CN107987154B - Ostrea gigas IgSF molecule CgCAICP1 gene recombinant protein, preparation method and application - Google Patents
Ostrea gigas IgSF molecule CgCAICP1 gene recombinant protein, preparation method and application Download PDFInfo
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Abstract
The invention discloses a crassostrea gigas IgSF moleculeCgCAICP1The amino acid sequence of the gene recombinant protein is shown in SEQ ID NO. 1. The preparation method comprises the following steps in sequence: the primers P1 and P2 are used for pairing the ostrea gigantea IgSF moleculeCgCAICP1Carrying out PCR amplification on the coding region segment; the PCR amplification product was ligated with pET32a vectorNcoI andHindIII, after enzyme digestion, connecting through T4 ligase, transforming, sequencing and identifying the recon; the recombinant is transferred into an escherichia coli Transetta (DE3) expression strain for induction culture, and then purification and renaturation are carried out. The crassostrea gigas IgSF moleculeCgCAICP1The gene recombinant protein can be applied to preparing medicaments for inhibiting gram-negative bacteria.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a crassostrea gigas IgSF moleculeCgCAICP1A gene recombinant protein, a preparation method and application.
Background
Immunoglobulin superfamily IgSF molecules, which are mainly produced by B lymphocytes, are important components of adaptive immunity (adaptive immunity) of vertebrates, can generate various antigen binding epitopes through a series of molecular mechanisms, and are specifically bound with corresponding antigenic determinants. Invertebrates are capable of developing an effective immune response to pathogenic invasion, although they do not possess the antibody-based adaptive immune system in vertebrates. The main ways in which IgSF molecules function have been found to include the following four types: a. pattern recognition; b. agglutination of microorganisms; c. phagocytic opsonization; d. cell agglutination and anti-agglutination. Such as hemolin in insects, are capable of binding to two binding sites on LPS and of agglutinating a variety of microorganisms; VCBP in amphioxus and DSCAM in crustaceans are also both able to recognize and bind large amounts of ligand; the Fenneropenaeus chinensis FcLec4 is combined with beta-integrin on the surface of hemolymph cells and has the function of conditioning.
However, no relevant reports on the crassostrea gigas IgSF molecule CgCAICP1 gene recombinant protein, a preparation method and application in preparing medicaments for inhibiting gram-negative bacteria exist so far.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a crassostrea gigas IgSF moleculeCgCAICP1A gene recombinant protein, a preparation method and application.
The technical solution of the invention is as follows: ostrea gigas IgSF moleculeCgCAICP1A recombinant protein characterized by: the amino acid sequence is shown as SEQ ID NO. 1.
The Concha Ostreae IgSF moleculeCgCAICP1The preparation method of the gene recombinant protein is characterized by comprising the following steps in sequence:
a. the primers P1 and P2 are used for pairing the ostrea gigantea IgSF moleculeCgCAICP1Carrying out PCR amplification on the coding region segment;
b. the PCR amplification product was ligated with pET32a vectorNcoI andHindIII, after enzyme digestion, connecting through T4 ligase, transforming, sequencing and identifying the recon;
c. the recombinant is transferred into an escherichia coli Transetta (DE3) expression strain for induction culture, and then purification and renaturation are carried out to obtain the recombinant protein with the amino acid sequence in the sequence table SEQ ID NO. 1.
The Concha Ostreae IgSF moleculeCgCAICP1The application of the gene recombinant protein in preparing the medicine for inhibiting gram-negative bacteria.
Crassostrea gigas IgSF molecules of the inventionCgCAICP1The gene recombinant protein is cloned from a crassostrea gigas cDNA library, has the binding activity of various microorganisms, can obviously improve the phagocytosis rate of crassostrea gigas blood lymphocytes to gram-negative bacteria, 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 shows IgSF molecules of examples of the inventionCgCAICP1Bacterial binding activity assay for recombinant proteins.
FIG. 2 shows IgSF molecules of examples of the inventionCgCAICP1The detection effect of the gene recombinant protein promoting phagocytosis is shown.
Detailed Description
The crassostrea gigas IgSF molecule of the inventionCgCAICP1A process for preparing the gene recombinant protein,
the method comprises the following steps of:
1. construction of recombinant vectors
The recombinant vector adopted in the embodiment of the invention is a pET-32a (+) prokaryotic expression vector of Novagen company. By PCR technique, respectively add at 5' endNcoI andHind 
primers P1 and P2 for restriction sites amplification from crassostrea gigasCgCAICP1 Coding region of mRNA.
