CN111087463B

CN111087463B - Recombinant human III-type collagen and prokaryotic expression method thereof

Info

Publication number: CN111087463B
Application number: CN201911384510.2A
Authority: CN
Inventors: 徐兰举; 齐磊; 刘鑫; 申翠美; 杜亚东
Original assignee: Hebei Naco Biotechnology Co ltd
Current assignee: Hebei Naco Biotechnology Co ltd; Shandong Furuida Biology Co Ltd
Priority date: 2019-12-28
Filing date: 2019-12-28
Publication date: 2021-02-19
Anticipated expiration: 2039-12-28
Also published as: CN111087463A

Abstract

The invention relates to a recombinant human type III collagen and a prokaryotic expression method thereof, wherein the amino acid sequence of the human type III collagen is shown as SEQ ID NO.1, the nucleotide sequence of a coding gene is shown as SEQ ID NO.2, a gene sequence is obtained by codon optimization and design, the gene sequence is inserted between enzyme cutting sites NdeI and XhoI of an expression vector PET30a (+) to construct a recombinant expression vector pET30-1230, competent cells of escherichia coli BL211(DE3) are transformed, positive cloning is selected, and efficient expression is carried out by culture induction.

Description

Recombinant human III-type collagen and prokaryotic expression method thereof

Technical Field

The invention belongs to the technical field of optimized coding genes, and particularly relates to a recombinant human type III collagen and a prokaryotic expression method thereof.

Background

Collagen is a glycoprotein, which is classified into more than 28 types of collagen according to differences in tissue sites, physiological functions and molecular structures, and the types of collagen, I, II and III, which are the most thoroughly studied, account for the most. Collagen in human skin is type I collagen and type III collagen, wherein the type I collagen is mainly present in adult skin, tendon and bone tissues, the type III collagen is mainly present in infant skin or blood vessel intima and intestinal tract, and the type I collagen and the type III collagen are closely related to the skin injury repair process and repair quality. The III type collagen is a three-strand spiral shape formed by twisting three peptide chains rightward, has excellent biological activity and histocompatibility, can promote fibroblast proliferation of a dermis layer, improves cell activity, is absorbed by fibroblast serving as a raw material for synthesizing the collagen, stimulates cells to synthesize more collagen, fills and repairs damaged and aged skin, reconstructs a reticular structure, enhances the expansion force of the damaged skin, and recovers the elasticity of the skin.

The complex structure of native collagen and the insolubility in water limit the development, utilization and production of native collagen. Collagen products sold in the market at present are all obtained from animal tissues such as pigs, cows, fish and the like, and are further processed by acid, alkali and enzymolysis methods, the biological activity of the collagen products is far lower than that of natural collagen of human bodies, and the collagen products cannot meet the industrial requirements of medicines, foods, cosmetics and the like. With the rapid development of DNA recombination technology, the recombination technology for type III human collagen in the prior art is reported as follows: CN110194795A discloses a recombinant human collagen and application thereof, wherein a section of sequence of a human three-type protein conserved region is selected and repeated for 8 times, the designed sequence is recombined to construct a prokaryotic expression vector pET22b, and the recombinant human collagen which can be efficiently expressed in escherichia coli and is soluble in water is finally obtained through induction expression. CN103122027A discloses a recombinant human collagen and a production method thereof, the structure is a single chain structure, the basic repeating unit is gergapgfrgpaggpngipgekgpagegap which is a human collagen III type peptide segment, the terminal sequence is GPPGPCCGGG which is a human collagen II type peptide segment. The above has the following disadvantages: the patent selects a short segment as a repeating unit, and the short segment is spliced together to express the recombinant human collagen with a long segment, so that the sequence of the human collagen cannot be well copied.

Disclosure of Invention

The invention aims to provide a recombinant human type III collagen with high biological activity and good water solubility, and simultaneously improve the prokaryotic expression efficiency.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

on the one hand, the invention provides a recombinant humanized III type collagen, the amino acid sequence of which is shown in SEQ ID NO. 1.

