CN100485036C

CN100485036C - Fennero penaeus chinensis antibacterial protein gene and recombinant expression and use

Info

Publication number: CN100485036C
Application number: CNB2005100472287A
Authority: CN
Inventors: 相建海; 张继泉; 李富花; 王在照
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2005-09-19
Filing date: 2005-09-19
Publication date: 2009-05-06
Anticipated expiration: 2025-09-19
Also published as: WO2007033529A1; CN1936000A

Abstract

The invention relates to a Fenneropenaeus chinensis antimicrobial protein gene and the recombination expression and the application. It has the amino acid sequence of SEQ ID No.2 and basic group sequence of SEQ ID No.1 in sequence table. It selects the carrier of the antimicrobial protein pCR T7/NT TOPO TA and host bacterium BL21(DE3)pLysS that could high efficiently express recombination protein. Aimed at the feature of plural disulfide bonds, using the oxidation reduction system made of oxidized form of glutathione and glutathione in separating and purification and protein recombination process, the recombination protein with antimicrobial ability would be gained. The protein has strong inhibition to the growth of gram positivebacteria, and some inhibition to gram negativebacteria.

Description

A kind of Crustin antibiotic protein gene and recombinant expressed and application

Technical field

The present invention relates to antibacterial protein in the genetically engineered field (crustin) expression of gene, specifically a kind of Crustin antibiotic protein gene and recombinant expressed and application.

Background technology

Prawn culturing is one of mainstay industry of sea farming, and along with the continuous growth of market to high-quality fishery products demand, shrimp culture occupies more and more important position in aquaculture.Since nineteen nineties, the large-scale outbreak of disease makes mariculture industry suffer tremendous loss, and it is fundamentally controlled that the deterioration of antibiotic abuse and environment makes that the disease of marine cultured animal is difficult to.

The sea farming kind moves in the water surrounding that is rich in various microorganisms, and secular environmental stress makes it form effective defense function.Antibacterial peptide (albumen) is considered to one of main component of systems of defense such as fish, shrimp, shellfish.The 26S Proteasome Structure and Function of the antibacterial peptide gene by research sea farming kind, and then carry out recombinant expressedly external by genetic engineering technique, will promise to be and can substitute antibiotic newtype drug.

The immune defense system is the basis of zoonosis power, particularly hangs down and waits invertebrates, and their immune response is much simpler than higher animal, mainly depends on endogenous nonspecific immune reaction.Studies show that in recent years; hang down and wait invertebrates when external source harmful microorganism (as bacterium, fungi or virus) injury is avoided self in protection, non-specific antimicrobial factors in its body such as antibacterial peptide (albumen), N,O-Diacetylmuramidase, Transferrins,iron complexes etc. play important effect.Wherein, antibacterial peptide (albumen) is to compile the small molecule polypeptide (albumen) that produces by animal and plant cell specific gene, has anti-microbial activity.20th century the mid-1970s, Sweden scientist Boman etc. have obtained first kind of antibacterial peptide one cecropin (cecropin) in the world inducing with E.coli to separate when cherishing guppy giant silkworm (Hyatophora cecropia).After this, people find to exist similar polypeptide (albumen) with anti-microbial activity in succession in organisms such as other insect, Amphibians, Mammals, people, plant, crustacean, mollusk.Crustin is a kind of small molecular protein with very strong inhibition gram-positive bacteria activity that is obtained by separation and purification in beach crab (Carcinusmaenas) body such as Relf the earliest, himself is difficult for inactivation, even after being boiled, still can keep very high reactivity (J.M.Relf, J.R.S.Chisholm, G.D.Kemp and V.J.Smith, Purification and characterization of acysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes ofthe shore crab, Carcinus maenas Eur.J.Biochem.1999; 264 (2): 350-357).After this, people have successively found the existence of crustin or antibacterial protein analogue (crustin-like) gene from shrimps such as Environment of Litopenaeus vannamei Low, white shore prawn, Japanese capsule prawn and tigar prawn.

The mechanism of action of crustin is different with traditional microbiotic, and its target site mainly is the pathogen cells film, therefore is difficult for producing resistance.At present, many pathogenic bacterias progressively produce resistance to existing microbiotic, and the discovery of new antibiotic is extremely difficult, and therefore, the research of crustin has been opened up wide prospect for the development of new antibacterials.

In a word, the research of crustin and its alloytype antibacterial peptide (albumen) not only has important significance for theories, also has huge application potential in practice, and therefore, it has become one of focus of current biological medicine research.

Summary of the invention

Purpose is to provide a kind of Crustin antibiotic protein gene and high efficiency recombinant expressed and application thereof.

