WO2007033529A1

WO2007033529A1 - A gene encoding antibacterial protein of fenneropenaeus chinensis and its recombination expression method and uses

Info

Publication number: WO2007033529A1
Application number: PCT/CN2005/001560
Authority: WO
Inventors: Jianhai Xiang; Jiquan Zhang; Huhua Li; Zaizhao Wang
Original assignee: Institute Of Oceanology Chinese Academy Of Sciences
Priority date: 2005-09-19
Filing date: 2005-09-23
Publication date: 2007-03-29
Also published as: CN100485036C; CN1936000A

Abstract

A gene encoding antibacterial protein of Fenneropenaeus chinensis, its recombination expression method and uses are disclosed. The gene has the base sequence of SEQ ID No. 1 and encodes the protein having the amino acid sequence shown in SEQ ID No. 2. A vector pCR®T7/NT TOPO®TA recombination and host bacterium BL21 (DE3) pLysS which can express the antibacterial protein in vitro using prokaryotic expression system are obtained. The recombination protein can be expressed in high efficiency by the host bacterium. On the basis of the characteristic of inclusion of several disulfide bonds in the recombination protein, oxidation-reduction system consisting of oxidative glutathion and reductive glutathion is used in the isolation, purification and renaturation of the expressed protein. Thus a recombination protein having antibiotic activity is acquired. The high efficiency expression and renaturation of the recombination protein can be achieved. In vitro verification of biological function of the recombination protein confirmed that it has strong inhibiting effect on gram-positive bacteria and has some inhibiting effect on gram-negative bacteria as well.

Description

Antibacterial protein gene of Chinese prawn and its recombinant expression and application

The invention relates to the expression of a crustin gene in the field of genetic engineering, in particular to an antibacterial protein gene of Chinese prawn and its recombinant expression and application. Background technique

Shrimp culture is one of the pillar industries of marine aquaculture. As the market demand for high quality aquatic products continues to grow, shrimp farming plays an increasingly important role in aquaculture. Since the 1990s, the large-scale outbreak of disease has caused huge losses to the marine aquaculture industry. The abuse of antibiotics and the deterioration of the environment have made it difficult to control the diseases of marine aquaculture animals.

Marine aquaculture species live in a water environment rich in various microorganisms, and long-term environmental pressures make them effective defense functions. Antibacterial peptides (proteins) are considered to be one of the main components of defense systems such as fish, shrimp, and shellfish. By studying the structure and function of the antimicrobial peptide gene of marine aquaculture species, and then performing recombinant expression in vitro through genetic engineering technology, it will hopefully become a new drug that can replace antibiotics.

The immune defense system is the basis of animal disease resistance, especially low invertebrates. Their immune response is much simpler than that of higher animals, mainly relying on endogenous non-specific immune responses. Studies in recent years have shown that low-level invertebrates protect themselves from harmful microorganisms (such as bacteria, fungi or viruses), and non-specific antibacterial factors such as antimicrobial peptides (proteins), lysozyme, Transferrin and the like play a very important role. Among them, the antimicrobial peptide (protein) is a small molecule polypeptide (protein) encoded by animal and plant cell specific genes, and has antibacterial activity. In the mid-1970s, Swedish scientist Boman et al. isolated the world's first antimicrobial peptide, CeC ropin, when using £ coli to induce _Hyatophora cecropia. Since then, people have found similar polypeptides (proteins) with antibacterial activity in other organisms such as insects, amphibians, mammals, humans, plants 1, crustaceans, and mollusks. Crustin is a small molecule protein that has been strongly isolated and purified from Relf et al. from Carcinus imems. It is not easy to inactivate itself and remains high even after being boiled. Activity (JM Relf, JRS Chisholm, GD Kemp and VJ Smith, Purification and characterization of a cysteine-rich 11. 5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas. Eur. J. Biochem. 1999; 264 (2) : 350-357). Since then, people have discovered the presence of crustin or antibacterial protein analogs (crustin-like) from shrimps such as vannamei, white prawn, prawn and prawns.