On the other hand, the invention provides that the nucleotide sequence of the encoding gene of the human type III collagen is shown as SEQ ID NO. 2.

In still another aspect, the invention provides a recombinant expression plasmid pET30-1230 containing the encoding gene of the human type III collagen.

In some embodiments of the invention, the gene encoding human type III collagen is inserted between the cleavage sites NdeI and XhoI of the expression vector PET30a (+).

The invention also provides a recombinant genetic engineering bacterium containing the recombinant expression plasmid pET30-1230, and the host cell of the recombinant genetic engineering bacterium is Escherichia coli BL21(DE 3).

The last aspect of the invention provides a prokaryotic expression method of the human type III collagen, which specifically comprises the following steps:

(1) gene design and synthesis: reversely designing a gene sequence according to the amino acid sequence SEQ ID NO:1 of the human type III collagen, carrying out codon optimization to obtain a gene sequence SEQ ID NO:2, and carrying out whole-gene synthesis according to the gene sequence shown in SEQ ID NO: 2;

(2) construction of recombinant expression vector pET 30-1230: 2, the coding gene of the human III type collagen is inserted between enzyme cutting sites NdeI and XhoI of an expression vector PET30a (+), and is transformed into host bacteria by a heat shock methodE.coliSelecting positive clones from DH5 alpha, and extracting plasmids by adopting a plasmid rapid extraction kit to obtain a recombinant expression vector pET 30-1230;

(3) construction of transformant BL21(DE3)/PET 30-1230: transforming the recombinant expression vector pET30-1230 into competent cells BL21(DE3) through heat shock, and screening to obtain recombinant gene engineering bacteria;

(4) inducing expression: inoculating the obtained recombinant gene engineering bacteria into an M9 culture medium, culturing overnight at 37 ℃, inoculating the obtained recombinant gene engineering bacteria into an M9 culture medium with the inoculation amount of 2%, culturing for 14h at 23 ℃, adding IPTG (0.05 mM) for induction expression, continuously culturing for 12h, and centrifugally collecting thalli.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:

the invention

(1) The type III collagen amino acid sequence selected by the invention is longer than other reports, but not repeated expression of specific short human type III collagen amino acid sequence.

(2) The amino acid sequence and the protein space structure of the recombinant human type III collagen obtained by the invention are closer to those of human type III collagen, and the recombinant human type III collagen has better bioactivity and the effects of promoting cell migration, adhesion and proliferation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a map of the recombinant expression vector pET30-1230 of the present invention;

FIG. 2 is a diagram showing the results of double digestion of the recombinant expression vector pET30-1230 of the present invention, wherein the first lane is Marker (15K), the second lane is recombinant expression vector pET30-1230 # 1, the third lane is recombinant expression vector # 1 digested with Nde I and Xho I, the fourth lane is recombinant expression vector # 2 pET30-1230, the fifth lane is recombinant expression vector # 2 digested with Nde I and Xho I, and the sixth lane is Marker (DL 2000).

FIG. 3 SDA-PAGE analysis of the electrophoresis pattern of the expression of human type III collagen protein, in which: 1. pre-staining a Marker with protein; 2. no.1 strain holoprotein is not induced by IPTG; 3. inducing the 1# strain holoprotein by IPTG; 4. no IPTG induction 3# strain holoprotein is not carried out; 5. inducing the 3# strain holoprotein by IPTG; 6. Blank; 7. no.1 strain supernatant was not induced with IPTG; 8. inducing the supernatant of the 1# strain by IPTG; 9. no. 3 strain supernatant was not induced with IPTG; 10. inducing the supernatant of the 3# strain by IPTG; 11. the protein is pre-stained with Marker.

FIG. 4 is a graph showing the results of recombinant human type III collagen identification, in which: m is Marker, 1, inducing 1# strain holoprotein by IPTG; 2. inducing the 1# strain holoprotein by IPTG; 3. no IPTG induction 3# strain holoprotein is not carried out; 4. inducing the 3# strain holoprotein by IPTG; 5. blank; 6. no.1 strain supernatant was not induced with IPTG; 7. inducing the supernatant of the 1# strain by IPTG; 8. no. 3 strain supernatant was not induced with IPTG; 9. the supernatant of the 3# strain was induced with IPTG.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail and fully with reference to the following embodiments.

