131.2 × 30; 131.2 in the formula is leucine relative to molecular mass, 5 is the coefficient of 0.2mL detection volume to 1mL total fluorescamine reaction volume, and 30 is GPA hydrolysis reaction time.

Enzyme activity (U/mL) × (U/mg enzyme amount involved in reaction) × (concentration of stock solution protein mg/mL)

Specific activity (U/mg) is U/amount of enzyme participating in the reaction mg

3. Results of the experiment

The leucine standard curve is shown in FIG. 6a, and the enzyme activity statistics are shown in FIG. 6 b. The rColH specific activity was calculated to be 179425U/mg according to the standard curve, with an RSD of 6.6%.

The above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention. The scope of the invention is to be determined by the appended claims.

Sequence listing

SEQ ID NO. 1: nucleotide sequence of clostridium histolyticum type II collagenase mature protein

GCCGTTGATAAAAATAATGCAACCGCAGCCGTGCAGAATGAAAGCAAACGTTATACCGTTAGTTATCTGAAAACCCTGAATTATTACGATCTGGTTGATCTGCTGGTGAAAACCGAAATTGAAAATCTGCCGGATCTGTTTCAGTATAGTAGCGATGCCAAAGAATTTTATGGCAATAAGACCCGTATGAGTTTTATTATGGATGAAATTGGCCGTCGTGCACCGCAGTATACCGAAATTGATCATAAAGGTATTCCGACCCTGGTGGAAGTGGTTCGTGCAGGTTTTTATCTGGGTTTTCATAATAAGGAACTGAATGAAATCAACAAGCGTAGTTTTAAAGAGCGTGTTATTCCGAGTATTCTGGCAATTCAGAAAAATCCGAATTTTAAACTGGGTACAGAAGTTCAGGATAAAATTGTGAGCGCCACCGGCCTGCTGGCCGGCAATGAAACCGCCCCGCCGGAAGTGGTGAATAATTTTACCCCGATTATTCAGGATTGCATTAAGAATatgGATCGTTATGCACTGGATGATCTGAAAAGCAAAGCACTGTTTAATGTTCTGGCAGCCCCGACCTATGATATTACCGAATATCTGCGTGCAACCAAAGAAAAACCGGAAAATACCCCGTGGTATGGCAAAATTGATGGTTTTATTAACGAGCTGAAGAAACTGGCACTGTATGGCAAAATCAATGATAATAATAGCTGGATTATCGACAACGGTATCTATCATATTGCACCGCTGGGTAAACTGCATAGCAATAATAAGATTGGTATCGAAACCCTGACCGAAGTGATGAAAATTTATCCGTATCTGAGTATGCAGCATCTGCAGAGTGCAGATCAGATTGAACGTCATTATGATAGTAAAGATGCAGAAGGTAATAAGATTCCGCTGGATAAATTCAAAAAGGAAGGCAAAGAAAAGTACTGCCCGAAAACCTATACCTTTGATGATGGCAAAGTGATTATTAAGGCCGGCGCCCGCGTTGAAGAAGAAAAAGTGAAACGTCTGTATTGGGCCAGCAAAGAAGTTAATAGCCAGTTTTTCCGCGTGTATGGTATTGATAAACCGCTGGAAGAAGGCAATCCGGATGATATTCTGACCATGGTTATCTATAATAGCCCGGAAGAATATAAACTGAATAGCGTGCTGTATGGCTATGATACCAATAATGGTGGTATGTATATCGAACCGGATGGCACCTTTTTCACCTATGAACGCAAAGCAGAAGAAAGCACCTATACCCTGGAAGAACTGTTTCGTCATGAATATACCCATTATCTGCAGGGTCGTTATGCAGTTCCGGGTCAGTGGGGTCGCACCAAACTGTATGATAATGATCGCCTGACCTGGTATGAAGAAGGTGGTGCAGAACTGTTTGCAGGCAGTACCCGTACCAGTGGCATTCTGCCGCGTAAAAGTATTGTTAGTAATATTCATAACACCACCCGCAATAATCGCTATAAACTGAGCGATACCGTTCATAGTAAATATGGTGCAAGCTTTGAATTTTACAACTATGCCTGCATGTTTATGGATTATATGTATAATAAGGACATGGGTATCCTGAATAAGCTGAATGATCTGGCCAAAAATAATGATGTGGATGGCTATGATAACTATATTCGTGATCTGAGCAGCAATCATGCACTGAATGATAAATATCAGGACCACATGCAGGAACGTATTGATAATTATGAAAACCTGACCGTGCCGTTTGTGGCAGATGATTATCTGGTGCGTCATGCCTATAAAAATCCGAACGAAATCTATAGCGAAATTAGTGAAGTTGCCAAACTGAAAGATGCAAAAAGCGAAGTGAAAAAATCACAGTATTTCAGTACCTTTACCCTGCGTGGCAGCTATACCGGCGGTGCAAGCAAAGGCAAACTGGAAGATCAGAAAGCAATGAATAAGTTTATCGATGATAGCCTGAAAAAGCTGGATACCTATAGTTGGAGTGGTTATAAAACCCTGACCGCCTATTTTACCAATTATAAAGTTGATAGCAGCAACCGCGTTACCTATGATGTTGTTTTTCATGGTTATCTGCCGAATGAAGGTGACAGCAAAAATAGTCTGCCGTATGGTAAAATTAACGGCACCTATAAAGGTACAGAAAAAGAAAAAATCAAGTTCAGTAGCGAAGGCAGCTTTGATCCGGATGGCAAAATTGTGAGTTATGAATGGGATTTTGGCGATGGCAATAAGAGTAATGAAGAAAATCCGGAACATAGTTATGATAAAGTTGGTACATACACCGTTAAACTGAAAGTTACCGATGATAAAGGTGAAAGTAGTGTGAGCACCACCACCGCAGAAATTAAGGATCTGAGTGAAAATAAGCTGCCGGTTATCTATATGCATGTGCCGAAAAGTGGCGCACTGAATCAGAAAGTTGTGTTTTATGGCAAAGGTACATACGATCCGGATGGTAGCATTGCAGGTTATCAGTGGGATTTTGGTGACGGCAGTGATTTTAGTAGTGAACAGAATCCGAGCCATGTGTATACCAAAAAAGGCGAATATACCGTGACCCTGCGCGTTATGGATAGTAGCGGTCAGATGAGCGAAAAAACCATGAAAATTAAGATCACCGATCCGGTTTATCCGATTGGCACCGAAAAAGAACCGAATAATAGCAAAGAAACCGCAAGCGGTCCGATTGTTCCGGGCATTCCGGTTAGCGGCACCATTGAAAATACCAGTGATCAGGATTATTTCTATTTCGATGTTATCACCCCGGGCGAAGTGAAAATTGATATTAATAAGCTGGGTTACGGCGGCGCCACCTGGGTGGTTTATGATGAAAATAATAATGCCGTGAGTTACGCCACCGATGATGGCCAGAATCTGAGTGGTAAATTCAAAGCCGATAAACCGGGCCGTTATTATATTCATCTGTATATGTTTAACGGTAGCTATATGCCGTATCGTATTAATATTGAAGGCAGTGTTGGCCGT

