CN108003233B - Oyster containing DM9 structural domain protein CgDM9CP-2, preparation method and application - Google Patents

Abstract

The invention discloses a crassostrea gigas protein CgDM9CP-2 containing DM9 structural domain, and an amino acid sequence is shown as SEQ ID NO. 1. The preparation method comprises the following steps in sequence: primers P1 and P2 are used for pairing crassostrea gigasCgDM9CP‑2Carrying out PCR amplification on the gene coding region segment; the PCR amplification product is mixed with pET-30a vectorNde1AndXho1after enzyme digestion, the recombinant is connected through ligase, transformed, sequenced and identified; transferring the recombinant into Escherichia coliTransetta(DE3) carrying out induction culture in the expression strain, and then purifying and renaturing to obtain the recombinant protein with the amino acid sequence in the sequence table SEQ ID NO. 1. The protein CgDM9CP-2 of the structural domain DM9 of the crassostrea gigas can be applied to the preparation of pichia pastoris inhibiting medicines.

Description

Oyster containing DM9 structural domain protein CgDM9CP-2, preparation method and application

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a DM9 structural domain-containing protein CgDM9CP-2 of crassostrea gigas, a preparation method and application.

Background

The crassostrea gigas is an important seawater cultured shellfish. Because crassostrea gigas lacks an adaptive immune defense system and mainly depends on an innate immune system to resist the infection of exogenous pathogenic microorganisms, various diseases caused by bacteria, fungi and viruses continuously burst in the breeding population of crassostrea gigas, and huge economic loss is caused. Proteins containing the DM9 domain were first found in drosophila. Later, Magalhaes et al found some toxins with the domain DM9 in fish and named "natterin" that contained an N-terminal domain DM9 and a C-terminal toxin/bacterial toxin (ETX/MTX 2) domain. natterin can produce cytotoxic effects in human tissues, resulting in cell necrosis, edema, localized pain, and the like. Recent studies have shown that natterin functions as a perforin. In recent decades, more and more proteins containing the domain of DM9 (DM 9 CPs) have been found in invertebrates including arthropods, platyhelminths, etc., and it has been demonstrated that DM9CPs are involved in immune responses of the body. For example, in anopheles gambiae, a DM9CPs named "cause salivary gland reaction of plasmodium (PRS1), the expression level on the lateral side of salivary glands was significantly increased after plasmodium invasion and increased with the increase in the number of infected sporozoites.

However, no reports on the DM9 domain-containing protein CgDM9CP-2 of crassostrea gigas, a preparation method and application in preparation of pichia pastoris inhibiting medicines are provided so far.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a protein CgDM9CP-2 containing DM9 structural domain of crassostrea gigas, a preparation method and application.

The technical solution of the invention is as follows: a crassostrea gigas protein CgDM9CP-2 containing DM9 domain is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.

A preparation method of the crassostrea gigas protein CgDM9CP-2 containing DM9 domain is characterized by comprising the following steps in sequence:

a. primers P1 and P2 are used for pairing crassostrea gigasCgDM9CP-2Carrying out PCR amplification on the gene coding region segment;

b. the PCR amplification product is mixed with pET-30a vectorNde1AndXho1after enzyme digestion, the recombinant is connected through ligase, transformed, sequenced and identified;

c. transferring the recombinant into Escherichia coliTransetta(DE3) carrying out induction culture in the expression strain, and then purifying and renaturing to obtain the recombinant protein with the amino acid sequence in the sequence table SEQ ID NO. 1.

An application of the protein CgDM9CP-2 containing DM9 domain of Crassostrea gigas in preparing medicine for inhibiting Pichia pastoris is provided.

The invention obtains the protein CgDM9CP-2 containing the DM9 structural domain of the crassostrea gigas by utilizing an in-vitro recombinant expression technology, the recombinant protein has the biological activity of combining Mannose, LPS and PGN, and simultaneously has stronger inhibiting effect on pichia pastoris outside an organism. The protein CgDM9CP-2 containing the DM9 structural domain of the crassostrea gigas is used as an effective mode recognition receptor and has application value in the aspects of preparing antibacterial drugs, novel immune preparations, feed additives and the like.

Drawings

FIG. 1 is a schematic diagram showing the binding activity of the DM9 domain-containing protein CgDM9CP-2 of the crassostrea gigas of the present invention to LPS, PGN and Mannose.

FIG. 2 is a schematic diagram showing the inhibitory effect of DM9 domain-containing protein CgDM9CP-2 on Pichia pastoris of crassostrea gigas of the invention.

Detailed Description

The crassostrea gigas of the invention contains a DM9 structural domain protein CgDM9CP-2, and the amino acid sequence is shown as SEQ ID NO. 1.