The mechanism of action of crustin is different from that of traditional antibiotics. Its target site is mainly the pathogen cell membrane, so it is not easy to produce drug resistance. At present, many pathogens gradually develop resistance to existing antibiotics, and the discovery of new antibiotics is extremely difficult. Therefore, crustin research has opened up broad prospects for the development of new antibacterial drugs.

In short, the study of crustin and other types of antimicrobial peptides (proteins) is not only of great theoretical significance,

1

Confirmation It also has great application potential in practice. Therefore, it has become one of the hotspots of current biomedical research.

The aim is to provide an antibacterial protein gene of Chinese prawn and its high efficient recombinant expression and application.

The technical scheme of the present invention is as follows:

Chinese prawn antibacterial protein gene: has the nucleotide sequence of SEQ ID No. 1 and the amino acid sequence of SEQ ID No. 2.

Recombinant expression of the antibacterial protein gene of the Chinese prawn: An antibacterial protein gene was cloned from the Chinese prawn cDNA library, and the mature peptide gene was used to construct the prokaryotic expression vector, and a high-efficiency expression of the antibacterial protein of P. chinensis was screened. The recombinant expression system of the gene obtains a buffer system for efficiently reconstituting the recombinant protein containing the disulfide bond, thereby obtaining an active recombinant antibacterial protein; specifically:

Gene cloning

According to the EST of Chinese prawn EST, the target gene was cloned from the blood cell cDNA library using RACE technology;

2. Selection of recombinant expression vector and host bacteria

The expression vector pCIf T7/NT T0P0* TA which is recombinantly expressed in the form of inclusion body and the host strain BL21 (DE3) pLysS capable of alleviating the toxicity of the target gene expression product are determined;

3. Construction of recombinant expression vector '

Using the expression vector as the characteristic of the T vector, a pair of specific primers were used to clone the mature peptide gene, and the PCR product was directly recovered and ligated to form a recombinant expression vector;

4. Conversion and screening

The constructed recombinant expression vector was transformed into Escherichia coli BL21 (DE3) pLysS, and the engineered bacteria were screened and induced to express;

5. Isolation and purification of the expressed product

表达 Inducing agent isopropyl-β-D-thiogalactoside induced expression of engineering bacteria, ultrasonic disruption of cells, purification of antibacterial proteins by metal chelate column chromatography, protein renaturation, fusion tag excision, obtained active Recombinant antibacterial protein;

The protein renaturation uses a disulfide-containing prokaryotic expression recombinant protein to efficiently refold the buffer system, and oxidized glutathione and reduced glutathione are added to the renaturation buffer solution; concentration ratio: renaturation Buffer solution (PBS): oxidized glutathione: reduced glutathione = 50: 1: 10; renaturation buffer solution pH 7. 2~7. 4; The pair of specific primers are:

CD-F: 5, CAG AAT AAA GAC GAT ACT CG 3 ';

CD-R: 5, CTA TCC CTC AGA ACC CAG 3, .

The antibacterial protein gene of the Chinese prawn has an inhibitory effect on the growth of Gram-positive bacteria and Gram-negative bacteria. The invention has the following advantages -

1. The present invention determines the entire coding sequence and non-coding sequence of an antibacterial protein gene of P. chinensis, thereby clarifying the amino acid sequence and its signal peptide cleavage site, and using the deduced amino acid sequence of the mature peptide. The gene is expressed recombinantly.

2. The present invention screens vectors and host bacteria which can effectively carry out recombinant expression of antibacterial egg white, and provides a basis for studying recombinant expression of other antibacterial functional proteins.

3. The present invention provides a suitable redox system for the separation and purification of recombinant proteins containing disulfide bonds and protein renaturation, which can effectively ensure the correct formation of disulfide bonds in recombinant proteins.

4. Meaningful. Through the present invention, an effective drug for preventing and treating bacterial diseases of shrimp can be developed. At the same time, new ideas for the separation and purification of recombinant proteins with antibacterial activity and easily oxidized proteins are provided. detailed description

The present invention will be further described in detail below with reference to the embodiments.

