CN110845594A - Recombinant serum amyloid protein A capable of enhancing immune response of crassostrea gigas and preparation method thereof - Google Patents
Recombinant serum amyloid protein A capable of enhancing immune response of crassostrea gigas and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a recombinant serum amyloid protein A capable of enhancing immune response of crassostrea gigas, which is characterized in that an amino acid sequence is shown as SEQ ID NO.1, and the recombinant serum amyloid protein A can remarkably promote cytokines in blood lymphocytes in vitro and in vivo of the crassostrea gigasCgIL17‑1、CgIL17-5 andCgTNF gene expression for enhancing the immune response of crassostrea gigasThe method has application value in the aspect of preparing medicaments such as novel immune preparations for breeding shellfish, in particular shellfish.
Description
Technical Field
The invention belongs to the technical field of biochemistry and molecular biology, and particularly relates to serum amyloid A capable of enhancing immune response of crassostrea gigas and a preparation method thereof.
Technical Field
Oyster (Chang mu Li)Crassostrea gigas，Cg) Is an important seawater culture economic shellfish in China. In recent years, various diseases caused by bacteria, fungi and viruses are continuously outbreaked in the breeding population of the crassostrea gigas, and huge economic losses are caused. Due to the lack of an adaptive immune defense system, crassostrea gigas relies primarily on innate immune responses to resist infestation by foreign pathogenic microorganisms. Recent research shows that the lack of the inherent immune response capability of the crassostrea gigas to cause imbalance of homeostasis caused by long-term latent infection of pathogens is one of the important reasons for large-scale death of the crassostrea gigas. In mammals, Serum Amyloid A (SAA) is an acute reactive phase protein of inflammatory diseases, whose concentration in plasma increases dramatically and is commonly used for evaluation of body health. To date, no report on the physiological function of crassostrea gigas SAA has been found, and no drug that can significantly promote the level of crassostrea gigas innate immune response has been found.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides serum amyloid A capable of enhancing the immune response of crassostrea gigas and a preparation method thereof.
The technical solution of the invention is as follows: a recombinant serum amyloid A capable of enhancing immune response of crassostrea gigas is characterized in that an amino acid sequence is shown as SEQ ID NO. 1.
The preparation method of the recombinant serum amyloid A capable of enhancing the immune response of the crassostrea gigas is characterized by sequentially comprising the following steps of:
a. carrying out PCR amplification on the coding region fragment of the crassostrea gigas serum amyloid A gene by using specific primers P1 and P2, wherein the DNA sequence of the specific primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the specific primer P2 is shown as SEQ ID NO. 3;
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes;
b. purifying and recovering the amplified fragment, connecting the amplified fragment with a pMD19-T vector, screening positive clones after transformation, and extracting plasmids; use ofBamH I andXhoi, enzyme digestion of plasmid, purification and recovery of a target fragment generated by the enzyme digestion of plasmid;
c. combining the recovered target fragment with the proteinBamHI andXhoi enzyme-digested expression vector pET-30a is connected and transferred into escherichia coliTransetta(DE 3);
d. a single clone was picked, inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured at 37 ℃ to OD600= 0.4-0.8, 1 mmol L of final concentration is added-1Culturing for 4 hours after the inducer IPTG is added, centrifuging for 10 minutes at 4 ℃ and 5000 rpm, and collecting thalli;
e. purifying and renaturing the obtained thallus to obtain the serum amyloid A capable of enhancing the immune response of the crassostrea gigas.
The step e is as follows in sequence:
(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20 cm and the volume of a column bed being 10 mL;
(2) using buffer solution I to balance 2-5 bed volumes, the flow rate is 2 mL min-1(ii) a The buffer I had the following composition: 50 mmol L-1Tris-HCl buffer, pH 7.4; 50 mmol L-1NaCl；8 mol L-1Urea;
(3) d, resuspending the thallus collected in the step d with a buffer solution I, carrying out 150W ultrasonic disruption for 30 minutes, carrying out 12000 rmp, centrifuging for 30 minutes at 4 ℃, taking the supernatant, filtering the supernatant with a 0.45-micron filter membrane, and passing through a column at the flow rate of 1 mL min-1；
(4) Washing with buffer solution I for 2-5 bed volumes at a flow rate of 2 mL min-1；
(5) Washing with buffer solution II for 2-5 bed volumes at a flow rate of 2 mLmin-1(ii) a The buffer solution II is prepared by adding 50 mmol L of buffer solution I-1Imidazole;
(6) eluting the target protein with buffer solution III, and collecting(ii) a The buffer solution III is prepared by adding 400 mmol L of buffer solution I-1Imidazole;
(7) the collected proteins were renatured by dialysis.