SEQ ID NO. 2: amino acid sequence of tag protein

MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSS

SEQ ID NO. 3: nucleotide sequence ATGCCGAGTGTTCAGGAAGTGGAAAAACTGCTGCATGTGCTGGATCGTAATGGTGACGGCAAAGTTAGCGCAGAAGAACTGAAAGCATTTGCAGATGATAGCAAATGCCCGCTGGATAGTAATAAGATTAAGGCATTCATTAAGGAGCATGATAAAAATAAGGACGGCAAACTGGATCTGAAAGAACTGGTTAGTATTCTGAGTAGC of tag protein

SEQ ID NO. 4: amino acid sequence of recombinant Clostridium histolyticum type II collagenase fusion protein

MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSSGSGSGHMHHHHHHSSGPDLDDDDKAVDKNNATAAVQNESKRYTVSYLKTLNYYDLVDLLVKTEIENLPDLFQYSSDAKEFYGNKTRMSFIMDEIGRRAPQYTEIDHKGIPTLVEVVRAGFYLGFHNKELNEINKRSFKERVIPSILAIQKNPNFKLGTEVQDKIVSATGLLAGNETAPPEVVNNFTPIIQDCIKNMDRYALDDLKSKALFNVLAAPTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYGKINDNNSWIIDNGIYHIAPLGKLHSNNKIGIETLTEVMKIYPYLSMQHLQSADQIERHYDSKDAEGNKIPLDKFKKEGKEKYCPKTYTFDDGKVIIKAGARVEEEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEEYKLNSVLYGYDTNNGGMYIEPDGTFFTYERKAEESTYTLEELFRHEYTHYLQGRYAVPGQWGRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVSNIHNTTRNNRYKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLNDLAKNNDVDGYDN008YIRDLSSNHALNDKYQDHMQERIDNYENLTVPFVADDYLVRHAYK NPNEIYSEISEVAKLKDAKSEVKKSQYFSTFTLRGSYTGGASKGKLEDQKAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNYKVDSSNRVTYDVVFHGYLPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEGSFDPDGKIVSYEWDFGDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIKDLSENKLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDFSSEQNPSHVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEPNNSKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGYGGATWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMPYRINIEGSVGR

SEQ ID No. 5: nucleotide sequence of recombinant Clostridium histolyticum type II collagenase fusion protein