Technical solution of the present invention is as follows:

Crustin antibiotic protein gene: have base sequence among the special tabulation SEQ ID No.1 and the aminoacid sequence shown in the SEQ ID No.2.

Described Crustin antibiotic protein gene recombinant expressed: from Crustin cDNA library, be cloned into a kind of antibiotic protein gene, utilize the gene constructed prokaryotic expression carrier of its mature peptide, filter out a kind of recombinant expression system that is used to efficiently express the Crustin antibiotic protein gene, acquisition contains the Laemmli buffer system Laemmli of the prokaryotic expression recombinant protein highly efficient renaturation of disulfide linkage, thereby obtains activated reorganization antibacterial protein; Be specially:

1. gene clone

According to pointing out of Crustin EST, utilize the RACE technology, from hemocyte cDNA library, clone goal gene;

2. the selection of recombinant expression vector and host bacterium

Determine to carry out recombinant expressed expression vector with the inclusion body form

T7/NT

TA and can alleviate the toxic host bacterium of destination gene expression product BL21 (DE3) pLysS;

3. the structure of recombinant expression vector

Utilize the feature of expression vector for the T carrier, adopt a pair of Auele Specific Primer clone mature peptide gene, the PCR product reclaims the back and directly is connected with expression, makes up recombinant expression vector;

4. transform and screening

With recombinant expression vector transformed into escherichia coli BL21 (DE3) pLysS that structure obtains, the screening engineering bacteria also carries out abduction delivering;

5. the separation and purification of expression product

Adopt inductor isopropyl-abduction delivering engineering bacteria, the ultrasonic disruption cell utilizes the method purifying antibacterial protein of metal-chelating column chromatography, protein renaturation, and the fusion tag excision obtains activated reorganization antibacterial protein;

Described protein renaturation adopts the Laemmli buffer system Laemmli of the prokaryotic expression recombinant protein highly efficient renaturation that contains disulfide linkage, adds Sleep-promoting factor B and reduced glutathion in renaturation buffered soln; Its concentration ratio: renaturation buffered soln (PBS): Sleep-promoting factor B: reduced glutathion=50:1:10; The renaturation pH value of buffer solution is 7.2 ~ 7.4;

Described a pair of Auele Specific Primer is:

CD-F：5’CAG?AAT?AAA?GAC?GAT?ACT?CG?3’；

CD-R：5’CTA?TCC?CTC?AGA?ACC?CAG?3’。

Described Crustin antibiotic protein gene is all inhibited to the growth of gram-positive microorganism and Gram-negative bacteria.

The present invention has following advantage:

1. the present invention has determined a kind of whole encoding sequences and non-coding sequence of Crustin antibiotic protein gene, thereby also understood fully amino acid preface and signal peptide cutting site thereof that it is coded, utilized the mature peptide aminoacid sequence of being derived to carry out recombinant expressed this gene.

2. the present invention filters out and can carry out recombinant expressed carrier of antibacterial protein and host bacterium effectively, has the proteic recombinant expressed foundation that provides of antibacterial to studying other.

3. the present invention provides a suitable redox system to the separation and purification and the protein renaturation of the recombinant protein that contains disulfide linkage, can effectively guarantee the correct formation of disulfide linkage in the recombinant protein.

4. of far-reaching significance.Can develop effective control medicine by the present invention to shrimp bacterial disease.Simultaneously, the research of separation and purification that other is had the recombinant expressed of antimicrobial active protein and be easy to the recombinant protein of oxidation provides new thinking.

Embodiment

Below in conjunction with embodiment the present invention is described in further detail.

Embodiment

A kind of clone's Crustin antibiotic protein gene has following sequence:

(1) information of SEQ ID No 1 (referring to sequence table)

(a) sequence signature:

* length: 523 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: cDNA

(c) suppose: not

(d) antisense: not

(e) initial source: Crustin (Fenneropenaeus chinensis)

Sequence description: SEQ ID No.1

GAGTCCGTTCATCGCACAGCCGAGAGAAACACTATCAAGATGTTGAAGTTTGTAGTATTATCCGTTGTCGCCGTG

GCTGTGGTACACGCCCAGAATAAAGACGATACTCGCTTCCTTGGCGGAGTTCCTGGGGTTGGGGGTGGCTTCGTT

CCAGGGGTTCCTGGGCATGGCGGCGTTGCTCCTGTAGGCGGTGGCCTTGTCCCTGGCGGCGGTGCCTTATCCCT

GGAGGTGGATTCGAGTGCAATTACTGCAGAACGAGGTACGGATACGTATGCTGCAAGCCCGGCAGGTGTCCACCG

GTTCGCGACGTCTGCCCAGGCCTCAGGCAGGCAGGCTGTCCCGATCTGCCGTCAGGACACCGACTGTCGGCTCCGAC

AAATGCTGCTACGACGTCTGCTTGAACGACACCGTCTGCAAACCCATCGTGCTGGGTTCTGAGGGATAGGCCTGC

ATGTTTAACCCTTCAATTCTACTTTATTAAATAAATACTATAACTGTTAATTGCTTAAAAAAAAAAAAAAAAA

(2) information of SEQ ID No.2 (referring to sequence table)

