CN107011424B

CN107011424B - Epinephelus coioides complement C1INH gene, vector, recombinant strain and protein and application thereof

Info

Publication number: CN107011424B
Application number: CN201710151088.0A
Authority: CN
Inventors: 王维娜; 罗盛伟; 谢仁崇; 刘媛; 康欢
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2017-03-14
Filing date: 2017-03-14
Publication date: 2020-07-03
Anticipated expiration: 2037-03-14
Also published as: CN107011424A

Abstract

The invention discloses a grouper rC1INH-Nonserpin protein, the amino acid sequence of which is shown as SEQ ID NO. 4, or the amino acid sequence shown as SEQ ID NO. 4 is a protein which is modified by substituting, deleting and/or adding one or more amino acids and/or the tail end and has the same or higher activity. The complement C1INH gene sequence of the grouper and the functional fragment rC1INH-Nonserpin nucleotide sequence on the C1INH sequence are obtained for the first time, so that the gene library of the grouper is enriched, the method can be applied to preparation of recombinant protein, fish immune preparations or feed additives, and a new theoretical and practical basis is provided for physiological immune research of the grouper.

Description

Epinephelus coioides complement C1INH gene, vector, recombinant strain and protein and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to rC1INH-Nonserpin protein, a gene for coding the protein, a vector containing the gene and application of the gene; also relates to a complement C1INH protein, a gene for coding the protein, a vector containing the gene and application thereof.

Background

Complement plays a regulatory center in the animal immune system regulated by macrophage activating factor, and can effectively enhance the immunity of the organism by improving the phagocytosis of immune cells to invading bacteria and stimulating the generation of superoxide dismutase. However, excessive complement activation can cause damage to the body. Among the complement systems, complement C1 inhibitor (C1INH) is the only negative regulatory element in complement elements. Complement C1INH is a single chain molecule, typically composed of 478 amino acid residues, with two pairs of disulfide bonds in the chain, a nonsperpin domain at the N-terminus and a serine protease inhibitor domain at the C-terminus. Functionally, C1INH is capable of negatively regulating three complement activation pathways, which is capable of inhibiting activity by forming covalent ester bonds with the active portions of C1r and C1s, thereby inhibiting the classical pathway of complement; c1INH can inhibit the lectin pathway of complement by inhibiting the activity of MBSP-1 and MBSP-2; c1INH may also inhibit the alternative complement pathway by binding factor B to C3B. The C1INH is a multifunctional macromolecule, can inhibit three complement pathways, can effectively antagonize the toxic and side effects of LPS due to the existence of the C1INH, can inhibit the activities of kallikrein, blood coagulation factor XII, blood coagulation factor XI and plasmin, and has important regulation and control effects on inflammatory response.

Epinephelus coioides (A) and (B)Epinepheluscoioides) Belonging to the order Perciformes (Perciformes) Bass family (Serranidae) Grouper subfamily (Epinephelinae) Genus Epinephelus (A)Epinephelus) The water-warming reef fish is widely distributed in tropical and subtropical regions of the Indian ocean and the Pacific ocean. The grouper belongs to one of rare marine fishes, has delicious meat quality, is popular among consumers in various regions, and has great economic value. However, the current problems of rapid climate change disasters and serious polluted water quality are faced, so that the morbidity and the mortality of the diseases are high, and the serious economic attack to the artificial culture of the grouper is also a problem which is difficult to solve. How to enhance the stress resistance of the groupers and improve the cultivation survival rate is a problem to be solved urgently at present.

Disclosure of Invention

An object of the present invention is to provide a rockfish rC1 INH-nonsperpin protein.

Another object of the present invention is to provide a gene encoding rC1INH-Nonserpin protein of grouper as described above.

Another objective of the invention is to provide a protein in the structural domain of rockfish complement C1INH, namely rockfish complement rC1INH-Nonserpin protein.

Another object of the present invention is to provide a gene encoding the complement C1INH protein of grouper as described above.

Another objective of the invention is to provide a cloning vector containing the rockfish complement C1INH protein gene and a functional fragment rC1 INH-Nonserpin.

Another objective of the invention is to provide an expression vector containing the grouper complement C1INH protein gene and a functional fragment rC1 INH-Nonserpin.

The invention also aims to provide a grouper complement C1INH functional fragment rC1INH-Nonserpin recombinant protein and a preparation method thereof.

The invention also aims to provide application of rockfish rC1INH-Nonserpin protein and/or C1INH protein in improving immunity of rockfish.

The technical scheme adopted by the invention is as follows:

the rockfish rC1INH-Nonserpin protein has the amino acid sequence shown in SEQ ID NO. 4, or the protein which has the same or higher activity after the amino acid sequence shown in SEQ ID NO. 4 is substituted, deleted and/or added with one or more amino acids and/or the tail end is modified.

The gene of rC1INH-Nonserpin protein of the grouper is coded. The nucleotide sequence is shown as SEQ ID NO: 3, respectively.

The amino acid sequence of the grouper complement C1INH protein is shown as SEQ ID NO: 2, or SEQ ID NO: 2 by substituting, deleting and/or adding one or more amino acids and/or modifying the tail end, and has the same or higher activity.