ATGCCGAGTGTTCAGGAAGTGGAAAAACTGCTGCATGTGCTGGATCGTAATGGTGACGGCAAAGTTAGCGCAGAAGAACTGAAAGCATTTGCAGATGATAGCAAATGCCCGCTGGATAGTAATAAGATTAAGGCATTCATTAAGGAGCATGATAAAAATAAGGACGGCAAACTGGATCTGAAAGAACTGGTTAGTATTCTGAGTAGCGGTTCTGGTTCTGGCCACATGCACCATCATCATCATCATTCTTCTGGTCCAGATCTGGATGATGATGATAAAGCCGTTGATAAAAATAATGCAACCGCAGCCGTGCAGAATGAAAGCAAACGTTATACCGTTAGTTATCTGAAAACCCTGAATTATTACGATCTGGTTGATCTGCTGGTGAAAACCGAAATTGAAAATCTGCCGGATCTGTTTCAGTATAGTAGCGATGCCAAAGAATTTTATGGCAATAAGACCCGTATGAGTTTTATTATGGATGAAATTGGCCGTCGTGCACCGCAGTATACCGAAATTGATCATAAAGGTATTCCGACCCTGGTGGAAGTGGTTCGTGCAGGTTTTTATCTGGGTTTTCATAATAAGGAACTGAATGAAATCAACAAGCGTAGTTTTAAAGAGCGTGTTATTCCGAGTATTCTGGCAATTCAGAAAAATCCGAATTTTAAACTGGGTACAGAAGTTCAGGATAAAATTGTGAGCGCCACCGGCCTGCTGGCCGGCAATGAAACCGCCCCGCCGGAAGTGGTGAATAATTTTACCCCGATTATTCAGGATTGCATTAAGAATatgGATCGTTATGCACTGGATGATCTGAAAAGCAAAGCACTGTTTAATGTTCTGGCAGCCCCGACCTATGATATTACCGAATATCTGCGTGCAACCAAAGAAAAACCGGAAAATACCCCGTGGTATGGCAAAATTGATGGTTTTATTAACGAGCTGAAGAAACTGGCACTGTATGGCAAAATCAATGATAATAATAGCTGGATTATCGACAACGGTATCTATCATATTGCACCGCTGGGTAAACTGCATAGCAATAATAAGATTGGTATCGAAACCCTGACCGAAGTGATGAAAATTTATCCGTATCTGAGTATGCAGCATCTGCAGAGTGCAGATCAGATTGAACGTCATTATGATAGTAAAGATGCAGAAGGTAATAAGATTCCGCTGGATAAATTCAAAAAGGAAGGCAAAGAAAAGTACTGCCCGAAAACCTATACCTTTGATGATGGCAAAGTGATTATTAAGGCCGGCGCCCGCGTTGAAGAAGAAAAAGTGAAACGTCTGTATTGGGCCAGCAAAGAAGTTAATAGCCAGTTTTTCCGCGTGTATGGTATTGATAAACCGCTGGAAGAAGGCAATCCGGATGATATTCTGACCATGGTTATCTATAATAGCCCGGAAGAATATAAACTGAATAGCGTGCTGTATGGCTATGATACCAATAATGGTGGTATGTATATCGAACCGGATGGCACCTTTTTCACCTATGAACGCAAAGCAGAAGAAAGCACCTATACCCTGGAAGAACTGTTTCGTCATGAATATACCCATTATCTGCAGGGTCGTTATGCAGTTCCGGGTCAGTGGGGTCGCACCAAACTGTATGATAATGATCGCCTGACCTGGTATGAAGAAGGTGGTGCAGAACTGTTTGCAGGCAGTACCCGTACCAGTGGCATTCTGCCGCGTAAAAGTATTGTTAGTAATATTCATAACACCACCCGCAATAATCGCTATAAACTGAGCGATACCGTTCATAGTAAATATGGTGCAAGCTTTGAATTTTACAACTATGCCTGCATGTTTATGGATTATATGTATAATAAGGACATGGGTATCCTGAATAAGCTGAATGATCTGGCCAAAAATAATGATGTGGATGGCTATGATAACTATATTCGTGATCTGAGCAGCAATCATGCACTGAATGATAAATATCAGGACCACATGCAGGAACGTATTGATAATTATGAAAACCTGACCGTGCCGTTTGTGGCAGATGATTATCTGGTGCGTCATGCCTATAAAAATCCGAACGAAATCTATAGCGAAATTAGTGAAGTTGCCAAACTGAAAGATGCAAAAAGCGAAGTGAAAAAATCACAGTATTTCAGTACCTTTACCCTGCGTGGCAGCTATACCGGCGGTGCAAGCAAAGGCAAACTGGAAGATCAGAAAGCAATGAATAAGTTTATCGATGATAGCCTGAAAAAGCTGGATACCTATAGTTGGAGTGGTTATAAAACCCTGACCGCCTATTTTACCAATTATAAAGTTGATAGCAGCAACCGCGTTACCTATGATGTTGTTTTTCATGGTTATCTGCCGAATGAAGGTGACAGCAAAAATAGTCTGCCGTATGGTAAAATTAACGGCACCTATAAAGGTACAGAAAAAGAAAAAATCAAGTTCAGTAGCGAAGGCAGCTTTGATCCGGATGGCAAAATTGTGAGTTATGAATGGGATTTTGGCGATGGCAATAAGAGTAATGAAGAAAATCCGGAACATAGTTATGATAAAGTTGGTACATACACCGTTAAACTGAAAGTTACCGATGATAAAGGTGAAAGTAGTGTGAGCACCACCACCGCAGAAATTAAGGATCTGAGTGAAAATAAGCTGCCGGTTATCTATATGCATGTGCCGAAAAGTGGCGCACTGAATCAGAAAGTTGTGTTTTATGGCAAAGGTACATACGATCCGGATGGTAGCATTGCAGGTTATCAGTGGGATTTTGGTGACGGCAGTGATTTTAGTAGTGAACAGAATCCGAGCCATGTGTATACCAAAAAAGGCGAATATACCGTGACCCTGCGCGTTATGGATAGTAGCGGTCAGATGAGCGAAAAAACCATGAAAATTAAGATCACCGATCCGGTTTATCCGATTGGCACCGAAAAAGAACCGAATAATAGCAAAGAAACCGCAAGCGGTCCGATTGTTCCGGGCATTCCGGTTAGCGGCACCATTGAAAATACCAGTGATCAGGATTATTTCTATTTCGATGTTATCACCCCGGGCGAAGTGAAAATTGATATTAATAAGCTGGGTTACGGCGGCGCCACCTGGGTGGTTTATGATGAAAATAATAATGCCGTGAGTTACGCCACCGATGATGGCCAGAATCTGAGTGGTAAATTCAAAGCCGATAAACCGGGCCGTTATTATATTCATCTGTATATGTTTAACGGTAGCTATATGCCGTATCGTATTAATATTGAAGGCAGTGTTGGCCGT