The preparation method of the DM9 domain-containing protein CgDM9CP-2 of the crassostrea gigas comprises the following steps in sequence:

1. construction of recombinant vectors

Primers P1 and P2 are used for pairing crassostrea gigasCgDM9CP-2Carrying out PCR amplification on the gene coding region segment;

the PCR reaction conditions are as follows: first, pre-denaturation at 94 ℃ for 5min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes. The amplified fragment was purified and recovered, and ligated with pMD19-T vector. Screening positive clones after transformation, and extracting plasmids; use ofNde1AndXho1performing enzyme digestion on plasmids by two enzymes, and purifying and recovering a target fragment generated by enzyme digestion by using a glue recovery and purification kit (Dalibao bioengineering Co., Ltd.); recovering the target fragment and channelNde1AndXho1and connecting the two enzyme-digested expression vectors pET-30a to complete the construction of the vector.

2. Expression of recombinant protein CgDM9CP-2

Transforming the constructed recombinant vector into expression host bacteriaTransetta(DE3) and screening for positive clonesAnd sequencing to confirm the correctness of the expression cassette. The single clone was picked and inoculated into 200 mL of LB liquid medium, cultured at 220rpm and 37 ℃ to OD₆₀₀And = 0.4-0.8. IPTG was added to a final concentration of 1 mmol L^-1The cultivation was continued for 4 hours. Centrifuging at 4 deg.C and 5000rpm for 10 min, collecting thallus, and freezing at-20 deg.C for use. 1mL of the bacterial liquid is taken for centrifugation, the supernatant is discarded, 80 mu L of water and 20 mu L of 5-fold protein loading buffer solution are added, the mixture is boiled at 100 ℃ for 10 minutes, the centrifugation is carried out, and the expression product is detected by SDS-PAGE.

3. Purification and renaturation of recombinant protein CgDM9CP-2

The recombinant protein is purified by adopting a nickel sepharose FF column to obtain a denatured recombinant protein, and dialysis renaturation is carried out by using dialysis buffer solution. The specific operation steps are as follows:

(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20cm and the volume of a column bed of 10 ml;

(2) using buffer I (50 mmol L)^-1Tris-HCl buffer, pH = 7.4, 50m mol L^-1 NaCl, 8 mol L^-1Urea) 2-5 bed volumes balanced at a flow rate of 2 ml min^-1；

(3) Taking IPTG induced expression cells, resuspending with buffer I, ultrasonicating at 150W for 30 min, centrifuging at 4 deg.C for 30 min at 12000 rmp, filtering the supernatant with 0.45 μm filter membrane, and passing through column at flow rate of 1ml min^-1；

(4) Washing with buffer solution 1 for 2-5 bed volumes at a flow rate of 2 ml min^-1；

(5) With a solution of 50 mmol L^-1The imidazole buffer solution I is washed for 2-5 column bed volumes again, and the flow rate is 2 ml min^-1；

(6) With 400 mmol L of^-1Eluting the target protein by using the imidazole buffer solution I and collecting.

(7) Detecting the expression of the fusion protein by SDS-PAGE;

(8) washing with pure water for 5 bed volumes, and washing with 20% ethanol for 3 bed volumes at flow rate of 2 ml/min^-1The purified recombinant protein, which is kept in a denatured state at 4 ℃ in the column, requires removal of urea by dialysis in a renaturation buffer to allow the protein to become aligned againFold correctly and return to the correct conformation. Dialyzing the denatured and purified product with 2 mM reduced glutathione, 0.4 mM oxidized glutathione, 1 mM EDTA, 50mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine and gradient-decreasing urea, gradually changing the concentration of urea from the initial 6M to 4M, 3M, 2M, 1M, 0M, and finally dialyzing to a dialysate without urea without adding glycerol for 12 h at 4 ℃. Thus obtaining the crassostrea gigas protein CgDM9CP-2 containing DM9 structural domain, and the amino acid sequence is shown as SEQ ID NO. 1.

Length: 143 amino acids

Type (2): amino acids

Chain type: single strand

The characteristics are as follows: the molecular weight is 16 kDa, the isoelectric point is 8.64, and the molecular weight has two conserved DM9 domains.

Experimental example 1: detection of binding activity of DM9 structural domain-containing protein CgDM9CP-2 of crassostrea gigas of the invention with Mannose, LPS and PGN

The method comprises the following steps:

1. mu.g of each PAMPs was dissolved in 50mM sodium carbonate-sodium bicarbonate buffer, 100. mu.l of each well coated with an enzyme plate, 4 ℃ overnight.

2. PBS-T was washed 3 times for 5min, 200. mu.L of 3% BSA was added to each well, and blocked at 37 ℃ for 1 h.

3. PBS-T washing for 3 times, each for 5min, adding 100 μ L of 2-fold gradient diluted protein CgDM9CP-2 of the DM9 domain of the crassostrea gigas of the invention into each well, and incubating for 3 h at 18 ℃.

4. PBS-T washing 3 times, each time for 5min, adding 100. mu.L diluted polyclonal antibody (1: 1000) against the target protein to each well, and incubating at 37 ℃ for 1 h.

5. PBS-T washing for 3 times, each time for 5min, adding 100. mu.L alkaline phosphatase labeled goat anti-rat IgG (1: 4000) per well, and incubating at 37 ℃ for 1 h.