The application of the recombinant serum amyloid protein A capable of enhancing the immune response of the crassostrea gigas in preparing a medicine for enhancing the immune response of the crassostrea gigas.
The recombinant ostrea gigas serum amyloid protein A (r) of the inventionCgSAA) capable of remarkably promoting cytokine in blood lymphocyte in vitro and in vivoCgIL17-1、CgIL17-5 andCgthe gene expression of TNF can enhance the immune response level of the crassostrea gigas, and has application value in the aspect of preparing medicaments such as novel immune preparations for shellfish, particularly breeding shellfish, and the like.
Drawings
FIG. 1 is a diagram showing the effects of recombinant ostrea gigas serum amyloid A before and after induction and purification.
FIG. 2 is a diagram showing the effect of optimizing the conditions for inducing expression of recombinant ostrea gigas serum amyloid A.
FIG. 3 is a schematic diagram showing the effect of recombinant serum amyloid A of the present invention on the promotion of cytokine gene expression.
Detailed Description
The recombinant serum amyloid A capable of enhancing the immune response of the crassostrea gigas is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
The preparation method of the recombinant serum amyloid A capable of enhancing the immune response of the crassostrea gigas is sequentially carried out according to the following steps:
a. specific primers P1 and P2 are used for treating oyster serum amyloid protein A (A)CgSAA) gene coding region segment is subjected to PCR amplification, the DNA sequence of the specific primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the specific primer P2 is shown as SEQ ID NO. 3;
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes;
b. will amplify the fragmentPurifying, recovering, connecting with pMD19-T vector, transforming, screening positive clone, and extracting plasmid; use ofBamH I andXhoi, enzyme digestion of plasmid, namely, purifying and recovering a target fragment generated by the enzyme digestion of plasmid by using a glue recovery and purification kit (Dalibao bioengineering Co., Ltd.);
c. combining the recovered target fragment with the proteinBamHI andXhoi enzyme-digested expression vector pET-30a is connected and transferred into escherichia coliTransetta(DE 3);
d. a single clone was picked, inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured at 37 ℃ to OD600=0.6, add final concentration 1 mmol L-1Culturing for 4 hours after the inducer IPTG is added, centrifuging for 10 minutes at 4 ℃ and 5000 rpm, and collecting thalli;
e. the obtained thalli is purified and renatured, and the specific steps are as follows in sequence:
(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20 cm and the volume of a column bed being 10 mL;
(2) using buffer solution I to balance 2-5 bed volumes, the flow rate is 2 mL min-1(ii) a The buffer I had the following composition: 50 mmol L-1Tris-HCl buffer, pH 7.4; 50 mmol L-1NaCl；8 mol L-1Urea;
(3) d, resuspending the thallus collected in the step d with a buffer solution I, carrying out 150W ultrasonic disruption for 30 minutes, carrying out 12000 rmp, centrifuging for 30 minutes at 4 ℃, taking the supernatant, filtering the supernatant with a 0.45-micron filter membrane, and passing through a column at the flow rate of 1 mL min-1；
(4) Washing with buffer solution I for 2-5 bed volumes at a flow rate of 2 mL min-1；
(5) Washing with buffer solution II for 2-5 bed volumes at a flow rate of 2 mLmin-1(ii) a The buffer solution II is prepared by adding 50 mmol L of buffer solution I-1Imidazole;
(6) eluting the target protein by using a buffer solution III and collecting; the buffer solution III is prepared by adding 400 mmol L of buffer solution I-1Imidazole;
(7) dialyzing the collected protein for renaturation: the collected denatured and purified product was subjected to 2 mM reduced glutathione and 0.4mM oxygenGlutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine and urea with reduced gradient were dialyzed repeatedly, and the urea concentration was gradually changed from 6M to 4M, 3M, 2M, 1M, 0M. Glycerol was not added when the dialysate was subjected to the last dialysis (urea 0M), and was dialyzed at 4 ℃ for 12 hours each time. Obtaining the recombinant serum amyloid protein A (r)CgSAA) with the amino acid sequence shown in SEQ ID NO. 1.
Length: 174 amino acids
Type (2): amino acids
Chain type: single strand
The characteristics are as follows: the molecular weight is 19.78 kDa, isoelectric point is 7.31, and has 1 conserved SAA domain.