SEQ ID NO. 6: nucleotide sequence of linker

GGTTCTGGTTCTGGCCACATGCACCATCATCATCATCATTCTTCTGGTCCAGATCTGGATGATGATGATAAA

SEQ ID NO. 7: amino acid sequence of linker

GSGSGHMHHHHHHSSGPDLDDDDK

Sequence listing

<110> Chongqing Kerun biological medicine research & development Co Ltd

<120> recombinant Clostridium histolyticum type II collagenase and preparation method and application thereof

<141> 2021-04-01

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2973

<212> DNA

<213> Clostridium histolyticum (C. histolyticum)

<400> 1

gccgttgata aaaataatgc aaccgcagcc gtgcagaatg aaagcaaacg ttataccgtt 60

agttatctga aaaccctgaa ttattacgat ctggttgatc tgctggtgaa aaccgaaatt 120

gaaaatctgc cggatctgtt tcagtatagt agcgatgcca aagaatttta tggcaataag 180

acccgtatga gttttattat ggatgaaatt ggccgtcgtg caccgcagta taccgaaatt 240

gatcataaag gtattccgac cctggtggaa gtggttcgtg caggttttta tctgggtttt 300

cataataagg aactgaatga aatcaacaag cgtagtttta aagagcgtgt tattccgagt 360

attctggcaa ttcagaaaaa tccgaatttt aaactgggta cagaagttca ggataaaatt 420

gtgagcgcca ccggcctgct ggccggcaat gaaaccgccc cgccggaagt ggtgaataat 480

tttaccccga ttattcagga ttgcattaag aatatggatc gttatgcact ggatgatctg 540

aaaagcaaag cactgtttaa tgttctggca gccccgacct atgatattac cgaatatctg 600

cgtgcaacca aagaaaaacc ggaaaatacc ccgtggtatg gcaaaattga tggttttatt 660

aacgagctga agaaactggc actgtatggc aaaatcaatg ataataatag ctggattatc 720

gacaacggta tctatcatat tgcaccgctg ggtaaactgc atagcaataa taagattggt 780

atcgaaaccc tgaccgaagt gatgaaaatt tatccgtatc tgagtatgca gcatctgcag 840

agtgcagatc agattgaacg tcattatgat agtaaagatg cagaaggtaa taagattccg 900

ctggataaat tcaaaaagga aggcaaagaa aagtactgcc cgaaaaccta tacctttgat 960

gatggcaaag tgattattaa ggccggcgcc cgcgttgaag aagaaaaagt gaaacgtctg 1020

tattgggcca gcaaagaagt taatagccag tttttccgcg tgtatggtat tgataaaccg 1080

ctggaagaag gcaatccgga tgatattctg accatggtta tctataatag cccggaagaa 1140

tataaactga atagcgtgct gtatggctat gataccaata atggtggtat gtatatcgaa 1200

ccggatggca cctttttcac ctatgaacgc aaagcagaag aaagcaccta taccctggaa 1260

gaactgtttc gtcatgaata tacccattat ctgcagggtc gttatgcagt tccgggtcag 1320

tggggtcgca ccaaactgta tgataatgat cgcctgacct ggtatgaaga aggtggtgca 1380

gaactgtttg caggcagtac ccgtaccagt ggcattctgc cgcgtaaaag tattgttagt 1440

aatattcata acaccacccg caataatcgc tataaactga gcgataccgt tcatagtaaa 1500

tatggtgcaa gctttgaatt ttacaactat gcctgcatgt ttatggatta tatgtataat 1560

aaggacatgg gtatcctgaa taagctgaat gatctggcca aaaataatga tgtggatggc 1620

tatgataact atattcgtga tctgagcagc aatcatgcac tgaatgataa atatcaggac 1680

cacatgcagg aacgtattga taattatgaa aacctgaccg tgccgtttgt ggcagatgat 1740

tatctggtgc gtcatgccta taaaaatccg aacgaaatct atagcgaaat tagtgaagtt 1800

gccaaactga aagatgcaaa aagcgaagtg aaaaaatcac agtatttcag tacctttacc 1860

ctgcgtggca gctataccgg cggtgcaagc aaaggcaaac tggaagatca gaaagcaatg 1920

aataagttta tcgatgatag cctgaaaaag ctggatacct atagttggag tggttataaa 1980

accctgaccg cctattttac caattataaa gttgatagca gcaaccgcgt tacctatgat 2040

gttgtttttc atggttatct gccgaatgaa ggtgacagca aaaatagtct gccgtatggt 2100

aaaattaacg gcacctataa aggtacagaa aaagaaaaaa tcaagttcag tagcgaaggc 2160

agctttgatc cggatggcaa aattgtgagt tatgaatggg attttggcga tggcaataag 2220

agtaatgaag aaaatccgga acatagttat gataaagttg gtacatacac cgttaaactg 2280

aaagttaccg atgataaagg tgaaagtagt gtgagcacca ccaccgcaga aattaaggat 2340

ctgagtgaaa ataagctgcc ggttatctat atgcatgtgc cgaaaagtgg cgcactgaat 2400

cagaaagttg tgttttatgg caaaggtaca tacgatccgg atggtagcat tgcaggttat 2460

cagtgggatt ttggtgacgg cagtgatttt agtagtgaac agaatccgag ccatgtgtat 2520

accaaaaaag gcgaatatac cgtgaccctg cgcgttatgg atagtagcgg tcagatgagc 2580

gaaaaaacca tgaaaattaa gatcaccgat ccggtttatc cgattggcac cgaaaaagaa 2640

ccgaataata gcaaagaaac cgcaagcggt ccgattgttc cgggcattcc ggttagcggc 2700

accattgaaa ataccagtga tcaggattat ttctatttcg atgttatcac cccgggcgaa 2760

gtgaaaattg atattaataa gctgggttac ggcggcgcca cctgggtggt ttatgatgaa 2820

aataataatg ccgtgagtta cgccaccgat gatggccaga atctgagtgg taaattcaaa 2880

gccgataaac cgggccgtta ttatattcat ctgtatatgt ttaacggtag ctatatgccg 2940

tatcgtatta atattgaagg cagtgttggc cgt 2973

<210> 2

<211> 69

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Pro Ser Val Gln Glu Val Glu Lys Leu Leu His Val Leu Asp Arg

1 5 10 15

Asn Gly Asp Gly Lys Val Ser Ala Glu Glu Leu Lys Ala Phe Ala Asp

20 25 30

Asp Ser Lys Cys Pro Leu Asp Ser Asn Lys Ile Lys Ala Phe Ile Lys

35 40 45

Glu His Asp Lys Asn Lys Asp Gly Lys Leu Asp Leu Lys Glu Leu Val

50 55 60

Ser Ile Leu Ser Ser

65

<210> 3

<211> 207

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgccgagtg ttcaggaagt ggaaaaactg ctgcatgtgc tggatcgtaa tggtgacggc 60

aaagttagcg cagaagaact gaaagcattt gcagatgata gcaaatgccc gctggatagt 120

aataagatta aggcattcat taaggagcat gataaaaata aggacggcaa actggatctg 180

aaagaactgg ttagtattct gagtagc 207

<210> 4

<211> 1084

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Pro Ser Val Gln Glu Val Glu Lys Leu Leu His Val Leu Asp Arg