The gene of the C1INH protein of the grouper complement. The nucleotide sequence is shown as SEQ ID NO: 1 is shown.

The method for producing rockfish rC1INH-Nonserpin protein or rockfish complement C1INH protein comprises the steps of introducing the expression vector into host cells, and expressing to obtain rC1INH-Nonserpin protein or C1INH protein.

The invention has the following beneficial effects:

1. according to the invention, the complement C1INH gene sequence of the grouper and the functional fragment rC1INH-Nonserpin nucleotide sequence on the C1INH sequence are obtained for the first time, so that the gene library of the grouper is enriched, and the method can be applied to preparation of recombinant protein, fish immune preparation or feed additive, and provides a new theoretical and practical basis for physiological immune research of the grouper;

2. the grouper rC1INH-Nonserpin nucleotide sequence is expressed by the recombinant strain, so that the grouper rC1INH-Nonserpin recombinant protein can be produced in large quantity, the production cost is greatly reduced, and the economic value is high;

3. experiments prove that the grouper rC1INH-Nonserpin recombinant protein provided by the invention has the functions of improving the immunity of the groupers and the like, can be applied to preparation of fish, particularly a grouper immune preparation or a feed additive, and has wide application prospects.

Drawings

FIG. 1 is a gel electrophoresis analysis of the intermediate fragment of the C1INH gene of Epinephelus coioides complement (M: DNA molecular weight standard; 1, 2: PCR product of the intermediate fragment of the C1INH gene of Epinephelus coioides);

FIG. 2 is a second PCR gel electrophoresis analysis of the synthesis of 5 'and 3' fragments of complement C1INH gene of Epinephelus coioides; (M: DNA molecular weight standard; 1-4: PCR product of 5 'end fragment of grouper; 6-8: PCR product of 3' end fragment of grouper; NC: negative control);

FIG. 3 is an electrophoretic identification chart of the PCR amplification product of the ORF of the C1INH gene (M: DNA molecular weight standard; 1: PCR product of the full-length sequence of the ORF of the C1INH gene of rockfish);

FIG. 4 is a gel electrophoresis analysis diagram of PCR amplification products of the C1INH functional fragment rC1INH-Nonserpin of Epinephelus coioides (M: DNA molecular weight standard; 1: PCR product of ORF full-length sequence of Epinephelus coioides rC1INH-Nonserpin gene; NC: negative control);

FIG. 5 is the cloning vector pMD18-T-rC1INH-Nonserpin double enzyme cut (II)EcoRI、XbaI) Verification graph (M: DNA molecular weight standard; 1: pMD18-T-rC1INH-Nonserpin plasmid, 2: double restriction enzyme pMD18-T-rC1INH-Nonserpin plasmid);

FIG. 6 isExpression vector pET32a-rC1INH-Nonserpin double enzyme digestion: (EcoRI、XbaI) Verification graph (M: DNA molecular weight standard; 1: double restriction enzyme pET32a-rC1INH-Nonserpin plasmid, 2: pET32a-rC1INH-Nonserpin plasmid, 3: double restriction enzyme pET32a plasmid, 4: the pET32a plasmid);

FIG. 7 is an SDS-PAGE analysis of recombinant protein rC1INH-Nonserpin expressed by recombinant strain pET32a-rC1INH-Nonserpin-BL21 (M: protein molecular weight standard; 1: pET32a-BL21 induction product; 2: pET32a-rC1INH-Nonserpin-BL21 induction product; 3: supernatant after disruption of pET32a-rC1INH-Nonserpin-BL21 induction product; 4: precipitation after disruption of pET32a-rC1INH-Nonserpin-BL21 induction product; 5: purified recombinant protein of rC1 INH-Nonserpin);

FIG. 8 is a western-blot identification chart of recombinant protein rC1INH-Nonserpin-BL21 expression product rC1INH-Nonserpin recombinant protein of recombinant strain pET32a-rC1INH-Nonserpin-BL21 (M: protein molecular weight standard; 1: pET32a-BL21 induction product; 2: pET32a-rC1INH-Nonserpin-BL21 induction product);

FIG. 9 is a schematic diagram of pET32a-rC1INH-Nonserpin expression vector construction;

FIG. 10 is a rC1INH-Nonserpin epitope prediction assay;

FIG. 11 is an electrophoretogram of RNA extracted from a sample;

FIG. 12 is the change in the levels of MyD88 gene in fish blood of three groups A, B and C in LPS stimulation (different letters indicate significant differences compared to control group, P < 0.0 ═ 5);

FIG. 13 is a graph of the change in the levels of TNF1 gene in A, B, C three groups of fish blood in LPS stimulation (different letters indicate significant differences compared to the control group, P < 0.05);

FIG. 14 is a graph of the change in the levels of TNF2 gene in A, B, C three groups of fish blood in LPS stimulation (different letters indicate significant differences compared to the control group, P < 0.05);

FIG. 15 is a graph of the change in the level of IL1b gene in A, B, C three groups of fish blood in LPS stimulation (different letters indicate significant difference compared to control group, P < 0.05).