Example

, a cloned antibacterial protein gene of Chinese prawn, having the following sequence:

(1) Information of SEQ ID No 1 (see sequence listing)

' (a) Sequence characteristics:

*Length: 523 base pairs

*Type: Nucleic acid

*chain type: double chain

*Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antisense: No

(e) Original source: Chinese Phyllostachys { Fenneropenaeus chinensis

Sequence Description: SEQ ID No. 1

AAAAAAAAAAAA (2) Information of SEQ ID No. 2 (see sequence listing)

(a) Sequence characteristics

*Length: 134 amino acids

*Type: Amino acid

*Chain type: single chain

*Topology: Linear

(b) Molecular type: Protein sequence description: SEQ ID No. 2

Recombinant expression, isolation, purification and biological activity analysis of EG Chinese prawn gene:

A crustin gene was screened from the cDNA library of P. chinensis, and the prokaryotic expression vector was constructed by using the mature peptide gene to screen a recombinant expression system for efficient expression of the antibacterial protein gene of P. chinensis. A buffer system for the efficient refolding of prokaryotic expression recombinant proteins containing disulfide bonds, and an active recombinant antibacterial protein is obtained. details as follows:

1) Gene cloning

2) Selection of recombinant expression vector and host bacteria

Since the recombinant expression product of the target gene has strong antibacterial activity, the present invention screens several commercial expression vectors and host bacteria, and determines the expression vector pCR ⁸ T7/NT which is recombinantly expressed in the form of inclusion bodies. T0P0* TA and host strain BL21 (DE3) pLysS capable of alleviating the toxicity of the gene expression product of interest.

The recombinant expression system can efficiently express antibacterial proteins with an expression efficiency of 845 mg/L.

3) Construction of recombinant expression vector

Using the expression vector as a feature of the T vector, a pair of specific primers (CD-F: 5' CAG AAT AAA GAC GAT ACT CG 3'; CD-R: 5, CTA TCC CTC AGA ACC CAG 3, ) was designed to clone the mature peptide. The gene, PCR product is recovered and directly ligated to the expression to construct a recombinant expression vector.

4) Induction of expression and screening of induced products

The recombinant expression vector was transformed into the expression host strain BL21 (DE3) pLysS, and the cell concentration was cultured to 0D ₆ in LB medium at a final concentration of 100 μg/tnL of ampicillin and 34 μg/mL of chloramphenicol. „ is about 0.6 (37 ° C, 250 r / min), adding a final concentration of lmmol / L IPTG for induction of expression for 4h; then using the inducer isopropyl - e-D-thiogalactoside (IPTG) was screened to induce expression of engineered bacteria.

5) Separation and purification

After the end of induction, the cells were disrupted by ultrasonication, and the antibacterial protein was purified by metal chelate column chromatography. The protein refolding was as follows: Centrifugal collection of the bacterial sludge, weighing the wet weight, adding buffer A (10ramol/L sodium phosphate) at 1:10 , 6mol / L guanidine hydrochloride, 300 awake ol / L sodium chloride, lmmol / L oxidized glutathione, 10mmol / L reduced glutathione, PH7. 2), ultrasonic disruption, 4. Centrifuge for 30 min at C, 12, OOOr/min and collect the supernatant. Separation and purification were carried out under denaturing conditions using Co-NTA-Agarose. The purified recombinant protein was isolated in 50 mmol/L PBS buffer (1 mmol/L oxidized glutathione) containing 8 mol/L, 4 raol/L, 2 mol/L, 1 mol/L and 0.5 mol/L urea. lOmraol/L reduced glutathione, pH 7. 4) dialysis for 8 h, replace guanidine hydrochloride in the lysate, remove urea in Sephadex G-75 column equilibrated in 50 mmol/L pH7.4 buffer , collect the eluent.

6) Enzymatic removal of fusion labels

The protein concentration of the eluate was diluted to 2 mg/mL, and the fusion label was excised (25, 24 h) using Irwitrogen's EKMaxTM kit.