1 5 10 15

Asn Gly Asp Gly Lys Val Ser Ala Glu Glu Leu Lys Ala Phe Ala Asp

20 25 30

Asp Ser Lys Cys Pro Leu Asp Ser Asn Lys Ile Lys Ala Phe Ile Lys

35 40 45

Glu His Asp Lys Asn Lys Asp Gly Lys Leu Asp Leu Lys Glu Leu Val

50 55 60

Ser Ile Leu Ser Ser Gly Ser Gly Ser Gly His Met His His His His

65 70 75 80

His His Ser Ser Gly Pro Asp Leu Asp Asp Asp Asp Lys Ala Val Asp

85 90 95

Lys Asn Asn Ala Thr Ala Ala Val Gln Asn Glu Ser Lys Arg Tyr Thr

100 105 110

Val Ser Tyr Leu Lys Thr Leu Asn Tyr Tyr Asp Leu Val Asp Leu Leu

115 120 125

Val Lys Thr Glu Ile Glu Asn Leu Pro Asp Leu Phe Gln Tyr Ser Ser

130 135 140

Asp Ala Lys Glu Phe Tyr Gly Asn Lys Thr Arg Met Ser Phe Ile Met

145 150 155 160

Asp Glu Ile Gly Arg Arg Ala Pro Gln Tyr Thr Glu Ile Asp His Lys

165 170 175

Gly Ile Pro Thr Leu Val Glu Val Val Arg Ala Gly Phe Tyr Leu Gly

180 185 190

Phe His Asn Lys Glu Leu Asn Glu Ile Asn Lys Arg Ser Phe Lys Glu

195 200 205

Arg Val Ile Pro Ser Ile Leu Ala Ile Gln Lys Asn Pro Asn Phe Lys

210 215 220

Leu Gly Thr Glu Val Gln Asp Lys Ile Val Ser Ala Thr Gly Leu Leu

225 230 235 240

Ala Gly Asn Glu Thr Ala Pro Pro Glu Val Val Asn Asn Phe Thr Pro

245 250 255

Ile Ile Gln Asp Cys Ile Lys Asn Met Asp Arg Tyr Ala Leu Asp Asp

260 265 270

Leu Lys Ser Lys Ala Leu Phe Asn Val Leu Ala Ala Pro Thr Tyr Asp

275 280 285

Ile Thr Glu Tyr Leu Arg Ala Thr Lys Glu Lys Pro Glu Asn Thr Pro

290 295 300

Trp Tyr Gly Lys Ile Asp Gly Phe Ile Asn Glu Leu Lys Lys Leu Ala

305 310 315 320

Leu Tyr Gly Lys Ile Asn Asp Asn Asn Ser Trp Ile Ile Asp Asn Gly

325 330 335

Ile Tyr His Ile Ala Pro Leu Gly Lys Leu His Ser Asn Asn Lys Ile

340 345 350

Gly Ile Glu Thr Leu Thr Glu Val Met Lys Ile Tyr Pro Tyr Leu Ser

355 360 365

Met Gln His Leu Gln Ser Ala Asp Gln Ile Glu Arg His Tyr Asp Ser

370 375 380

Lys Asp Ala Glu Gly Asn Lys Ile Pro Leu Asp Lys Phe Lys Lys Glu

385 390 395 400

Gly Lys Glu Lys Tyr Cys Pro Lys Thr Tyr Thr Phe Asp Asp Gly Lys

405 410 415

Val Ile Ile Lys Ala Gly Ala Arg Val Glu Glu Glu Lys Val Lys Arg

420 425 430

Leu Tyr Trp Ala Ser Lys Glu Val Asn Ser Gln Phe Phe Arg Val Tyr

435 440 445

Gly Ile Asp Lys Pro Leu Glu Glu Gly Asn Pro Asp Asp Ile Leu Thr

450 455 460

Met Val Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn Ser Val Leu

465 470 475 480

Tyr Gly Tyr Asp Thr Asn Asn Gly Gly Met Tyr Ile Glu Pro Asp Gly

485 490 495

Thr Phe Phe Thr Tyr Glu Arg Lys Ala Glu Glu Ser Thr Tyr Thr Leu

500 505 510

Glu Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Gly Arg Tyr

515 520 525

Ala Val Pro Gly Gln Trp Gly Arg Thr Lys Leu Tyr Asp Asn Asp Arg

530 535 540

Leu Thr Trp Tyr Glu Glu Gly Gly Ala Glu Leu Phe Ala Gly Ser Thr

545 550 555 560