The recombinant crassostrea gigas serum amyloid A before and after induction and the purification effect are shown in figure 1. M in FIG. 1: marker; 1: inducing a pre-mycoprotein; 2: induced mycoprotein; 3: after purification rCgSAA。
As can be seen, the monoclonal cultures of the examples of the present invention were induced to obtain clear bands.
Examples of the experiments
The recombinant protein r of the inventionCgComparison of different inducible expression of SAA
According to the preparation methods a, b and c of the present invention, after that, single clones were picked up and inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured to OD at different temperatures (37 ℃, 28 ℃, 16 ℃), respectively600= 0.6. Different final concentrations (1.5 mmol L) were added-1，1 mmol L-1，0.5 mmol L-1) The culture was continued for 4 hours after the induction agent IPTG was added, and then the cells were centrifuged at 4 ℃ and 5000 rpm for 10 minutes, respectively, and the cells were collected and frozen at-20 ℃ for further use. 1 mL of the cells were taken, added with 80. mu.L of water and 20. mu.L of a 5-fold protein-loading buffer, boiled at 100 ℃ for 10 minutes, centrifuged slightly, and the expression product was detected by SDS-PAGE. The results are shown in FIG. 1. In FIG. 2, A: different culture temperatures (37 ℃, 28 ℃, 16 ℃) and IPTG inducer concentrations (1.5 mmol L)-1，1 mmol L-1，0.5 mmol L-1) SDS-PAGE analysis of total protein of DE3 strain under the conditions (white arrow)And inducible expression of rCgSAA). B: under different conditions rCgQuantitative analysis of SAA-induced expression (according to r in graph A)CgSAA stripe grayscale computation statistics).
The results show that: preparation method r of the embodiment of the inventionCgThe highest SAA induction expression level.
The recombinant serum amyloid protein A (r) capable of enhancing the immune response of the crassostrea gigas of the inventionCgSAA) detection of the in vivo and in vitro Regulation of cytokine Gene expression in blood lymphocytes from Crassostrea gigas
The method comprises the following specific steps:
1. stimulation of blood lymphocytes in vivo and in vitro
The in vivo stimulation experiment procedure was as follows: dividing temporarily cultured adult species of Concha Ostreae into two groups, wherein each Concha Ostreae in one group is injected with 100 μ L of 500 μ g/mL rCgSAA, injecting 100 μ L PBS into each crassostrea gigas in the other group as negative control, injecting no individual as blank control, randomly sampling in each group at 6 hours and 12 hours after injection, extracting crassostrea gigas blood lymphocytes, and extracting RNA according to subsequent method to detect gene expression level.
The in vitro stimulation experiment procedure was as follows: collecting Concha Ostreae hemolymph at ratio of hemolymph: ALS anticoagulant = 1: 1, 800gThe blood cells were collected by centrifugation for 10 minutes. Washing collected blood lymphocytes for 2 times by using a salted L-15 culture medium, finally re-suspending by using a salted L-15 culture medium, and adjusting the cell concentration to 4-8 multiplied by 105cell mL-1The cells were plated in 12-well plates (1 mL per well), and the plated cells were incubated at 18 ℃. ALS anticoagulant formula: 20.8 g L-1glucose, 8 g L-1sodiumcitrate, 3.36 g L-1EDTA2Na，22.5 g L-1NaCl, pH 7.0; the formula of the L-15 culture medium added with salt comprises the following components: 20.2 gL-1NaCl, 0.54 g L-1KCl, 0.6 g L-1CaCl2, 1 g L-1MgSO4, 3.9 g L-1MgCl2, 20.8 gL-1glucose, 10% FCS. After the inoculated hemolymphocytes were cultured in vitro for 6 hours, 0. mu.g, 5. mu.g, 10. mu.g or 50. mu.g of the recombinant protein r was added to each wellCgSAA, after further culturing at 18 ℃ for 6 hours, was taken out and culturedPlate, remove the supernatant culture solution and wash with PBS once,
2. recombinant protein rCgAssay for SAA modulation of cytokine gene expression in blood lymphocytes
After the inoculated hemolymphocytes were cultured in vitro for 6 hours, 0. mu.g, 5. mu.g, 10. mu.g or 50. mu.g of the recombinant protein r was added to each wellCgSAA, incubation was continued for 6 hours at 18 ℃. The cells were lysed by directly adding 1 mL of Trizol lysate, total RNA was extracted according to the Trizol method, and then a cDNA template was prepared using a reverse transcription kit. Detecting with ABI 7500 gene detection instrument by using specific RT-PCR primer P3-P8 (the DNA sequence of the primer P3-P8 is shown as SEQ ID NO. 4-9)CgIL17-1、CgIL17-5 andCgexpression levels of TNF genes vary. Three replicates were set for each sample, the mean values were plotted based on the 3 test results, and the study data were statistically analyzed using SPSS 6.0 software with significant differences (p<0.05) with a mark.