Detailed Description

The present invention will be further illustrated by the following examples, but is not limited thereto. The molecular biological experimental techniques used in the following examples include PCR amplification, plasmid extraction, plasmid transformation, ligation of DNA fragments, digestion, gel electrophoresis, etc., and, unless otherwise specified, are generally performed according to conventional methods, specifically, see molecular cloning, A.C. (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peyer, 2002, Beijing: science publishers), or according to the conditions recommended by the manufacturers.

Synthesis of intermediate fragment of Epinephelus coioides complement C1INH gene

A section of highly homologous fragment of the human C1INH gene sequence is analyzed by sequence analysis software, a degenerate primer pair of C1INH-F and C1INH-R is designed, an upstream primer C1INH-F is [ 5 '-TGTAACCGCTCAGTCGCC ] (SEQ ID NO. 5) and a downstream primer C1INH-R [ 5' -GCATTATCCCTCGCACCTA ] (SEQ ID NO. 6), and the liver cDNA of the Epinephelus coioides obliquus obtained by reverse transcription of M-MLV retrotranscriptase kit (Promega, Madison, Wis., USA) is taken as a template, and the intermediate fragment of the Epinephelus coioides complement C1INH is obtained by the PCR descent amplification, wherein the total length is 753bp. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; (1) denaturation at 94 ℃ for 30 seconds, annealing at 66 ℃ for 30 seconds, and extension at 72 ℃ for 45 seconds for 5 cycles; (2) denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 45 seconds for 5 cycles; finally, denaturation at 94 ℃ for 30 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 45 seconds, 30 cycles in total, and finally extension at 72 ℃ for 10 minutes. The electrophoretic identification of the amplification product is shown in FIG. 1.

Synthesis of 5 'end fragment and 3' end fragment of Epinephelus coioides complement C1INH gene

The operation was carried out according to the BD SMART RACE cDNA Ampli ﬁ location Kit (BD Bioscience Clontech, Calif., USA) Protocol. According to the requirements of the kit, two primers, namely C1INH5-1 and C1INH5-2 are designed according to the sequenced sequence of the intermediate fragment of the complement C1INH for 5 'end fragment nested PCR, and two primers, namely C1INH3-1 and C1INH3-2 are designed for 3' end fragment nested PCR. In the 5 ' end fragment nested PCR reaction, the first primer C1INH5-1 is [ 5 ' -GCACCCCGCTGATACTGATAGGAG ] (SEQ ID NO. 7), and the second primer C1INH5-2 is [ 5 ' -GGGTTCCCCTGTGCGACTGTTC ] (SEQ ID NO. 8). In the 3 ' end fragment nested PCR reaction, the first upstream primer C1INH3-1 is [ 5 ' -AACACCCGTAGAGCCATTGAGACAG ] (SEQ ID NO. 9), and the second upstream primer C1INH3-2 is [ 5 ' -GTTTTATGAAGCAAAGCCCACCAGG ] (SEQ ID NO. 10).

In the first 5 'end and 3' end nested PCR reaction, the liver cDNA of the Epinephelus coioides obtained by reverse transcription of M-MLV reverse transcriptase kit (Promega, Madison, Wis., USA) is used as a template and is amplified by a touchdown PCR method. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; (1) denaturation at 94 ℃ for 30 seconds, annealing at 72 ℃ for 30 seconds, 5 cycles in total, and extension at 72 ℃ for 2 minutes; (2) denaturation at 94 ℃ for 30 seconds, annealing at 70 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes for 5 cycles; final denaturation at 94 ℃ for 30 seconds and annealing at 68 ℃ for 30 seconds and extension at 72 ℃ for 2 minutes for 30 cycles. Performing amplification by a touchdown PCR method by using the first PCR product as a template in the second PCR, (1) performing denaturation at 94 ℃ for 30 seconds, performing annealing at 72 ℃ for 30 seconds, performing 5 cycles in total, and performing extension at 72 ℃ for 2 minutes; (2) denaturation at 94 ℃ for 30 seconds, annealing at 70 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes for 5 cycles; final denaturation at 94 ℃ for 30 seconds and annealing at 68 ℃ for 30 seconds and extension at 72 ℃ for 2 minutes for 30 cycles. The first PCR showed no detectable band on the agarose gel, and the electrophoretic identification of the second PCR amplification product is shown in FIG. 2.

Splicing of the full-length sequence of the Epinephelus coioides complement C1INH gene

Splicing sequenced complement C1INH intermediate fragments, 5 'RACE fragments and 3' RACE fragments to obtain the ORF full length of the Epinephelus coioides complement C1INH gene, namely the 1-2219 bp of the complete complement C1INH cDNA sequence (SEQ ID NO. 1) of Epinephelus coioides, the total length is 2219bp, and the amino acid sequence (SEQ ID NO. 2) is deduced.