Arg Thr Ser Gly Ile Leu Pro Arg Lys Ser Ile Val Ser Asn Ile His

565 570 575

Asn Thr Thr Arg Asn Asn Arg Tyr Lys Leu Ser Asp Thr Val His Ser

580 585 590

Lys Tyr Gly Ala Ser Phe Glu Phe Tyr Asn Tyr Ala Cys Met Phe Met

595 600 605

Asp Tyr Met Tyr Asn Lys Asp Met Gly Ile Leu Asn Lys Leu Asn Asp

610 615 620

Leu Ala Lys Asn Asn Asp Val Asp Gly Tyr Asp Asn Tyr Ile Arg Asp

625 630 635 640

Leu Ser Ser Asn His Ala Leu Asn Asp Lys Tyr Gln Asp His Met Gln

645 650 655

Glu Arg Ile Asp Asn Tyr Glu Asn Leu Thr Val Pro Phe Val Ala Asp

660 665 670

Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr Ser

675 680 685

Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val Lys

690 695 700

Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr Gly

705 710 715 720

Gly Ala Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys Phe

725 730 735

Ile Asp Asp Ser Leu Lys Lys Leu Asp Thr Tyr Ser Trp Ser Gly Tyr

740 745 750

Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser Asn

755 760 765

Arg Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Asn Glu Gly

770 775 780

Asp Ser Lys Asn Ser Leu Pro Tyr Gly Lys Ile Asn Gly Thr Tyr Lys

785 790 795 800

Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Phe Asp

805 810 815

Pro Asp Gly Lys Ile Val Ser Tyr Glu Trp Asp Phe Gly Asp Gly Asn

820 825 830

Lys Ser Asn Glu Glu Asn Pro Glu His Ser Tyr Asp Lys Val Gly Thr

835 840 845

Tyr Thr Val Lys Leu Lys Val Thr Asp Asp Lys Gly Glu Ser Ser Val

850 855 860

Ser Thr Thr Thr Ala Glu Ile Lys Asp Leu Ser Glu Asn Lys Leu Pro

865 870 875 880

Val Ile Tyr Met His Val Pro Lys Ser Gly Ala Leu Asn Gln Lys Val

885 890 895

Val Phe Tyr Gly Lys Gly Thr Tyr Asp Pro Asp Gly Ser Ile Ala Gly

900 905 910

Tyr Gln Trp Asp Phe Gly Asp Gly Ser Asp Phe Ser Ser Glu Gln Asn

915 920 925

Pro Ser His Val Tyr Thr Lys Lys Gly Glu Tyr Thr Val Thr Leu Arg

930 935 940

Val Met Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met Lys Ile Lys

945 950 955 960

Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn

965 970 975

Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser

980 985 990

Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val

995 1000 1005

Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly

1010 1015 1020

Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr

1025 1030 1035 1040

Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys

1045 1050 1055

Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met

1060 1065 1070

Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg

1075 1080

<210> 5

<211> 3252

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgccgagtg ttcaggaagt ggaaaaactg ctgcatgtgc tggatcgtaa tggtgacggc 60