The regulation effect of the recombinant serum amyloid A on the cytokine gene expression is shown in figure 3.
A, B, C in FIG. 3 respectively shows the r prepared by the present inventionCgSAA in the Pacific oyster bodyCgIL17-1、CgIL17-5 andCgpromotion of TNF expression; d represents r prepared by the inventionCgSAA in Pacific oyster in vitroCgIL17-1、CgIL17-5 andCgpromotion of TNF expression.
The experimental result shows that the recombinant serum amyloid protein A (r) capable of enhancing the immune response of the crassostrea gigas of the inventionCgSAA) can promote cytokine in Concha Ostreae blood lymphocyteCgIL17-1、CgIL17-5 andCgthe gene expression of TNF has the function of regulating the level of innate immune response, and can be used as a medicament for enhancing the immune response of crassostrea gigas.
Sequence listing
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<120> recombinant serum amyloid protein A capable of enhancing immune response of crassostrea gigas and preparation method thereof
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Claims (4)
1. A recombinant serum amyloid A capable of enhancing immune response of crassostrea gigas is characterized in that the amino acid sequence is shown as SEQID NO. 1.
2. A method for preparing recombinant serum amyloid a according to claim 1, which can enhance immune response of crassostrea gigas, comprising the steps of:
a. carrying out PCR amplification on the coding region fragment of the crassostrea gigas serum amyloid A gene by using specific primers P1 and P2, wherein the DNA sequence of the specific primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the specific primer P2 is shown as SEQ ID NO. 3;
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes;
b. purifying and recovering the amplified fragment, connecting the amplified fragment with a pMD19-T vector, screening positive clones after transformation, and extracting plasmids; use ofBamH I andXhoi, enzyme digestion of plasmid, purification and recovery of a target fragment generated by the enzyme digestion of plasmid;
c. combining the recovered target fragment with the proteinBamHI andXhoi enzyme-digested expression vector pET-30a is connected and transferred into escherichia coliTransetta(DE 3);
d. a single clone was picked, inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured at 37 ℃ to OD600= 0.4-0.8, 1 mmol L of final concentration is added-1Culturing for 4 hours after the inducer IPTG is added, centrifuging for 10 minutes at 4 ℃ and 5000 rpm, and collecting thalli;
e. purifying and renaturing the obtained thallus to obtain the serum amyloid A capable of enhancing the immune response of the crassostrea gigas.
3. The method for preparing serum amyloid A capable of enhancing the immune response of crassostrea gigas according to claim 2, wherein the step e is as follows in sequence:
(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20 cm and the volume of a column bed being 10 mL;
(2) using buffer solution I to balance 2-5 bed volumes, the flow rate is 2 mL min-1(ii) a The buffer I had the following composition: 50 mmol L-1Tris-HCl buffer, pH 7.4; 50 mmol L-1NaCl；8 mol L-1Urea;
(3) d, resuspending the thallus collected in the step d with a buffer solution I, carrying out 150W ultrasonic disruption for 30 minutes, carrying out 12000 rmp, centrifuging for 30 minutes at 4 ℃, taking the supernatant, filtering the supernatant with a 0.45-micron filter membrane, and passing through a column at the flow rate of 1 mL min-1；
(4) Washing with buffer solution I for 2-5 bed volumes at a flow rate of 2 mL min-1；
(5) Washing with buffer solution II for 2-5 bed volumes at a flow rate of 2 mLmin-1(ii) a The buffer solution II is prepared by adding 50 mmol L of buffer solution I-1Imidazole;
(6) eluting the target protein by using a buffer solution III and collecting; the buffer solution III is prepared by adding 400 mmol L of buffer solution I-1Imidazole;
(7) the collected proteins were renatured by dialysis.
4. The recombinant serum amyloid protein A according to claim 1, which is used for enhancing the immune response of crassostrea gigas, and is characterized by being used for preparing a medicament for enhancing the immune response of crassostrea gigas.
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Cited By (2)
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| CN113717268A (en) * | 2021-09-27 | 2021-11-30 | 北京市水产科学研究所（国家淡水渔业工程技术研究中心） | Application of koi serum amyloid A5 or encoding gene thereof in regulation and control of koi against pathogenic bacteria infection |
| CN114470160A (en) * | 2022-03-18 | 2022-05-13 | 山东农业大学 | Inhibitors of viral replication and uses thereof |
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