And designing a primer C1INHqF (SEQ ID NO. 11) and a primer C1INHqA (SEQ ID NO. 5-TGGGTTGGTCACTCTGCCAACAA) according to the full length of the ORF of the C1INH gene obtained by splicing (SEQ ID NO. 12). The amplified length was 1797 bp. The complement C1INH sequence of the grouper is amplified by a PCR method by taking liver cDNA of the grouper as a template, wherein the liver cDNA is obtained by reverse transcription of M-MLV reverse transcriptase transcription kit (Promega, Madison, Wis., USA), and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; the following are 40 cycles: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes; final extension at 72 ℃ for 10 min. The electrophoretic identification of the PCR amplification product is shown in FIG. 3.

Fourthly, analyzing the structural sequence of the C1INH complement Nonserpin of the Epinephelus coioides active segment by a bioinformatics method

In addition. The epitope of complement C1INH gene of grouper was analyzed by software (fig. 10). Further, the active segment grouper C1INH-Nonserpin structural sequence comprising a plurality of binding sites is determined.

Synthesis of Epinephelus coioides functional fragment rC1INH-Nonserpin sequence gene

Designing and synthesizing two primers according to the analyzed cDNA sequence (SEQ ID NO. 3) of the functional fragment rC1INH-Nonserpin of the epinephelus coioides, wherein an upstream primer (5' -CGCGGATCCATGAAACTACAGGCCGTACT) (SEQ ID NO. 13) is synthesized; the downstream primer [ 5' -CCCAAGCTTATACAGCTTCATTGAAAACT ] (SEQ ID NO. 14). The method comprises the following steps of (1) amplifying a complement C1INH functional fragment rC1INH-Nonserpin sequence of the grouper by a PCR method by taking liver cDNA of the grouper obliquus obtained by reverse transcription of M-MLVrevrse transcription kit (Promega, Madison, Wis., USA) as a template, wherein the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; the following are 40 cycles: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds; final extension at 72 ℃ for 10 min. The electrophoretic identification of the PCR amplification product is shown in FIG. 4.

Sixthly, cloning vector containing functional fragment rC1INH-Nonserpin sequence of epinephelus coioides

Separating and purifying the obtained rC1INH-Nonserpin sequence fragment, then connecting the rC1INH-Nonserpin sequence fragment with a pMD18-T (TAKARA) vector at 4 ℃ overnight (about 16 h) according to a system provided by the kit, transforming the ligation product into Escherichia coli DH5 α, screening out positive clones through Amp + resistance and blue white spots, extracting plasmids through a plasmid extraction kit, verifying double digestion and sequencing by the plasmids, and proving that the functional fragment rC1INH-Nonserpin sequence of the Epinephelus coioides is cloned into the vector and named as pMD18-T-rC1INH-Nonserpin, and obtaining a recombinant strain named as pMD18-T-rC1INH-Nonserpin-DH5 α by transforming the Escherichia coli DH5 α by the cloning vectorBamHI 、HindIIIFor cloning vector pMD18-T-rC1INH-Nonserpin is subjected to double enzyme digestion, and the enzyme digestion map is shown in figure 5.

Seventhly, construction of expression vector pET32a-rC1INH-Nonserpin containing epinephelus coioides functional fragment rC1INH-Nonserpin sequence

Restriction enzyme for cloning vector pMD18-T-rC1INH-NonserpinBamHIAndHindIIIafter double enzyme digestion，The product of the digestion is E.Z.N.A.^®Recovering Gel Extraction Kit, performing agarose Gel electrophoresis, and separating and purifying the functional fragment rC1INH-Nonserpin sequence of the Epinephelus coioides;

the vector plasmid pET32a was restricted by restriction endonucleaseBamHIAndHindIIIafter double enzyme digestion, the fragment is mixed with a functional fragment rC1INH-Nonserpin sequence of the Epinephelus coioides in a ratio of 1:3, and the mixture is connected with T4 ligase at 16 ℃ for 16 hours and then is transformed into Escherichia coli DH5a by a calcium chloride conversion method. Screening transformants having Amp resistance with LB plate, extracting plasmid, and using restriction enzymeEcoRIAndXbaIthe recombinant plasmid was identified by double digestion and named pET32a-rC1 INH-Nonserpin. The plasmid construction scheme is shown in FIG. 9. Use ofBamHI 、HindIIIThe expression vector pET32a-rC1INH-Nonserpin was subjected to double digestion, and the digestion analysis chart is shown in FIG. 6.

Eighth, can express the Escherichia coli recombinant strain pET32a-rC1INH-Nonserpin-BL21 construction of the functional fragment rC1INH-Nonserpin protein of Epinephelus coioides

The objective recombinant plasmid obtained in example 7 was transformed into E.coli BL21 by calcium chloride transformation, and transformants resistant to Amp were selected on LB plates as pET32a-rC1INH-Nonserpin-BL 21.