aaagttagcg cagaagaact gaaagcattt gcagatgata gcaaatgccc gctggatagt 120

aataagatta aggcattcat taaggagcat gataaaaata aggacggcaa actggatctg 180

aaagaactgg ttagtattct gagtagcggt tctggttctg gccacatgca ccatcatcat 240

catcattctt ctggtccaga tctggatgat gatgataaag ccgttgataa aaataatgca 300

accgcagccg tgcagaatga aagcaaacgt tataccgtta gttatctgaa aaccctgaat 360

tattacgatc tggttgatct gctggtgaaa accgaaattg aaaatctgcc ggatctgttt 420

cagtatagta gcgatgccaa agaattttat ggcaataaga cccgtatgag ttttattatg 480

gatgaaattg gccgtcgtgc accgcagtat accgaaattg atcataaagg tattccgacc 540

ctggtggaag tggttcgtgc aggtttttat ctgggttttc ataataagga actgaatgaa 600

atcaacaagc gtagttttaa agagcgtgtt attccgagta ttctggcaat tcagaaaaat 660

ccgaatttta aactgggtac agaagttcag gataaaattg tgagcgccac cggcctgctg 720

gccggcaatg aaaccgcccc gccggaagtg gtgaataatt ttaccccgat tattcaggat 780

tgcattaaga atatggatcg ttatgcactg gatgatctga aaagcaaagc actgtttaat 840

gttctggcag ccccgaccta tgatattacc gaatatctgc gtgcaaccaa agaaaaaccg 900

gaaaataccc cgtggtatgg caaaattgat ggttttatta acgagctgaa gaaactggca 960

ctgtatggca aaatcaatga taataatagc tggattatcg acaacggtat ctatcatatt 1020

gcaccgctgg gtaaactgca tagcaataat aagattggta tcgaaaccct gaccgaagtg 1080

atgaaaattt atccgtatct gagtatgcag catctgcaga gtgcagatca gattgaacgt 1140

cattatgata gtaaagatgc agaaggtaat aagattccgc tggataaatt caaaaaggaa 1200

ggcaaagaaa agtactgccc gaaaacctat acctttgatg atggcaaagt gattattaag 1260

gccggcgccc gcgttgaaga agaaaaagtg aaacgtctgt attgggccag caaagaagtt 1320

aatagccagt ttttccgcgt gtatggtatt gataaaccgc tggaagaagg caatccggat 1380

gatattctga ccatggttat ctataatagc ccggaagaat ataaactgaa tagcgtgctg 1440

tatggctatg ataccaataa tggtggtatg tatatcgaac cggatggcac ctttttcacc 1500

tatgaacgca aagcagaaga aagcacctat accctggaag aactgtttcg tcatgaatat 1560

acccattatc tgcagggtcg ttatgcagtt ccgggtcagt ggggtcgcac caaactgtat 1620

gataatgatc gcctgacctg gtatgaagaa ggtggtgcag aactgtttgc aggcagtacc 1680

cgtaccagtg gcattctgcc gcgtaaaagt attgttagta atattcataa caccacccgc 1740

aataatcgct ataaactgag cgataccgtt catagtaaat atggtgcaag ctttgaattt 1800

tacaactatg cctgcatgtt tatggattat atgtataata aggacatggg tatcctgaat 1860

aagctgaatg atctggccaa aaataatgat gtggatggct atgataacta tattcgtgat 1920

ctgagcagca atcatgcact gaatgataaa tatcaggacc acatgcagga acgtattgat 1980

aattatgaaa acctgaccgt gccgtttgtg gcagatgatt atctggtgcg tcatgcctat 2040

aaaaatccga acgaaatcta tagcgaaatt agtgaagttg ccaaactgaa agatgcaaaa 2100

agcgaagtga aaaaatcaca gtatttcagt acctttaccc tgcgtggcag ctataccggc 2160

ggtgcaagca aaggcaaact ggaagatcag aaagcaatga ataagtttat cgatgatagc 2220

ctgaaaaagc tggataccta tagttggagt ggttataaaa ccctgaccgc ctattttacc 2280

aattataaag ttgatagcag caaccgcgtt acctatgatg ttgtttttca tggttatctg 2340

ccgaatgaag gtgacagcaa aaatagtctg ccgtatggta aaattaacgg cacctataaa 2400

ggtacagaaa aagaaaaaat caagttcagt agcgaaggca gctttgatcc ggatggcaaa 2460

attgtgagtt atgaatggga ttttggcgat ggcaataaga gtaatgaaga aaatccggaa 2520

catagttatg ataaagttgg tacatacacc gttaaactga aagttaccga tgataaaggt 2580

gaaagtagtg tgagcaccac caccgcagaa attaaggatc tgagtgaaaa taagctgccg 2640

gttatctata tgcatgtgcc gaaaagtggc gcactgaatc agaaagttgt gttttatggc 2700

aaaggtacat acgatccgga tggtagcatt gcaggttatc agtgggattt tggtgacggc 2760

agtgatttta gtagtgaaca gaatccgagc catgtgtata ccaaaaaagg cgaatatacc 2820

gtgaccctgc gcgttatgga tagtagcggt cagatgagcg aaaaaaccat gaaaattaag 2880

atcaccgatc cggtttatcc gattggcacc gaaaaagaac cgaataatag caaagaaacc 2940

gcaagcggtc cgattgttcc gggcattccg gttagcggca ccattgaaaa taccagtgat 3000

caggattatt tctatttcga tgttatcacc ccgggcgaag tgaaaattga tattaataag 3060

ctgggttacg gcggcgccac ctgggtggtt tatgatgaaa ataataatgc cgtgagttac 3120