(a) sequence signature

* length: 134 amino acid

* type: amino acid

* chain: strand

* topological framework: linearity

(b) molecule type: albumen

Sequence description: SEQ ID No.2

MLKFVVLSVVAVAVVHAQNKDDTRFLGGVPGVGGGFVPGVPGHGGVAPVGGGLVPGGGGLIPGGGFECN

YCRTRYGYVCCKPGRCPPVRDVCPGLRQGVPICRQDTDCFGSDKCCYDVCLNDTVCKPIVLGSEG

The Crustin gene in vitro is recombinant expressed, separation and purification and biological activity analysis:

From Crustin cDNA library, screen a kind of antibacterial protein (crustin) gene, utilize the gene constructed prokaryotic expression carrier of its mature peptide, filter out a kind of recombinant expression system that is used to efficiently express the Crustin antibiotic protein gene, find a kind of Laemmli buffer system Laemmli that is used to contain the prokaryotic expression recombinant protein highly efficient renaturation of disulfide linkage, obtained activated reorganization antibacterial protein.Specific as follows:

1) gene clone

According to the prompting of Crustin EST, utilize the RACE technology, from hemocyte cDNA library, clone goal gene;

2) selection of recombinant expression vector and host bacterium

Because the recombination expression product of goal gene has stronger bacteriostatic activity, the present invention screens present several business-like expression vectors and host bacterium, and has determined to carry out recombinant expressed carrier with the inclusion body form

T7/NT

TA and can alleviate the toxic host bacterium of destination gene expression product BL21 (DE3) pLysS.

Described recombinant expression system can be expressed antibacterial protein efficiently, and expression efficiency reaches 845mg/L.

3) structure of recombinant expression vector

Utilize the feature of expression vector for the T carrier, design a pair of Auele Specific Primer (CD-F:5 ' CAG AAT AAA GAC GAT ACT CG 3 '; CD-R:5 ' CTA TCC CTC AGA ACC CAG 3 ') clone's mature peptide gene, the PCR product reclaims the back and directly is connected with expression, makes up recombinant expression vector.

4) screening of abduction delivering and induced product

Recombinant expression vector is transformed expressive host bacterium BL21 (DE3) pLysS, in final concentration is the LB substratum of the penbritin of 100 μ g/mL and 34 μ g/mL paraxin, cultivate cell concentration to 0D ₆₀₀Be about 0.6 (37 ℃ 250r/min), are added final concentration and carry out abduction delivering 4h for 1mmol/L IPTG; Adopt inductor isopropyl-(being called for short IPTG) screening abduction delivering engineering bacteria then.

5) separation and purification

After inducing end, the ultrasonic disruption cell utilizes the method purifying antibacterial protein of metal-chelating column chromatography, protein renaturation is specifically: centrifugal collection bacterium mud, and the weighing weight in wet base is by adding buffer A (50mmol/L sodium phosphate at 1: 10, the 6mol/L Guanidinium hydrochloride, 300mmol/L sodium-chlor, 1mmol/L Sleep-promoting factor B, the 10mmol/L reduced glutathion, pH7.2), ultrasonic disruption, 4 ℃, 12, centrifugal 30min under the 000r/min collects supernatant.Utilize Co-NTA-Agarose under the sex change condition, to carry out separation and purification.Recombinant protein after the separation and purification, containing 50mmol/L PBS damping fluid (the 1mmol/L Sleep-promoting factor B of 8mol/L, 4mol/L, 2mol/L, 1mol/L and 0.5mol/L urea successively, the 10mmol/L reduced glutathion, pH7.4) each 8h of dialysis in, Guanidinium hydrochloride in the lysate is replaced out, remove urea at 50mmol/L pH7.4 PBS damping fluid equilibrated Sephadex G-75 chromatography column, collect elutriant.

6) enzyme cuts except that fusion tag

The protein concentration of elutriant is diluted to 2mg/mL, utilizes the EKMax of Invitrogen company ^TMTest kit carry out fusion tag excision (25 ℃, 24h).