Ninth, the Escherichia coli recombinant bacterium pET32a-rC1INH-Nonserpin-BL21 is used for producing recombinant Epinephelus coioides functional fragment rC1INH-Nonserpin protein

Respectively picking recombinant BL21 monoclonal engineering bacteria, inoculating in LB culture medium containing AMP resistance, culturing OD600 to 2-6 at 37 ℃ and 200rpm, expanding the strain, replacing fresh LB culture medium containing AMP resistance, stopping when OD600 is 1.0, and adding about 1mM IPTG to induce for 4 h. After induction, the cells were collected at 2000rpm and 4 ℃. Adding TEB buffer to suspend the thallus, adding a proper amount of lysozyme, standing overnight at 4 ℃, and then ultrasonically crushing in an ice bath until the thallus is clear. Resuspend it with Buffer A containing 30% TritioX-100, let stand on ice for 30 min, add Buffer B containing urea to dissolve the protein at room temperature. The solubilized protein was purified according to the method of His bind Ni-NTA (Novagan). The results are shown in FIG. 7.

Ten, grouper rC1INH-Nonserpin protein antigen activity identification experiment

And (3) carrying out immunological activity identification on the recombinant Epinephelus coioides functional fragment rC1INH-Nonserpin protein by adopting an immunoblotting (Westernblot) method. The primary antibody was murine his monoclonal antibody (Novagen) and the secondary antibody was goat anti-mouse IgG-HRP (Dingguo). The result is shown in figure 8, and shows that the murine his monoclonal antibody can identify the recombinant Epinephelus coioides functional fragment rC1INH-Nonserpin protein expressed by Pichia pastoris, and the obtained protein is proved to be the recombinant Epinephelus coioides functional fragment rC1INH-Nonserpin protein.

Eleven, animal experiments

1. Preparation before experiment

Mixing the purified grouper rC1INH-Nonserpin protein and LPS according to the mass-volume ratio rC1 INH-Nonserpin: LPS = 3: mixing evenly on 10 ice, and shaking gently at 180rpm for 2h at 4 ℃ to obtain the LPS + rC1INH-Nonserpin mixed solution.

2. Design of experiments

The experimental components are 3 groups, group A is injection purified 30 mug rC1INH-Nonserpin protein liquid/tail, and PBS is used for complementing the volume; group B was injected with 100. mu.g LPS lysate/tail, and the volume was made up with PBS; group C was injected with 100. mu.g LPS + 30. mu.g rC1INH-Nonserpin protein cocktail/tail.

3. Conditions of the experiment

Selecting the Epinephelus coioides of the same batch, healthy physique and similar specifications to breed in prepared storage boxes of 1.0 m × 0.5.5 m × 0.5.5 m, filling 40L of seawater, 25-30 ℃ of water temperature, 28-32 per thousand of water salinity, 8.1-8.3 of pH value and more than 5.0 mg/L in each storage box, sampling at 0 hour, 6 hours, 12 hours and 24 hours respectively after injecting each group of fishes, taking 5 fishes in each group, separating fish blood of each group, adding Trizol, quickly freezing by liquid nitrogen, immediately putting the fishes at-80 ℃ for storage.

RNA extraction and cDNA reverse transcription

According to the RNA extraction method, total RNA was extracted using Trizol reagent (Invitrogen, USA). The quality and purity of the extracted RNA was determined by the values of 260nm and 260/280 nm. The integrity of total RNA was judged by 1% agarose gel, see fig. 11. Reverse transcription experiments were performed using 1000ng of mRNA according to the method of the Revert Aid ™ M-MuLV Reverse Transcriptase Kit (MBI Fermentas, USA).

5. Real-time fluorescent quantitative qRT-PCR

The inverted cDNA was subjected to a Real-time quantitative fluorescence qRT-PCR experiment in which the total volume of the system was 20ml, 0.8ml of diluted upstream and downstream primers (Myd 88, TNF1, TNF2, il1 b), 0.4ml of ROX reference dye (50 ×), 10ml of SYBR premix Ex Taq II (perfect read time) (TaKaRa, Dalian, China), 0.4ml of cDNA template diluted 1 to 10 and 6ml of ultrapure water, the qRT-PCR reaction conditions included pre-denaturation at 95 ℃ for 2 minutes followed by 40 cycles of denaturation at 95 ℃ for 30 seconds and annealing at 65 ℃ for 30 seconds, and after the amplification was completed, 1 cycle of denaturation at 95 ℃ for 30 seconds and annealing at 60 ℃ for 30 seconds, and the amplified products were subjected to dissolution curve analysis, each qRT-PCR experiment was repeated 3 times, and the results were analyzed by 7500 SDS software (Applied Biosystems, USA) for 15-15 minutes.

The above experiments show that: in experimental group B, LPS was able to activate myd 88-dependent signaling pathway-activation 24 hours after LPS injection and up-regulate the relevant inflammatory factors downstream of the pathway. Experimental group a, injected with rC1 INH-noserpin recombinant protein, which failed to activate the LPS-dependent myd88 signaling pathway, served as a negative control. In the experimental group C, after LPS + rC1INH-Nonserpin injection, myd88 signal channel and its downstream related inflammatory factors can not be activated. Therefore, the recombinant protein rC1INH-Nonserpin can effectively interact with LPS (lipopolysaccharide) to reduce the stress effect on fish bodies, and the recombinant protein can be used for preparing fish immune preparations and improving the immunity of fishes, particularly groupers.

