CN103724412A - Fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and application thereof - Google Patents
Fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to genetic engineering and particularly to a fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and an application thereof. A gene of the FcALF (fenneropenaeus chinensiss anti-lipopolysaccharide factor) is (a) protein represented by an amino acid sequence in SEQ ID NO.1 or (b) protein which is derived from (a) through substitution, deletion or addition of one or more amino acids in the amino acid sequence limited by (a) and has the anti-lipopolysaccharide factor activity. According to the factor, pichia pastoris is used as a eukaryon expression system, and recombinant expression is performed on the FcALF. In addition, the antibacterial activity of the FcALF is analyzed, so that guidance is provided for screening of antibacterial agents and immunopotentiators . Specifically, recombinant protein of the FcALF has obvious growth-inhibition and bactericidal effects on Gram-negative bacterium escherichia coli.
Description
Technical field
The present invention relates to genetically engineered, especially relate to a kind of Fenneropenaeus chinensis Anti-LPS factor and preparation thereof and application.
Background technology
Enter 21 century, culture fishery is to the direction fast development of many kinds, high investment, high-density, intensive style, batch production, but the contagious disease moment of frequently breaking out is threatening the healthy sustainable development of this industry.At present, aquaculture organisms disease control main method has medical treatment, immune protection and bionomic control etc., and progressively to efficient, low toxicity, noresidue and non-harmful future development.And chemicals is still current disease control of aquatic animal the most directly and the most effective a kind of means.The factors such as China's fishing medicine Industry Foundation weakness, aquaculture organisms disease control technological lag and raiser's medication knowledge scarcity, cause most of raiser to use cheap, residual serious agricultural chemicals and industrial chemicals, have caused the severe contamination of water body.In addition, the application of the medicine such as microbiotic, hormones in aquaculture process, although control plays a role to disease, but destroyed to a great extent the microecological balance in water surrounding and animal body, make aquatic animal lose normal flora barrier and Ant agonism, and then cause exogenous effect bacterium and endogenous pathogenic bacterium to breed in a large number, caused thus potential larger disease.Drug abuse will cause pathogenic strains to produce resistance, pathogenic stronger, the pathogenic microorganism that hazardness is larger, popular wider that makes a variation out, and life-time service pharmaceutical chemicals certainly will cause the drug-fast increase of causal organism.Along with the raising of scientific and technological development and human economy educational level, people have higher requirement to food safety and environmental safety, and chemicals is substituted by institute of Biological Products just gradually.
As a kind of invertebrates, Crustin relies on its only innate immune system to realize the immunity of pathogenic micro-organism to external world.Normal growth, the breeding of Crustin in this complicated microbial environment of seawater, illustrates that its immunity system can effectively resist the invasion and attack of extraneous pathogenic micro-organism.Coagulogen (anti-lipopolysaccharide factor, ALF) be a kind of lipopolysaccharide binding protein, it is the class antibacterial peptide extensively existing in the crustacean bodies such as king crab, crab shrimps, by in conjunction with lipopolysaccharides, regulate the degranulation of cell, cause a series of cascade reactions, thereby play an important role in the humoral immunization of crustacean.In early-stage Study, applicant place project team is cloned into ALF full length cDNA sequence (FcALF) from Crustin hemocyte, research is found under the cause of disease such as Vibrio anguillarum stimulates, the expression generation acute variation of FcALF in mRNA level, and prompting FcALF is prawn to external world in pathogen infection process, play an important role (Liu, FS, etc., Marine Biotechnology, 2005,7(6): 600-608).
Summary of the invention
The object of the invention is to provide a kind of Fenneropenaeus chinensis Anti-LPS factor and preparation and application.
For achieving the above object, the technical solution used in the present invention is:
A kind of Fenneropenaeus chinensis Anti-LPS factor FcALF, Fenneropenaeus chinensis Anti-LPS factor FcALF gene is
(a) by the protein shown in aminoacid sequence in SEQ ID NO.1, or
(b) (a) limit aminoacid sequence in through replacement, lack or add one or several amino acid and have coagulogen activity by (a) derivative protein.
The preparation method of Fenneropenaeus chinensis Anti-LPS factor FcALF recombinant protein:
(a) build Fenneropenaeus chinensis Anti-LPS factor recombinant expression vector pPIC9k-FcALF;
(b) step (a) gained recombinant expression vector is imported to host cell after linearizing, the method for combination antibiotic G418 and PCR is carried out the screening of positive colony, and the positive colony obtaining is carried out to abduction delivering, obtains expression product;
(c) expression product of purification procedures (b) gained, obtain shown in the SEQ ID NO.1 sequence of restructuring in the amino acid shown in amino acid whose albumen (being rFcALF) or SEQ ID NO.1 sequence through replacement, lack or add one or several amino acid and have coagulogen activity by the derivative protein shown in aminoacid sequence in SEQID NO.1.