7) bacteriostatic experiment

Albumen behind the removal fusion tag is concentrated into 64 μ mol/L, simultaneously with the negative contrast of the PBS of 50mmol/L pH7.4.Utilize Oxford cup agar diffusion method to detect its bacteriostatic activity to gram-positive microorganism streptococcus aureus (Staphylococcusaureu), subtilis (Bacillus subtilis), micrococcus luteus (Micrococcus luteus) and Gram-negative bacteria intestinal bacteria (Escherichia coli), Vibrio anguillarum (Vibrio anguillarum).That is: with bacterial strain to be detected respectively at 37 ℃, overnight incubation under the 200r/min; Next day, centrifugal thalline also washed thalline 2 times with the PBS of the 50mmol/L pH7.4 that sterilizes, and utilized PBS dilution thalline to 10 ⁶Cells/mL, get 100 μ l and evenly coat the LB planar surface, after in 37 ℃ of incubators, cultivating 2h, take out dull and stereotyped at the equidistant placement of agar surface Oxford cup, in the cup of Oxford, add the recombinant protein of different concns gradient, while as negative control, continues to cultivate 22h with PBS in 37 ℃ of incubators, take out observations.

The result shows that the recombinant protein of 4 μ mol/L can suppress the growth of the gram-positive microorganism (streptococcus aureus, subtilis and micrococcus luteus) that detected effectively, and the recombinant protein of 16 μ mol/L can suppress the growth of the Gram-negative bacteria (intestinal bacteria and Vibrio anguillarum) that detected effectively.

The successful clone of this gene and the bioactive evaluation of recombinant protein have the important application prospect in the exploitation of novel antibacterial medicine.

Sequence table

SEQUENCE?LISTING

＜110〉Institute of Oceanology of the Chinese Academy of Sciences

＜120〉a kind of Crustin antibiotic protein gene and recombinant expressed and application

<130>

<160>2

<170>PatentIn?version?3.1

<210>1

<211>523

<212>DNA

＜213〉Crustin (Fenneropenaeus chinensis)

<220>

<221>ORF

<222> (40)..(444)

<223>

<400>1

<210>2

<211>134

<212>PRT

＜213〉Crustin (Fenneropenaeus chinensis)

<400>2

Claims

1. Crustin antibiotic protein gene, the base sequence that it is characterized in that this gene is the SEQ ID No.1 in the sequence table.

2. by the Crustin antibacterial protein of the described genes encoding of claim 1, it is characterized in that this proteic aminoacid sequence is the SEQ ID No.2 in the sequence table.

3. recombinant expressed method according to the described Crustin antibiotic protein gene of claim 1, it is characterized in that: from Crustin cDNA library, be cloned into a kind of antibiotic protein gene, utilize the gene constructed prokaryotic expression carrier of its mature peptide, filter out a kind of recombinant expression system that is used to efficiently express the Crustin antibiotic protein gene, acquisition contains the Laemmli buffer system Laemmli of the prokaryotic expression recombinant protein highly efficient renaturation of disulfide linkage, thereby obtains activated reorganization antibacterial protein; Be specially:

1) gene clone: according to the prompting of Crustin EST, utilize the RACE technology, from hemocyte cDNA library, clone goal gene;

2) selection of recombinant expression vector and host bacterium: determine to carry out recombinant expressed expression vector with the inclusion body form

T7/NT

3) structure of recombinant expression vector: utilize the feature of expression vector for the T carrier, adopt a pair of Auele Specific Primer clone mature peptide gene, the PCR product reclaims the back and directly is connected with expression, makes up recombinant expression vector;

4) transform and screening: with recombinant expression vector transformed into escherichia coli BL21 (DE3) pLysS that structure obtains, the screening engineering bacteria also carries out abduction delivering;

5) separation and purification of expression product: adopt inductor isopropyl-abduction delivering engineering bacteria, the ultrasonic disruption cell utilizes the method purifying antibacterial protein of metal-chelating column chromatography, protein renaturation, the fusion tag excision obtains activated reorganization antibacterial protein.

4. in accordance with the method for claim 3, it is characterized in that: described protein renaturation adopts the Laemmli buffer system Laemmli of the prokaryotic expression recombinant protein highly efficient renaturation that contains disulfide linkage, and Laemmli buffer system Laemmli is to add Sleep-promoting factor B and reduced glutathion in PBS renaturation buffered soln; PBS, Sleep-promoting factor B and reduced glutathion concentration ratio are 50:1:10 in the Laemmli buffer system Laemmli; PBS renaturation pH value of buffer solution is 7.2-7.4, and concentration is 50mmol/L.

5. it is characterized in that in accordance with the method for claim 3: described a pair of Auele Specific Primer is:

CD-F：5’?CAG?AAT?AAA?GAC?GAT?ACT?CG3’；

CD-R：5’?CTA?TCC?CTC?AGA?ACC?CAG3’。