SEQUENCE LISTING

<110> university of south China

<120> Epinephelus coioides complement C1INH gene, vector, recombinant strain and protein and application thereof

<130>

<160>14

<170>PatentIn version 3.5

<210>1

<211>2219

<212>DNA

<213>Epinepheluscoioides

<400>1

atggggctgc agtttagagg agcagacagt cagaggtggt gaacgggtgt ggcagaaata 60

ttactgctaa aaactggtga ctgaatttgg atacatttgg aaatccaaga ggtgtgagat 120

gaaactacag gccgtactct gcctcttgct gctgctcgtt ttcgggcttt cttcatgcag 180

acatttcgag gtgatacctg gttccactct ggagctgccc tgtctctcgt ttcaagatgt 240

ctacactgga gggaccatca cctggaaatt caatggtgaa gatctaagtc tcagcgctca 300

gtcgcctggc tcgccaagaa tgaaaaaggg tggcatggtt ctctctgtat cccctgttac 360

tgccgccagc cagggcgaat acagttgtac gatagaggag aacgatctgg agatgtccag 420

tacatacacc attaacgttg tacacatgat ctataccatg aaggtgaccc aaggctccac 480

agctcaactc cagtgccatt tcccaacttc cagccaagtc acagccaatg cactttggtt 540

caaagaggac aataatggca caaggacaca gctggacact gaagacgagt caagtgataa 600

cagagtgaac ctgctttatc cactcggcct tgatcagacc atcatgatca aaaacattgt 660

catggaggat tctggaaatt acttgtgtga atctgctggg gggcaaacac tgagctcagt 720

atatatcaat gcccaagttg ctcctactcc tgtccctcac tcgtgccttg gcttcaccgc 780

agcatgggag ccctgccagg atgagaacag tcgcacaggg gaacccattt tgcaggagtc 840

catcacggag ttttcaatga agctgtattc ttacctcaga gaatcacaac cctccagcaa 900

tctactcttc tctcctatca gtatcagcgg ggtgctgtcc catttgttgc taggtgccag 960

ggataacacc cgtagagcca ttgagacagc tgtcagtgtg cctcatgact tccactgtgt 1020

tcacctccac atgaagaagc ttagagagaa gttggccggc tccttgcaga tggcttctca 1080

gatctactat aaccctcata atatgaatct gagcgagtcc tttaccaacc agtccatcca 1140

gttttatgaa gcaaagccca ccaggctgct ggaaaccagt gaggaaaaca cacagatgat 1200

caacagctgg gtggcaaata agaccaacaa taagatccaa catttggttg actctgtatc 1260

acccaataca cagatgatac tgctcaacgc cgtctccttt agcggtgagt ggaaggtcaa 1320

gtatgatatg aagccccgga aaggactttt cacaaaactg gatggtgata tggtgtcggt 1380

gccagtcctc tatcattcgg gatacatggc agctactaag tttgtagtcg agctgaaggc 1440

acacgtggca aggttggctc tcacgggtga caacagtctt tacatcctgc tgcctcgctc 1500

ccacaaagtg agtgacctgc agcagctgga ggagaagatg acggacacag ctgtgcgtca 1560

agtgattgaa caactgaaaa caacaactcc tcagcttgtt gaggtcactc tgcccaaaat 1620

caagctgcag gttgagccag acatgaactt actcattaag aaattaggac tatcattact 1680

ctttgagaat cccaacctgt gtggtctcta ctcggaagac aggatagctt tgatgatgcc 1740

agacacagag ccttcctcgc actgtaccga acaaggagtt gaggctgggg ccgccaccag 1800

catgaccttc tctcgttcct tcacttcctt ctctgccctg cggcctttca tcatgctgct 1860

gtggagtgac caggctaatg taccgctctt tgttggcaga gtgaccaacc catgagagag 1920

agaaacggag agagggagtg cagatgtaac agatgggcaa aagggcagga aagaaacaaa 1980

gatattataa acaaagccca tctggtagaa tggaaaatac atgtaatgat cttactgtgc 2040

tttaacactg tacattcagt atcagtattg cttcaggtag ttactaaagt gagcagtgac 2100

cctcacagaa acatccagtt ctgaatgtgg attgttttca aggatgatga tgaataaact 2160

catattagca caactaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2219

<210>2

<211>598

<212>PRT

<213>Epinepheluscoioides

<400>2

Met Lys Leu Gln Ala Val Leu Cys Leu Leu Leu Leu Leu Val Phe Gly

1 5 10 15

Leu Ser Ser Cys Arg His Phe Glu Val Ile Pro Gly Ser Thr Leu Glu

20 25 30

Leu Pro Cys Leu Ser Phe Gln Asp Val Tyr Thr Gly Gly Thr Ile Thr

35 40 45

Trp Lys Phe Asn Gly Glu Asp Leu Ser Leu Ser Ala Gln Ser Pro Gly

50 55 60

Ser Pro Arg Met Lys Lys Gly Gly Met Val Leu Ser Val Ser Pro Val

65 70 75 80