The PCR reaction conditions are as follows: first, pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 2 min for a total of 30 cycles, and final extension at 72 ℃ for 10 min. The amplified fragment was recovered by agarose gel electrophoresis and ligated with pMD19-T vector. Screening positive clone after transformation, extracting plasmid, and usingNcoI andHind 
carrying out double enzyme digestion on the plasmid; recovering the target fragment and passing throughNcoI andHind 
and connecting the enzyme-cut expression vector pET-32a (+) to complete the construction of the recombinant plasmid.
2. Recombinant protein rCgExpression of CAICP1
Transforming the constructed recombinant plasmid into an expression host bacterium Escherichia coli Transetta (DE3), selecting a single clone, inoculating the single clone into 200 ml LB liquid culture medium, culturing at 220 rpm and 37 ℃ to OD 600And = 0.4-0.8. IPTG (final concentration 1 mmol L) was added-1) Continuously culturing for 4 hours, centrifuging at 4 ℃ and 10000 rpm for 5 min, collecting thalli, and freezing and storing at-80 ℃ for later use; meanwhile, 1 ml of bacterial liquid is taken for centrifugation, after the supernatant is discarded, 80 mul of water and 20 mul of 5 Xprotein loading buffer solution are added, boiling is carried out for 10min at 99 ℃, and the solution is slightly centrifuged, and SDS-PAGE detects the expression product.
3. Recombinant protein rCgPurification and renaturation of CAICP1
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, 50 m 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 1 ml 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, so that the protein is correctly folded again and the correct conformation is restored. Dialyzing the denatured and purified product with 2 mM reduced glutathione, 0.4 mM oxidized glutathione, 1 mM EDTA, 50 mM 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 ℃. Obtaining the crassostrea gigasCgCAICP1 gene recombination protein (r)CgCAICP1）。
The obtained Concha OstreaeCgCAICP1The amino acid sequence of the gene recombinant protein is shown in SEQ ID NO. 1.
Length: 574 amino acids
Type (2): amino acids
Chain type: single strand
The characteristics are as follows: has a molecular weight of 63.4 kDa, an isoelectric point of 5.91, a cysteine-rich region and three immunoglobulin domains.
Experimental example 1: concha Ostreae recombinant protein rCgDetection of binding activity of CAICP1 bacterium
The binding activity of the recombinant protein to two gram-negative bacteria (vibrio splendidus and escherichia coli), two gram-positive bacteria (staphylococcus aureus and micrococcus luteus) and a fungus pichia pastoris is detected based on a western blotting method. The bacterial sources used were as follows: vibrio splendidus (Vibrio splendidus JZ6) Escherichia coli (E.coli) from Beijing culture CollectionEscherichia coli) Staphylococcus aureus (available from Beijing Quanji Co.) (Staphylococcus aureus) Micrococcus luteus (purchased from Beijing culture Collection of microorganisms)Micrococcus luteus) Pichia pastoris (available from Beijing culture Collection)Pichia pastoris GS115) Purchased from Invitrogen corporationAnd (4) a driver.
The specific operation is as follows:
(1) the 5 microorganisms were cultured overnight, the culture method: culturing micrococcus luteus and Escherichia coli in LB culture medium at 37 deg.C for 20 hr, culturing Staphylococcus aureus in LB culture medium at 28 deg.C for 20 hr, culturing Vibrio splendidus in 2216E culture medium at 28 deg.C for 20 hr, and culturing Pichia pastoris in YPD culture medium at 28 deg.C for 20 hr;
(2) the cells were collected by centrifugation, resuspended in TBS buffer and adjusted to a cell concentration of 1X 108 CFU；
(3) Sucking 100 mul of microorganism suspension and equal volume of crassostrea gigas obtained in the embodiment of the inventionCgMixing CAICP1 gene recombinant proteins, and performing rotary incubation at room temperature for 30 min;
(4) centrifuging at 10000 rpm for 2 min, collecting thalli, and washing the thalli four times by TBS buffer solution;
(5) after washing, collecting the thalli, and using 40 mul of sterile water for resuspension;
(6) adding 10 μ l of 5 × protein electrophoresis buffer solution, heating at 99 deg.C for 10min, and separating protein sample by SDS-PAGE electrophoresis;
(7) after electrophoresis is finished, taking down the gel, cutting an NC membrane and filter paper with the same size, immersing the NC membrane and the filter paper into an electric transfer buffer solution, and standing for 10 min;
(8) sequentially placing filter paper, an NC membrane, gel and the filter paper into an electrotransformation instrument from top to bottom, setting corresponding current according to the area of a gel block, and performing membrane transfer for 65 min;
(9) taking out the NC membrane, washing three times by using TBS buffer solution, and each time for 5 min;
(10) washing three times with buffer TBST for 5 min each time;
(11) placing the NC membrane into 5% skimmed milk powder (dissolved in TBST), and sealing for 2 hours at room temperature;
(12) taking out the NC membrane, and washing the NC membrane for three times for 5 min each time by using TBST buffer solution;
(13) immersing the NC membrane into a labeled monoclonal antibody solution (5% skimmed milk powder, TBST buffer solution) diluted in proportion, and incubating for 1 hour at room temperature;
(14) the NC membrane was removed and washed three times with TBST buffer for 5 min each.