The sources of reagents and materials in the examples of the invention are as follows:

the plasmid PET30a (+) was supplied by Novagen;

the DNA gel recovery kit is provided by Biotechnology engineering (Shanghai) Co., Ltd;

the plasmid rapid extraction kit is provided by biological engineering (Shanghai) Co.

Example 1 Gene design and Synthesis

(1) Gene design: based on the amino acid sequence of human type III collagen (UniProtKB/Swiss-Prot: P02461.4), SEQ ID NO: 1:

MYDSYDVKSGVAVGGLAGYPGPPGPPPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESPGGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYHHHHHH

the gene Sequence is reversely designed by using an online design tool Jcat (http:// www.jcat.de /), NdeI and XhoI enzyme cutting sites are removed in the design process aiming at the preferable codon required by the expression of host Escherichia coli, the later gene operation is facilitated, the optimized gene Sequence is shown as SEQ ID NO:2, and compared with the original gene Sequence (NCBI Reference Sequence: NM-000090.3): the rare codon of the escherichia coli is reduced from 29 wild type codons to 1, which is beneficial to the efficient expression of the gene by the escherichia coli of a host bacterium.

The optimized gene sequence is shown as SEQ ID NO:

ATGTACGACTCTTACGACGTTAAATCTGGTGTTGCTGTTGGTGGTCTGGCTGGTTACCCGGGTCCGCCGGGTCCGCCGCCGGGTCCGGCTGGTCCGCCGGGTCCGCCGGGTCCGCCGGGTACCTCTGGTCACCCGGGTTCTCCGGGTTCTCCGGGTTACCAGGGTCCGCCGGGTGAACCGGGTCAGGCTGGTCCGTCTGGTCCGCCGGGTCCGCCGGGTGCTATCGGTCCGTCTGGTCCGGCTGGTAAAGACGGTGAATCTCCGGGTGGTCGTCCGGGTCGTCCGGGTGAACGTGGTCTGCCGGGTCCGCCGGGTATCAAAGGTCCGGCGGGTATACCGGGCTTCCCGGGTATGAAGGGTCACCGTGGTTTCGACGGTCGTAACGGTGAAAAAGGTGAAACCGGTGCTCCGGGTCTGAAAGGTGAAAACGGTCTGCCGGGTGAAAACGGTGCTCCGGGTCCGATGGGTCCGCGTGGTGCTCCGGGTGAACGTGGTCGTCCGGGTCTGCCGGGTGCTGCTGGTGCTCGTGGTAACGACGGTGCTCGTGGTTCTGACGGTCAGCCGGGTCCGCCGGGTCCGCCAGGTACTGCTGGCTTCCCGGGTTCTCCAGGTGCTAAAGGTGAAGTTGGTCCGGCTGGTTCTCCGGGTTCTAACGGTGCTCCGGGTCAGCGTGGTGAACCGGGTCCGCAGGGTCACGCTGGTCCGCCGGGTCCGGTTGGTCCGGCTGGTAAATCTGGTGACCGTGGTGAATCTGGTCCGGCTGGTCCGGCTGGTGCTCCGGGTCCGGCTGGTTCTCGTGGTGCTCCGGGTCCGCAGGGTCCGCGTGGTGACAAAGGTGAAACCGGTGAACGTGGTGCTGCTGGTATCAAAGGTCACCGTGGTTTCCCGGGTAACCCGGGTGCTCCGGGTTCTCCGGGTCCGGCTGGTCAGCAGGGTGCTATCGGTTCTCCGGGTCCGGCTGGTCCGCGTGGTCCGGTTGGTCCGTCTGGTCCGCCGGGTAAAGACGGTACCTCTGGTCACCCGGGTCCGATCGGTCCGCCGGGTCCGCGTGGTAACCGTGGTGAACGTGGTTCTGAAGGTTCTCCGGGTCACCCGGGTCAGCCGGGTCCGCCGGGTCCGCCGGGTGCTCCGGGTCCGTGCTGCGGTGGTGTTGGTGCTGCTGCTATCGCTGGTATCGGTGGTGAAAAAGCTGGTGGTTTCGCTCCGTACTACCACCACCACCACCACCAC。

(2) gene synthesis:

the 1230 fragment, which is the gene sequence shown in SEQ ID NO.2, was submitted to Kinsley Biotechnology GmbH for total gene synthesis.