In described step (b), host cell is pichia spp.
The application of Fenneropenaeus chinensis Anti-LPS factor FcALF: described Fenneropenaeus chinensis Anti-LPS factor FcALF can be used for preparing broad-spectrum antimicrobial class medicine.
Described Fenneropenaeus chinensis Anti-LPS factor FcALF can be used for preparing the anti-microbial type medicine of Gram-negative bacteria.
Described Fenneropenaeus chinensis Anti-LPS factor FcALF can be used for preparing immunostimulant, healthcare products or fodder additives.
The present invention has advantages of: the present invention chooses pichia spp as eukaryotic expression system, to Fenneropenaeus chinensis Anti-LPS factor (FcALF), carries out recombinant expressed.Analyzed its anti-microbial activity, for the screening of antibacterials and immunostimulant provides guidance simultaneously.Fenneropenaeus chinensis Anti-LPS factor recombinant protein of the present invention has obvious inhibition growth and germicidal action to Gram-negative bacteria intestinal bacteria specifically.Recombinant protein of the present invention has very strong inhibition growth to intestinal bacteria, and minimum inhibition concentration is 3.12 μ g/mL.
Accompanying drawing explanation
Fig. 1 is the experimental result of Sal I restriction enzyme digestion recombinant expression vector pPIC9k-FcALF and pPIC9k.Wherein, M is DNA Marker; 1 cuts result for pPIC9k-FcALF through Sal I enzyme; 2 is pPIC9k-FcALF; 3 cut result for pPIC9k through Sal I enzyme; 4 is pPIC9k.
The recombinant protein rFcALF cation-exchange chromatography color atlas of the abduction delivering that Fig. 2 provides for the embodiment of the present invention.Wherein, in figure, curve 1 represents the ultraviolet detection result under 280nm in protein purification process; In figure, curve 2 represents the Changing Pattern of specific conductivity in protein purification process; In figure, curve 3 represents the change in concentration trend of NaCl in gradient elution process.In figure, the obvious absorption peak of curve 1 is the rFcALF obtaining by NaCl wash-out.
16.5% Tricine-SDS-PAGE analytical results figure of the recombinant expressed rFcALF separation and purification sample that Fig. 3 provides for the embodiment of the present invention, wherein M: protein standard molecular weight; 1: product before cationic exchange loading; 2:NaCl elution peak ascent stage is collected liquid; The collection liquid of 3:NaCl elution peak.
Fig. 4 is recombinant protein rFcALF Mass Spectrometric Identification result figure.
6 multiple copied clones that Fig. 5 provides for the embodiment of the present invention carry out the SDS-PAGE analytical results figure of methanol induction expression secondary fermentation supernatant liquor, wherein M: protein standard molecular weight; 1-6 is 6 multiple copied clones.
Fig. 6 is the albumen desalination result after cation-exchange chromatography.
Fig. 7 is for carrying out anti-microbial activity detected result with Oxford agar diffusion method to recombinant protein rFcALF.G (-) is E.coli, and its FcALF detectable level is 1mg/L; G (+) is Staphylococcus aureus, its rFcALF detectable level 50mg/L; Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of C:20mM pH7.2 as a control group.
Fig. 8 is the impact of different concns rFcALF on Escherichia coli Growth.Wherein Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 20mM pH7.2 as a control group.
Embodiment
In the following examples, the invention will be further elaborated, but the invention is not restricted to this.
The technical solution used in the present invention comprises the following steps:
1) build Fenneropenaeus chinensis Anti-LPS factor recombinant expression vector;
2) by step 1) gained recombinant expression vector is after Sal I linearizing, and importing yeast host cell, after positive colony screening, carries out abduction delivering by host cell, obtains expression product;
3) purification procedures 2) recombinant protein that obtains, i.e. Fenneropenaeus chinensis Anti-LPS factor.
In step 1) in, described expression vector can be selected pPIC9k, pPIC9, pPIC3.5K etc.
In step 2) in, described host cell can be selected pichia spp KM71, GS115 etc.
In step 3) in, described separation purification method can be ion exchange chromatography.