(15) Placing the NC membrane into a proportionally diluted goat anti-mouse HRP secondary antibody (purchased from Shanghai Biotechnology) (5% skimmed milk powder TBST buffer), and incubating for 1 hour at room temperature;
(16) taking out the NC membrane, and washing the NC membrane for three times for 10min each time by using TBST buffer solution;
(17) ECL method development, imager record western blotting results, as shown.
The results show that the crassostrea gigas of the present inventionCgThe CAICP1 gene recombinant protein shows stronger binding activity with two kinds of gram and staphylococcus aureus, and has weaker binding capacity with micrococcus luteus and pichia pastoris. No bands were evident in the TRX negative control and the wash blank.
Experimental example 2: concha Ostreae recombinant protein rCgDetection of phagocytosis promoting Activity of CAICP1
Labeling three microorganisms (vibrio splendidus, micrococcus luteus and pichia pastoris) by using Fluorescein Isothiocyanate (FITC), and detecting the phagocytic efficiency of oyster blood lymphocytes by using a fluorescence microscope counting method.
The strain is from the above.
The specific operation is as follows:
(1) culturing the three microorganisms respectively and collecting thalli;
(2) mixing formaldehyde with microorganism, and fixing for 10 min;
(3) collecting the bacteria by centrifugation at 4000 Xg for 10 min. 0.1M NaHCO3After three washes, incubate in 0.1M NaHCO with 0.1 mg/ml FITC3Medium, incubation at 25 ℃ for 2 h with gentle shaking;
(4) centrifuging and removing a supernatant, and washing the microorganisms to be colorless by using TBS buffer solution;
(5) the suspended cells were adjusted to 10 concentration using TBS8 CFU ml-1；
(6) Adding anticoagulant in equal proportion to extract hemolymph, centrifuging at 4 deg.C and 800 Xg for 10min, and collecting blood cells;
(7) cell concentration was adjusted to 10 using L15 saline cell culture medium6 cells ml-1;
(8) The crassostrea gigas obtained in the embodiment of the inventionCgThe CAICP1 gene recombinant protein and the cells are incubated for 30 min at room temperature in a dark place according to the volume ratio of 1: 1;
(9) adding the marked bacteria with the same volume, and incubating for 30 min at room temperature in a dark place;
(10) dropping 80 mul of tablets;
(11) settling for 1 h at normal temperature in a wet box;
(12) the residual liquid was decanted off and sucked dry with 2 mg ml1 Trypan blue quenchingFluorescence for 10 min;
(13) fixing in acetone for 20 min to ensure that the acetone does not overload the glass slide;
(14) washing with PBS for three times, 5 min each time;
(15) DAPI staining for 3 min;
(16) washing with PBS for three times;
(17) phagocytic cells and non-phagocytic cells were observed under a fluorescence microscope and counted, and the results are shown in fig. 2.
The results show that the crassostrea gigas of the embodiment of the inventionCgCAICP1The gene recombinant protein has obvious phagocytosis promoting effect on vibrio splendidus, has certain effect on phagocytosis of micrococcus luteus, and has no obvious phagocytosis promoting effect on pichia pastoris. No significant phagocytic effect was detected for the three microorganisms in the control rTRX.