Thr Ala Ala Ser Gln Gly Glu Tyr Ser Cys Thr Ile Glu Glu Asn Asp

85 90 95

Leu Glu Met Ser Ser ThrTyr Thr Ile Asn Val Val His Met Ile Tyr

100 105 110

Thr Met Lys Val Thr Gln Gly Ser Thr Ala Gln Leu Gln Cys His Phe

115 120 125

Pro Thr Ser Ser Gln Val Thr Ala Asn Ala Leu Trp Phe Lys Glu Asp

130 135 140

Asn Asn Gly Thr Arg Thr Gln Leu Asp Thr Glu Asp Glu Ser Ser Asp

145 150 155 160

Asn Arg Val Asn Leu Leu Tyr Pro Leu Gly Leu Asp Gln Thr Ile Met

165 170 175

Ile Lys Asn Ile Val Met Glu Asp Ser Gly Asn Tyr Leu Cys Glu Ser

180 185 190

Ala Gly Gly Gln Thr Leu Ser Ser Val Tyr Ile Asn Ala Gln Val Ala

195 200 205

Pro Thr Pro Val Pro His Ser Cys Leu Gly Phe Thr Ala Ala Trp Glu

210 215 220

Pro Cys Gln Asp Glu Asn Ser Arg Thr Gly Glu Pro Ile Leu Gln Glu

225 230 235 240

Ser Ile Thr Glu Phe Ser Met Lys Leu Tyr Ser Tyr Leu Arg Glu Ser

245 250 255

Gln Pro Ser Ser Asn Leu Leu PheSer Pro Ile Ser Ile Ser Gly Val

260 265 270

Leu Ser His Leu Leu Leu Gly Ala Arg Asp Asn Thr Arg Arg Ala Ile

275 280 285

Glu Thr Ala Val Ser Val Pro His Asp Phe His Cys Val His Leu His

290 295 300

Met Lys Lys Leu Arg Glu Lys Leu Ala Gly Ser Leu Gln Met Ala Ser

305 310 315 320

Gln Ile Tyr Tyr Asn Pro His Asn Met Asn Leu Ser Glu Ser Phe Thr

325 330 335

Asn Gln Ser Ile Gln Phe Tyr Glu Ala Lys Pro Thr Arg Leu Leu Glu

340 345 350

Thr Ser Glu Glu Asn Thr Gln Met Ile Asn Ser Trp Val Ala Asn Lys

355 360 365

Thr Asn Asn Lys Ile Gln His Leu Val Asp Ser Val Ser Pro Asn Thr

370 375 380

Gln Met Ile Leu Leu Asn Ala Val Ser Phe Ser Gly Glu Trp Lys Val

385 390 395 400

Lys Tyr Asp Met Lys Pro Arg Lys Gly Leu Phe Thr Lys Leu Asp Gly

405 410 415

Asp Met Val Ser Val Pro Val Leu Tyr HisSer Gly Tyr Met Ala Ala

420 425 430

Thr Lys Phe Val Val Glu Leu Lys Ala His Val Ala Arg Leu Ala Leu

435 440 445

Thr Gly Asp Asn Ser Leu Tyr Ile Leu Leu Pro Arg Ser His Lys Val

450 455 460

Ser Asp Leu Gln Gln Leu Glu Glu Lys Met Thr Asp Thr Ala Val Arg

465 470 475 480

Gln Val Ile Glu Gln Leu Lys Thr Thr Thr Pro Gln Leu Val Glu Val

485 490 495

Thr Leu Pro Lys Ile Lys Leu Gln Val Glu Pro Asp Met Asn Leu Leu

500 505 510

Ile Lys Lys Leu Gly Leu Ser Leu Leu Phe Glu Asn Pro Asn Leu Cys

515 520 525

Gly Leu Tyr Ser Glu Asp Arg Ile Ala Leu Met Met Pro Asp Thr Glu

530 535 540

Pro Ser Ser His Cys Thr Glu Gln Gly Val Glu Ala Gly Ala Ala Thr

545 550 555 560

Ser Met Thr Phe Ser Arg Ser Phe Thr Ser Phe Ser Ala Leu Arg Pro

565 570 575

Phe Ile Met Leu Leu Trp Ser Asp Gln Ala Asn ValPro Leu Phe Val

580 585 590

Gly Arg Val Thr Asn Pro

595

<210>3

<211>753

<212>DNA

<213>Epinepheluscoioides

<400>3

atgaaactac aggccgtact ctgcctcttg ctgctgctcg ttttcgggct ttcttcatgc 60

agacatttcg aggtgatacc tggttccact ctggagctgc cctgtctctc gtttcaagat 120

gtctacactg gagggaccat cacctggaaa ttcaatggtg aagatctaag tctcagcgct 180

cagtcgcctg gctcgccaag aatgaaaaag ggtggcatgg ttctctctgt atcccctgtt 240

actgccgcca gccagggcga atacagttgt acgatagagg agaacgatct ggagatgtcc 300

agtacataca ccattaacgt tgtacacatg atctatacca tgaaggtgac ccaaggctcc 360

acagctcaac tccagtgcca tttcccaact tccagccaag tcacagccaa tgcactttgg 420

ttcaaagagg acaataatgg cacaaggaca cagctggaca ctgaagacga gtcaagtgat 480

aacagagtga acctgcttta tccactcggc cttgatcaga ccatcatgat caaaaacatt 540

gtcatggagg attctggaaa ttacttgtgt gaatctgctg gggggcaaac actgagctca 600

gtatatatca atgcccaagt tgctcctact cctgtccctc actcgtgcct tggcttcacc 660