6. PBS-T washes 3 times for 5min, 100. mu.L of a mixture containing 0.1% (w/v) p-nitrophenylphosphate (pNPP) and 0.5 mM MgCl₂And (3) adding 50 mu l of 2M NaOH into each hole to terminate color development, and reading and recording numerical values at 405 nm of an enzyme-labeling instrument, wherein the color development is performed for 10-30 min in a dark place by using 50mM sodium carbonate-sodium bicarbonate buffer solution (pH 9.8).

Each well was replicated 3 times in the experiment, recombinant Trx protein was used as protein control and TBS buffer was used as blank for reading. When data were analyzed, wells in experimental group (P)/negative control group (N) >2.1 were considered positive. The results are shown in FIG. 1. The results show that the protein CgDM9CP-2 containing DM9 domain of the crassostrea gigas has biological activity of binding Mannose, LPS and PGN.

Experimental example 2: the method for detecting the bacteriostatic activity of DM9 structural domain-containing protein CgDM9CP-2 of crassostrea gigas of the invention

The method comprises the following specific steps:

1. culture and preparation of microorganisms

Pichia pastoris (laboratory-maintained species) was cultured in YPD medium at 28 ℃ and 220rpm until logarithmic growth phase, and then in Tris-HCl (50 mmol L)^-1pH = 8.0) was diluted so that the number of colonies per ml of the bacterial solution was about 1X 10³CFU；

2. Determination of antibacterial activity of recombinant protein CgDM9CP-2

After centrifugation and collection of Pichia pastoris in logarithmic growth phase, it was washed with TBS (50 mM Tris-HCl, 150 mM NaCl) and resuspended (10 mM NaCl)⁴CFU). 50 μ L of the crassostrea gigas of the example of the invention containing the DM9 domain protein CgDM9CP-2 was incubated with an equal volume of Pichia pastoris for 2 h at room temperature. 20 mu L of the mixture is put into a flat-bottom 96-well plate (Costar, Fisher), 200 mu L of YPD and 2216E liquid culture medium is added into each well, the mixture is subjected to shaking culture in a microplate reader at 28 ℃, and the OD600 value of each well is detected and recorded every 1 h. A rTrx protein control group was also provided. Three replicates were set for each sample, the mean values were plotted based on the 3 measurements, and the study data were statistically analyzed using SPSS6.0 software for significant differences (P <0.05) with a mark. The results are shown in FIG. 2. Experiments show that the protein CgDM9CP-2 containing the DM9 domain of the crassostrea gigas can inhibit the growth of pichia pastoris.

Sequence listing

<110> university of Dalian ocean

<120> crassostrea gigas protein CgDM9CP-2 containing DM9 structural domain, preparation method and application

<160>1

<170>SIP0SequenceListing 1.0

<210>1

<211>143

<212>PRT

<213> Crassostrea gigas (Crassostra gigas)

<400>1

Met Ala Glu Trp Lys Lys Thr Ser Gly Ser Lys Ile Pro Asp Asn Ala

Ile Arg Ala Gly Tyr Glu Lys Asp Gly Lys Pro Leu Phe Ile Ala Arg

Ala Lys Met Gly Gly Leu Trp Thr Ser Gly Lys Cys Gly Thr His Leu

Pro Gly Ala His Ile Pro Tyr Asp Cys Lys Glu Leu Ile Val Lys Asp

Tyr Glu Val Leu Val Tyr Pro Ile Thr Ala Val Gly Phe Leu Asp Trp

Lys Gln Ala Ser Gly Gly Lys Val Pro Asp Lys Ala Phe Lys Thr Asp

Thr Asp Leu Tyr Val Gly Arg Ala Asn Tyr Thr Gly Ser Leu Val Pro

Cys Lys Ile Ser Thr Ser His Lys Cys Ala Tyr Met Gly Tyr Cys Glu

Lys Glu His Asn Ala Lys Glu Tyr Glu Val Leu Cys Gln Ile Lys 143

Claims (3)

1. A crassostrea gigas protein CgDM9CP-2 containing DM9 domain is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.

2. The preparation method of the crassostrea gigas protein CgDM9CP-2 containing DM9 domain according to claim 1, which is characterized by comprising the following steps in sequence:

a. primers P1 and P2 are used for pairing crassostrea gigasCgDM9CP-2Carrying out PCR amplification on the gene coding region segment;

b. the PCR amplification product is mixed with pET-30a vectorNde1AndXho1after enzyme digestion, the recombinant is connected through ligase, transformed, sequenced and identified;

c. transferring the recombinant into Escherichia coliTransetta(DE3) expression strain, purifying and renaturing to obtain the amino acid sequence in SEQ ID NO.1 of the sequence tableThe recombinant proteins listed.

3. The application of the protein CgDM9CP-2 containing DM9 domain of the crassostrea gigas of claim 1 in preparing a medicament for inhibiting Pichia pastoris.

Images