EXAMPLE 2 construction of recombinant expression vector pET30-1230

The gene SEQ ID NO.2 fragment obtained in example 1 was digested with NdeI and XhoI in the following manner: the digestion system was 50. mu.L, 5. mu.L (2. mu.g) of the 2-fragment of SEQ ID NO, 1. mu.L each of NdeI and XhoI enzymes, 5. mu.L of buffer, 50. mu.L of sterile double distilled water, and incubation at 37 ℃ for 4 hours. The plasmid PET30a (+) was digested with the same system. Obtaining linear fragments by enzyme digestion, and purifying by adopting a DNA gel recovery kitAnd (3) carrying out a ligation reaction on the target fragment by using T4 ligase, wherein the ligation system is as follows: t4 DNA ligase 1 uL, 1230 cleavage fragment 3 uL, PET30a (+) cleavage fragment 2 uL, ligation buffer 1 uL, sterilized double distilled water to make up 10 uL. Keeping the temperature at 16 ℃ for 4 h. Transforming the product after heat shock into host bacteriaE.coliDH5 alpha, spread on LB-culture resistant plates and incubated overnight at 37 ℃. Positive clones were randomly picked and cultured in LB liquid medium at 37 ℃ and 220rpm overnight. The plasmid is extracted by a plasmid rapid extraction kit, and the constructed plasmid PET30-1230 is obtained, as shown in figure 1 and figure 2.

EXAMPLE 3 construction of the engineered bacteria

Picking out single colony of Escherichia coli BL21(DE3) to inoculate in LB test tube, and culturing overnight at 37 deg.C under shaking; adding 0.5mL of overnight culture solution into a triangular flask containing 50mL of LB, and carrying out vigorous shaking culture at 37 ℃ for about 2h to enable the thalli to grow to the prophase of logarithm; transferring the bacteria into a 50mL polypropylene tube precooled by ice under an aseptic condition, and standing the tube on the ice for 10 min; centrifuging at 4 ℃ and 4000rpm, pouring out supernatant, and inverting the tube to enable residual liquid to flow out as much as possible; 6mL of 0.1mol/L CaCl precooled with ice are added₂Resuspending the pellet, and placing on ice for 30 min; centrifuging at 4 deg.C and 3000rpm, pouring out supernatant, and inverting the tube to allow the residual liquid to flow out as much as possible; 1.2mL of 0.1mol/L CaCl precooled with ice was added₂Resuspending the precipitate (if preparing competent cells to be preserved at-70 ℃, adding 0.1mol/L CaCl2 suspension thallus containing 20% glycerol), standing at 4 ℃ for 5-24 h, and transforming; aspirate 200. mu.L of competent cell suspension and add DNA (volume)<10μL, DNA<50 ng) and mixing gently, and standing on ice for 30 min; standing in 42 deg.C water bath, heat-shocking for 90s, immediately cooling on ice; adding 500 μ L liquid LB culture solution, mixing, placing into 37 deg.C shaking table, shaking at low speed for recovery for 45min (or directly placing into 37 deg.C water bath for recovery for 1h after adding LB, and shaking the tube to suspend the cells); the transformed cells were pipetted and spread on a plate containing antibiotics, and placed in a 37 ℃ incubator for inverted culture, and the grown colony was the transformant PET30-1230/BL21(DE 3).

Example 4 inducible expression of the engineered bacteria

A single colony is picked up in LB liquid culture medium containing 50 mu g/mL Kan, cultured at 37 ℃ and 200rpm overnight to become activated seeds, then inoculated into M9 culture medium with the inoculum size of 2 percent, cultured at 23 ℃ and 200rpm for 14h, added with IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.05mM for induction expression, cultured for 12h continuously, and centrifuged to collect thalli.