gcagcatggg agccctgcca ggatgagaac agtcgcacag gggaacccat tttgcaggag 720

tccatcacgg agttttcaat gaagctgtat taa 753

<210>4

<211>250

<212>PRT

<213>Epinepheluscoioides

<400>4

Met Lys Leu Gln Ala Val Leu Cys Leu Leu Leu Leu Leu Val Phe Gly

1 5 10 15

Leu Ser Ser Cys Arg His Phe Glu Val Ile Pro Gly Ser Thr Leu Glu

20 25 30

Leu Pro Cys Leu Ser Phe Gln Asp Val Tyr Thr Gly Gly Thr Ile Thr

35 40 45

Trp Lys Phe Asn Gly Glu Asp Leu Ser Leu Ser Ala Gln Ser Pro Gly

50 55 60

Ser Pro Arg Met Lys Lys Gly Gly Met Val Leu Ser Val Ser Pro Val

65 70 75 80

Thr Ala Ala Ser Gln Gly Glu Tyr Ser Cys Thr Ile Glu Glu Asn Asp

85 90 95

Leu Glu Met Ser Ser Thr Tyr Thr Ile Asn Val Val His Met Ile Tyr

100 105 110

Thr Met Lys Val Thr Gln Gly Ser Thr Ala Gln Leu Gln Cys His Phe

115 120 125

Pro Thr Ser Ser Gln Val Thr Ala Asn Ala Leu Trp Phe Lys Glu Asp

130 135 140

Asn Asn Gly Thr Arg Thr Gln Leu Asp Thr Glu Asp Glu Ser Ser Asp

145 150 155 160

Asn Arg Val Asn Leu Leu Tyr Pro Leu Gly Leu Asp Gln Thr Ile Met

165 170 175

Ile Lys Asn Ile Val Met Glu Asp Ser Gly Asn Tyr Leu Cys Glu Ser

180 185 190

Ala Gly Gly Gln Thr Leu Ser Ser Val Tyr Ile Asn Ala Gln Val Ala

195 200 205

Pro Thr Pro Val Pro His Ser Cys Leu Gly Phe Thr Ala Ala Trp Glu

210 215 220

Pro Cys Gln Asp Glu Asn Ser Arg Thr Gly Glu Pro Ile Leu Gln Glu

225 230 235 240

Ser Ile Thr Glu Phe Ser Met Lys Leu Tyr

245 250

<210>5

<211>18

<212>DNA

<213> Artificial sequence

<400>5

tgtaaccgct cagtcgcc 18

<210>6

<211>19

<212>DNA

<213> Artificial sequence

<400>6

gcattatccc tcgcaccta 19

<210>7

<211>24

<212>DNA

<213> Artificial sequence

<400>7

gcaccccgct gatactgata ggag 24

<210>8

<211>22

<212>DNA

<213> Artificial sequence

<400>8

gggttcccct gtgcgactgt tc 22

<210>9

<211>25

<212>DNA

<213> Artificial sequence

<400>9

aacacccgta gagccattga gacag 25

<210>10

<211>25

<212>DNA

<213> Artificial sequence

<400>10

gttttatgaa gcaaagccca ccagg 25

<210>11

<211>20

<212>DNA

<213> Artificial sequence

<400>11

atgaaactac aggccgtact 20

<210>12

<211>23

<212>DNA

<213> Artificial sequence

<400>12

tgggttggtc actctgccaa caa 23

<210>13

<211>29

<212>DNA

<213> Artificial sequence

<400>13

cgcggatcca tgaaactaca ggccgtact 29

<210>14

<211>29

<212>DNA

<213> Artificial sequence

<400>14

cccaagctta tacagcttca ttgaaaact 29

Claims

1. The amino acid sequence of the rockfish rC1INH-Nonserpin protein is shown in SEQ ID NO. 4.

2. A gene encoding the rockfish rC1 INH-noserpin protein of claim 1.

3. The gene of claim 2, having a nucleotide sequence as set forth in SEQ ID NO: 3, respectively.

4. The amino acid sequence of the grouper complement C1INH protein is shown as SEQ ID NO: 2, respectively.

5. A gene encoding the complement C1INH protein of rockfish according to claim 4.

6. The gene of claim 5, having a nucleotide sequence as set forth in SEQ ID NO: 1 is shown.

7. A cloning vector comprising the gene of claim 2, 3, 5 or 6.

8. An expression vector comprising the gene of claim 2, 3, 5 or 6.

9. A method for producing rC1INH-Nonserpin protein of rockfish or C1INH protein of rockfish complement, which comprises introducing the expression vector of claim 8 into host cells, and expressing to obtain rC1INH-Nonserpin protein or C1INH protein.

10. Use of the protein of claim 1 or 4 for the preparation of a formulation for enhancing fish immunity.