gccaccgatg atggccagaa tctgagtggt aaattcaaag ccgataaacc gggccgttat 3180

tatattcatc tgtatatgtt taacggtagc tatatgccgt atcgtattaa tattgaaggc 3240

agtgttggcc gt 3252

<210> 6

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggttctggtt ctggccacat gcaccatcat catcatcatt cttctggtcc agatctggat 60

gatgatgata aa 72

<210> 7

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Ser Gly Ser Gly His Met His His His His His His Ser Ser Gly

1 5 10 15

Pro Asp Leu Asp Asp Asp Asp Lys

20

Claims

1. A recombinant clostridium histolyticum type II collagenase, comprising clostridium histolyticum type II collagenase and a tag protein linked to the N-terminus of the clostridium histolyticum type II collagenase; the tag protein comprises hepcidium calcium binding protein (Fh 8); preferably, the amino acid sequence of the Fasciola hepatica calcium binding protein is SEQ ID NO. 2.

2. The recombinant Clostridium histolyticum collagenase type II according to claim 1, wherein said tag protein further comprises one of transcription termination anti-termination factor (NusA), Escherichia coli thioredoxin A (TrxA), disulfide oxidoreductase (DsbA, DsbC) or glutathione S-transferase (GST).

3. The recombinant Clostridium histolyticum collagenase type II according to claim 1, wherein the tag protein is linked to the N-terminus of Clostridium histolyticum collagenase type II via a flexible linker peptide; preferably, the amino acid sequence of the flexible connecting peptide is SEQ ID NO. 7.

4. The recombinant Clostridium histolyticum collagenase type II according to claim 1, having the amino acid sequence SEQ ID NO 4.

5. A recombinant gene encoding the recombinant Clostridium histolyticum collagenase type II according to any one of claims 1 to 4, comprising Clostridium histolyticum collagenase type II encoding gene SEQ ID NO:1 and a signature protein encoding gene upstream of the Clostridium histolyticum collagenase type II encoding gene, wherein the signature protein encoding gene comprises a gene encoding Fasciola hepatica calcium binding protein (Fh 8); preferably, the encoding gene of the Fasciola hepatica calcium binding protein (Fh8) is SEQ ID NO. 3.

6. The recombinant gene encoding recombinant Clostridium histolyticum collagenase type II according to claim 5, wherein the nucleotide sequence of said recombinant gene is SEQ ID NO 5.

7. A recombinant vector for expressing recombinant Clostridium histolyticum collagenase type II, comprising the recombinant gene of claim 5 or 6; preferably, the backbone plasmid of the recombinant vector is selected from any one of pET-28a-c (+), pET29a, pET-30a-c (+), pET39b (+), pET-40b (+), pET-41a (+) or pET-43.1a (+).

8. A recombinant engineered bacterium for expressing recombinant clostridium histolyticum type II collagenase, comprising the recombinant vector of claim 7; preferably, the host bacterium of the recombinant engineering bacterium is a bacterium or a fungus; preferably, the host bacterium is escherichia coli, and more preferably any one of BL21(DE3), BL21(DE3) P1ysS, or TB 1.

9. A method for preparing Clostridium histolyticum collagenase type II, comprising:

1) ligating a gene encoding a tag protein upstream of a collagenase type II encoding gene of clostridium histolyticum to obtain a recombinant gene encoding the recombinant clostridium histolyticum collagenase type II of any one of claims 1 to 4;

6) enzymolyzing the recombinant Clostridium histolyticum collagenase II;

10. Use of a recombinant Clostridium histolyticum type II collagenase according to any one of claims 1-4 for the preparation of a medicament for the treatment of Dupuytren's contracture, Pelonie's disease or Cellulite.