7) Antibacterial experiment

The protein after removal of the fusion tag was concentrated to 64 μmol/L while using 50 mmol/L pH 7.4 PBS as a negative control. Using the Oxford Cup agar diffusion method to detect Gram-positive bacteria Staphylococcus aureii, Bacillus subtil is^, Micrococcus luteus and Gram-negative Escherichia coli ί Escherichia Coif), Vibrio anguillarum's antibacterial activity. SP: The strain to be tested was cultured overnight at 37 ° C, 200 r/niin; the cells were centrifuged the next day and the cells were washed twice with sterilized 50 mmol/L pH 7.4 PBS, and the cells were diluted with PBS to 10 6113/1! ^, 100 μ ΐ was applied to the surface of the LB plate, and after culturing for 2 hours in a 37 ° C incubator, the plate was taken out and placed on the agar surface at an equal distance to the Oxford Cup, and the recombinant white of different concentration gradients was added to the Oxford Cup. At the same time, PBS was used as a negative control, and culture was continued for 22 hours in a 37 ° C incubator, and the observation results were taken out.

The results showed that 4 mol/L recombinant protein could effectively inhibit the growth of Gram-positive bacteria (S. aureus, Bacillus subtilis and Micrococcus luteus), and 16 mol/L recombinant protein could effectively inhibit Growth of Gram-negative bacteria (E. coli and Vibrio anguillarum) detected.

The successful cloning of this gene and the identification of the biological activity of recombinant proteins have important application prospects in the development of new antibacterial drugs.

Claims

Claim

An antibacterial protein gene of P. chinensis having the nucleotide sequence of SEQ ID No. 1 of the Sequence Listing and the amino acid sequence of SEQ ID No. 2.

2. A recombinant expression of an antibacterial protein gene of the Chinese prawn according to claim 1, wherein: an antibacterial protein gene is cloned from a cDNA library of P. chinensis, and a prokaryotic expression vector is constructed using the mature peptide gene to screen A recombinant expression system for efficiently expressing the antibacterial protein gene of P. chinensis is obtained, and a buffer system for efficiently reconstituting the prokaryotic expression recombinant protein containing disulfide bond is obtained, thereby obtaining an active recombinant antibacterial protein; specifically:

1) Gene cloning

According to the EST of Chinese prawn EST, the RACE technology was used to clone the target base from the blood cell cDNA library.

'cause;

2) Selection of recombinant expression vector and host bacteria

The expression vector pCR ^e T7/NT Τ0Ρ0 ^β ΤΑ which is recombinantly expressed in the form of inclusion body and the host strain BL21 (DE3) pLysS capable of alleviating the toxicity of the expression product of the target gene are determined;

3) Construction of recombinant expression vector

4) Conversion and screening

5) Separation and purification of expression products

The engineered bacteria were induced by the inducer isopropyl-thiogalactoside, the cells were disrupted by ultrasonic wave, and the antibacterial protein was purified by metal chelate column chromatography. The protein was renatured and the fusion tag was excised to obtain the active recombinant antibacterial protein.

3. The recombinant expression of the antibacterial protein gene of the Chinese prawn according to claim 2, wherein the protein is renatured using a disulfide-containing prokaryotic expression recombinant protein, a refolding buffer system, in a renaturation buffer Oxidized glutathione and reduced glutathione are added to the solution at a concentration ratio: renaturation buffer solution: oxidized glutathione: reduced glutathione = 50: 1: 10; renaturation buffer 5〜7. 4。 The pH of the solution is 7. 2~7. 4.

4. The recombinant expression of the antibacterial protein gene of the Chinese prawn according to claim 2, wherein: the pair of specific primers are:

CD-F: 5, CAG AAT AAA GAC GAT ACT CG 3, ;

CD-R: 5' CTA TCC CTC AGA ACC CAG 3 '.

An application of the antibacterial protein gene of the Chinese prawn according to claim 1, characterized in that it has an inhibitory effect on the growth of Gram-positive bacteria and Gram-negative bacteria.