Sequence listing
<110> university of Dalian ocean
<120> Ostrea gigas IgSF molecule CgCAICP1 gene recombinant protein, preparation method and application
<160>1
<170>SIP0SequenceListing 1.0
<210>1
<211>574
<212>PRT
<213> Crassostrea gigas (Crassostra gigas)
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Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
Ala Gln Asp Ser Ser Gln Met Pro Pro Asn Met Thr Ile Thr Phe Asn
Asn Glu Tyr Phe Phe Pro Arg Gly Ser Ile Leu Ala Asp Ala Ser Phe
Asn Cys Thr Ala Glu Asn Gly Arg Val Leu Tyr Glu Trp Lys Lys Asp
Gly Asn Val Val Gln Asn Thr Gln Ser Val Thr Val Asp Asn Ser Thr
Gly Ile Leu Lys Phe His Gln Met Gln Asn Gly Asp Tyr Gly Thr Tyr
Gln Cys Phe Ala Thr Asn Gly Tyr Gly Thr Ser Leu Ser Lys Pro Phe
Lys Ile Met Glu Ala Arg Leu Gly Ser Phe Pro Thr Ser Gln Thr Gln
Glu Ile Lys Cys Glu Glu Phe Lys His Cys Lys Val Glu Cys Arg Tyr
Lys Pro Thr Cys Leu Pro Glu Ser Gln Cys Lys Val Glu Trp Lys Ile
Gly Glu Gly Thr Lys Thr Asn Val Glu Ile Asn Lys Arg Val Gly Val
Asp Gly Asn Gly Gly Leu His Phe Leu Trp Thr Ser Met Ser Asp Trp
Thr Gly Gln Gln Tyr Arg Cys Gly Val Trp His Glu Gln Leu Lys Thr
Leu Val Val Gly Ser Gln Thr Ser Leu Lys Ile Asp Ser Ala Thr Ala
Val Pro Lys Val Asp Pro Met Leu Val Phe Lys Glu Asn Gly Lys Ala
Leu Ile Gly Glu Arg Gly Val Leu Arg Cys Met Phe Ser Gly Tyr Pro
Val Pro Asp Ile Thr Trp Ile Ser Pro Gln Lys Met Asn Ile Asp Asp
Ala Asp Gly Lys Tyr Glu Ile Ser Asp Phe Gly Arg Val Leu Thr Ile
Leu Arg Ala Glu Ser Lys His Glu Gly Thr Tyr Thr Cys Lys His Ser
Gly Lys Asn Glu Thr Val Phe Leu Asn Ala Thr Ser Ala Pro Phe Leu
Asn Gly Ser Asn Gln Met Gln Asp Leu Val Leu Pro Glu Gly Gln Glu
Ala Thr Phe Arg Cys Glu Ala Glu Ser Ser Pro Asp Glu Leu Pro Pro
Thr Arg Pro Thr Trp Lys Lys Asn Gly Val Asp Leu Lys Ile Asp Gly
Gly Lys Tyr Leu Leu Gly Glu Asn Ser Gln Val Leu Ser Leu Lys Asp
Val Gln Lys Ser Asp Ser Gly Val Tyr Gln Cys Met Ser Glu Asn Ser
Glu Gly Val Leu Leu Lys Glu Ala Ile Leu Lys Val Thr Asp Pro Ile
Lys Lys Leu Ala Ala Ala Leu Glu His His His His His His 574
Claims (3)
1. Ostrea gigas IgSF moleculeCgCAICP1A recombinant protein characterized by: the amino acid sequence is shown as SEQ ID NO. 1.
2. The crassostrea gigas IgSF molecule of claim 1CgCAICP1The preparation method of the gene recombinant protein is characterized by comprising the following steps in sequence:
a. with 5' ends respectively addedNcoI andHind 
primers P1 and P2 of restriction sites for Pacific oyster IgSF moleculesCgCAICP1Carrying out PCR amplification on the coding region segment;
b. the PCR amplification product was ligated with pET32a vectorNcoI andHindIII, after enzyme digestion, connecting through T4 ligase, transforming, sequencing and identifying the recon;
c. the recombinant is transferred into an escherichia coli Transetta (DE3) expression strain for induction culture, and then purification and renaturation are carried out to obtain the recombinant protein with the amino acid sequence in the sequence table SEQ ID NO. 1.
3. The crassostrea gigas IgSF molecule of claim 1CgCAICP1The application of the gene recombinant protein in preparing medicaments for inhibiting vibrio splendidus.
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| CN104861056A (en) * | 2015-06-16 | 2015-08-26 | 中国科学院海洋研究所 | Recombinant protein CgC1qDC-1 of pacific oyster complement molecule, as well as preparation and application of recombinant protein CgC1qDC-1 |
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| CN104861056A (en) * | 2015-06-16 | 2015-08-26 | 中国科学院海洋研究所 | Recombinant protein CgC1qDC-1 of pacific oyster complement molecule, as well as preparation and application of recombinant protein CgC1qDC-1 |
Non-Patent Citations (3)
| Title |
|---|
| A hypervariable immunoglo bulin superfamily member from Crassostrea gigas functions as pattern recognition receptor with opsonic activity;Dongyang Liu等;《Developmental and Comparative Immunology》;20180505;第96-108页 * |
| PREDICTED: Crassostrea gigas neurofascin (LOC1 05322290), transcript variant X1 , mRNA;Genbank Database;《Genbank Database》;20170202;Accession：XM_011420933 * |
| 长牡蛎免疫球蛋白超家族（IgSF）成员结构与功能的研究;刘聪辉;《中国博士学位论文全文数据库（电子期刊）农业科技辑》;20160815;D052-13 * |
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