The preparation method of the M9 culture medium comprises the following steps:

(1) preparation of 1M MgSO₄: MgSO₄·7H₂Dissolving O2.46 g in 10mL of double distilled water, and sterilizing at high pressure for later use;

(2) preparation of 1M CaCl₂: CaCl₂·6H₂Dissolving O2.191 g in 10mL of double distilled water, and sterilizing at high pressure for later use;

(3) preparation of 5 × M9 salt solution: na (Na)₂PO₄·7H₂O 12.8g；KH₂PO₄ 3.0g；NaCl 0.5g；NH₄Cl 1.0 g; adding 200mL of double distilled water for dissolution, and sterilizing at 121 ℃ for 15 min;

(4) preparing a 20% glucose solution: dissolving 4g of glucose in 20mL of double distilled water, and filtering and sterilizing by using a 0.22 mu M filter;

(5) sterile procedure M9 medium was prepared: 200mL of 5 XM 9 salt solution; 1M MgSO ₄2 mL; 20mL of 20% glucose solution; 1M CaCl₂0.1 mL; sterile double distilled water was added to 1000 mL.

Experimental example 1: SDS-PAGE protein gel assay

See, J. SammBruk et al, methods of the third edition of the molecular cloning guidelines.

Sample treatment: the cells prepared in example 3 were collected, added to loadingbuffer and mixed well, boiled in a water bath for 10min, and cooled naturally for use.

A Kimura SurePAGE preformed gel (4-12%) is selected for sample loading, and electrophoresis is carried out for 45-55min under 140V voltage until a bromophenol blue band runs to the bottom of the gel.

Coomassie Brilliant blue R-250 was stained using a microwave oven: 1) preparing a dyeing solution: coomassie Brilliant blue R250 was dissolved in 40% ethanol and 10% acetic acid to a final concentration of 0.1% (W/V). 2) Preparing a decoloring solution: the final concentration of 10% (V/V) ethanol and 7.5% (V/V) acetic acid were dissolved together. 3) After electrophoresis, the gel plate is pried open to take out the gel, and then the gel is placed in a staining container filled with 100mL of staining solution. 4) Cover the container and put into a microwave oven to heat for 8min with high heat level. To avoid danger, care should be taken not to boil the solution. 5) Taking out the dyeing container from the microwave oven, and placing on a decolorizing shaking table to shake gently for 5min at normal temperature. 6) The staining solution was decanted and the gel carefully washed with deionized water. 7) The deionized water was decanted and 100mL of destaining solution was added. 8) Covering the cover, and heating in a microwave oven for 8min at high temperature. 9) And (4) pouring off the destaining solution, adding a new destaining solution, and repeating the step 8. 10) Taking out the mixture from the microwave oven, and placing the mixture on a decoloring shaking table to lightly shake the mixture at normal temperature until the background is clear. The results are shown in FIG. 3.

Experimental example 2 WesternBlot validation

1 protein Membrane transfer Using eBlot Rapid Wet transfer Instrument

1) Cutting 1 piece of filter paper and 1 piece of PVDF membrane with scissors, and marking one corner of the PVDF membrane with a pencil.

2) After activation of the PVDF membrane with methanol, the filter paper and PVDF membrane were soaked with transfer buffer.

3) Transfer buffer was added to the tray, and the sponge, PVDF membrane, filter paper and transfer clip were placed.

4) A piece of sponge is firstly paved on the black plate of the film transferring clamp, and then filter paper and gel are paved. After alignment, the bubbles were removed with a glass rod.

5) A small amount of transfer buffer solution is taken by a micropipette and placed on the gel, then a PVDF membrane is laid on the gel, and then filter paper and sponge are laid on the gel. After alignment, the bubbles were removed with a glass rod.

6) After the film transferring clamp is clamped and fixed, the black surface is put into a film transferring fixing device.

7) And placing the film rotating fixing device and the ice box filled with ice blocks in the film rotating groove. The transfer chamber was filled with transfer buffer.

8) The power supply is switched on, the voltage is generally constant at 100V-110V for about 1 h; the time can be properly shortened due to the small molecular weight; or constant pressure 30V, 4 ℃ film-transferring overnight; or constant current 300mA for 1h or so.

2. Blocking and antibody incubation

1) And (4) turning off the power supply, opening the membrane rotating clamp to take out the PVDF membrane, and flushing with double distilled water.

2) The PVDF membrane is placed in a sealing solution and sealed for 1h at 37 ℃ on a shaking table.

3) The blocking solution was discarded, washed with PBST buffer, incubated with primary antibody working solution, and incubated on a shaker at 37 ℃ for 1 h.

4) The primary antibody working solution was discarded, washed with PBST buffer, incubated with secondary antibody working solution, and incubated on a shaker at 37 ℃ for 1 h.

5) The secondary antibody working solution was discarded and washed with PBST buffer. The membrane was washed on a shaker for 4 times, 5min each time.

3. Development exposure

1) Residual liquid on the membrane was removed by blotting with flat paper, and the PVDF membrane was laid flat.

2) Equal volumes of liquid A and liquid B in the ECL reagent were removed by a micropipette and returned to room temperature in an EP tube.

3) Mixing, adding onto the membrane, and reacting in dark for 30-60 s.

4) The ECL mixed solution is discarded and placed in a dark box for exposure and development, and the exposure time is controlled to be about 30 s.

The results are shown in FIG. 4, and a specific band appears around 40kD after induction, which indicates that the SEQ ID NO.2 sequence fragment is specifically expressed due to induction, and the codon encoding 6 XHis fused to the 3' end is in the correct reading frame. Therefore, pET30-1230 constructed was correctly expressed, and the supernatant was substantially identical to the total protein in terms of the amount expressed, indicating that the expression product of 1230 was soluble.

It will be appreciated by those skilled in the art that the heat shock method of the present invention may be used in conjunction with scheme 25 or scheme 26 of chapter 1, scheme 25 or scheme 26, of sambrook et al, third edition of the molecular cloning, a laboratory manual; the formula of the LB culture resistant plate is shown in the chapter I of molecular cloning test guideline, third edition; the LB liquid medium formula is shown in chapter I of molecular cloning test manual, third edition, which is prior art and is not described in detail in this application.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Hubei Nake Biotech Co., Ltd

<120> recombinant human III type collagen and prokaryotic expression method thereof

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 410

<212> PRT

<213> type III collagen

<400> 1

Met Tyr Asp Ser Tyr Asp Val Lys Ser Gly Val Ala Val Gly Gly Leu

1 5 10 15

Ala Gly Tyr Pro Gly Pro Pro Gly Pro Pro Pro Gly Pro Ala Gly Pro

20 25 30

Pro Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly Ser Pro

35 40 45

Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly

50 55 60

Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro

65 70 75 80

Ala Gly Lys Asp Gly Glu Ser Pro Gly Gly Arg Pro Gly Arg Pro Gly

85 90 95

Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile

100 105 110

Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn

115 120 125

Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly

130 135 140

Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala

145 150 155 160

Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg

165 170 175

Gly Asn Asp Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly

180 185 190

Pro Pro Gly Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu

195 200 205

Val Gly Pro Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gln Arg

210 215 220

Gly Glu Pro Gly Pro Gln Gly His Ala Gly Pro Pro Gly Pro Val Gly

225 230 235 240

Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro

245 250 255

Ala Gly Ala Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pro Gln

260 265 270

Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Ala Gly

275 280 285

Ile Lys Gly His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser

290 295 300

Pro Gly Pro Ala Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pro Ala

305 310 315 320

Gly Pro Arg Gly Pro Val Gly Pro Ser Gly Pro Pro Gly Lys Asp Gly

325 330 335

Thr Ser Gly His Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Asn

340 345 350

Arg Gly Glu Arg Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gln Pro

355 360 365

Gly Pro Pro Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val

370 375 380

Gly Ala Ala Ala Ile Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly Phe

385 390 395 400

Ala Pro Tyr Tyr His His His His His His

405 410

<210> 2

<211> 1230

<212> DNA

<213> optimized Gene sequence (1230)

<400> 2

atgtacgact cttacgacgt taaatctggt gttgctgttg gtggtctggc tggttacccg 60

ggtccgccgg gtccgccgcc gggtccggct ggtccgccgg gtccgccggg tccgccgggt 120

acctctggtc acccgggttc tccgggttct ccgggttacc agggtccgcc gggtgaaccg 180

ggtcaggctg gtccgtctgg tccgccgggt ccgccgggtg ctatcggtcc gtctggtccg 240

gctggtaaag acggtgaatc tccgggtggt cgtccgggtc gtccgggtga acgtggtctg 300

ccgggtccgc cgggtatcaa aggtccggcg ggtataccgg gcttcccggg tatgaagggt 360

caccgtggtt tcgacggtcg taacggtgaa aaaggtgaaa ccggtgctcc gggtctgaaa 420

ggtgaaaacg gtctgccggg tgaaaacggt gctccgggtc cgatgggtcc gcgtggtgct 480

ccgggtgaac gtggtcgtcc gggtctgccg ggtgctgctg gtgctcgtgg taacgacggt 540

gctcgtggtt ctgacggtca gccgggtccg ccgggtccgc caggtactgc tggcttcccg 600

ggttctccag gtgctaaagg tgaagttggt ccggctggtt ctccgggttc taacggtgct 660

ccgggtcagc gtggtgaacc gggtccgcag ggtcacgctg gtccgccggg tccggttggt 720

ccggctggta aatctggtga ccgtggtgaa tctggtccgg ctggtccggc tggtgctccg 780

ggtccggctg gttctcgtgg tgctccgggt ccgcagggtc cgcgtggtga caaaggtgaa 840

accggtgaac gtggtgctgc tggtatcaaa ggtcaccgtg gtttcccggg taacccgggt 900

gctccgggtt ctccgggtcc ggctggtcag cagggtgcta tcggttctcc gggtccggct 960

ggtccgcgtg gtccggttgg tccgtctggt ccgccgggta aagacggtac ctctggtcac 1020

ccgggtccga tcggtccgcc gggtccgcgt ggtaaccgtg gtgaacgtgg ttctgaaggt 1080

tctccgggtc acccgggtca gccgggtccg ccgggtccgc cgggtgctcc gggtccgtgc 1140

tgcggtggtg ttggtgctgc tgctatcgct ggtatcggtg gtgaaaaagc tggtggtttc 1200

gctccgtact accaccacca ccaccaccac 1230

Claims

1. A recombinant human type III collagen is characterized in that the amino acid sequence is shown as SEQ ID NO 1.

2. The encoding gene of human type III collagen of claim 1, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID NO. 2.

3. A recombinant expression plasmid pET30-1230 comprising the gene encoding the human type III collagen of claim 2.

4. The recombinant expression plasmid pET30-1230 according to claim 3, wherein the gene encoding human type III collagen is inserted between the NdeI and XhoI cleavage sites of the expression vector PET30a (+).

5. A recombinant genetically engineered bacterium containing the recombinant expression vector pET30-1230 according to claim 3 or 4, wherein the host cell of the recombinant genetically engineered bacterium is Escherichia coli BL21(DE 3).

6. The human type III collagen prokaryotic expression method according to claim 1, characterized by comprising the following steps:

(2) construction of recombinant expression vector pET 30-1230: the encoding gene SEQ ID NO 2 of the human type III collagen is inserted between enzyme cutting sites NdeI and XhoI of an expression vector pET30a (+), and is transformed into host bacteria by a heat shock methodE .coliIn DH 5. alpha. positive clones were selected and usedExtracting plasmids by using the plasmid rapid extraction kit to obtain a recombinant expression vector pET 30-1230;

(3) construction of transformant BL21(DE3)/pET 30-1230: transforming the recombinant plasmid pET30-1230 into a competent cell BL21(DE3) through heat shock, and screening to obtain recombinant gene engineering bacteria;