Embodiment 1:
Fenneropenaeus chinensis Anti-LPS factor mature peptide FcALF, as shown in aminoacid sequence in SEQ ID NO.1,
QGWEAVAAAVAVKIVGLWRNEKTELLGHECKFTVKPYIKRFQLYYKGRMWCPGWTAIRGEAKTRSRSGVAGRTAKDFVRKAFQQGLISQQQANQWLNS
The nucleotide sequence of this gene is as shown in SEQ No.2, and its 5 ' end and 3 ' end has respectively initiator codon (ATG) and terminator codon (TAG).
ATGCGAGTTTCCGTGTTGGCAAGCCTGGTGCTGGTGGTGTCCCTGGTGGCACTCTTCGCCCCGCAGTGCCAGGCTCAAGGGTGGGAGGCTGTGGCAGCGGCCGTCGCCGTCAAGATTGTTGGGCTGTGGAGGAACGAGAAAACCGAACTCCTCGGCCACGAGTGCAAGTTCACCGTCAAGCCTTACATTAAGAGGTTCCAGTTGTACTACAAGGGGAGGATGTGGTGCCCAGGCTGGACGGCCATCAGAGGAGAAGCCAAAACACGCAGTCGGTCCGGGGTGGCTGGAAGGACAGCCAAAGACTTCGTCCGGAAAGCTTTCCAGCAAGGTCTCATCTCTCAACAGCAGGCTAACCAGTGGCTTAACTCATAG
(a) sequence signature:
● length: 372bp, wherein useful length 76-369bp
● type: base sequence
● chain: strand
● topological framework: linearity
(b) molecule type: double-stranded DNA
(c) suppose: no
(d) antisense: no
(e) originate at first: Crustin
(f) specificity title: gene
1) structure of Fenneropenaeus chinensis Anti-LPS factor FcALF recombinant expression vector
According to cDNA sequence (NCBI sequence number of registration: the AY859500) sequence information of encoding mature peptide and the multiple clone site feature of secreted expression carrier pPIC9k of Crustin ALF, design primer, upstream primer PRI-A1 is containing EcoR I restriction enzyme site; Downstream primer PRI-A2 is containing Not I restriction enzyme site.
PRI-A1：
GC
gAATTcCAAGGGTGGGAGGCTGTGGCA(underscore is EcoR I restriction enzyme site)
PRI-A2：
TA
g+GGCCGCcTATGAGTTAAGCCACTGG(underscore is Not I restriction enzyme site)
PCR reaction system is 20ul, as follows:
Ex Taq buffer 2μl
dNTPs（10mM） 0.4μl
PRI-A1(10μM) 0.4μl
PRI-A2(10μM) 0.4μl
Ex Taq 0.2μl
ddH
2O 15.6μl
Pcr amplification program: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 10min.
PCR product utilization sepharose purification kit is reclaimed, and the PCR product of recovery connects pMD19-T carrier (carrying out according to working instructions), and 16 ℃ connect 60min.To connect product and transform bacillus coli DH 5 alpha competent cell, the LB solid plate that coating contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation, take M13-F/M13-R as primer pair, utilize PCR method to detect the mono-clonal on LB flat board, PCR is detected after positive colony shakes bacterium and carries out DNA sequencing, screen thus recombinant plasmid pMD19-FcALF(referring to Fig. 1).
Then extracting plasmid pMD19-FcALF from the positive clone of measurement result, and with EcoR I and Not I double digestion, the object fragment reclaiming, with the pPIC9k fragment with EcoR I and Not I double digestion, utilize T4DNA ligase enzyme to spend the night 16 ℃ of connections, to connect product and transform bacillus coli DH 5 alpha competent cell, the LB solid plate that coating contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation, take 5 ' AOX1/3 ' AOX1 as primer pair, utilize PCR method to detect the mono-clonal on LB flat board, PCR is detected after positive colony shakes bacterium and carries out DNA sequencing, thereby build yeast recombinant expression plasmid pPIC9k-FcALF.
2) recombinant plasmid pPIC9k-FcALF is converted into pichia spp KM71
Recombinant vectors pPIC9k-FcALF enzyme is cut with Sal I, carry out plasmid linearization processing, reclaim linearization plasmid, utilize the method for PEG1000 to be converted in pichia spp KM71 competence, with the method screening positive clone of bacterium colony PCR, and the positive colony that utilizes microbiotic G418 concentration gradient screening height to copy, concrete grammar carries out (Multi-Copy Pichia Expression Kit with reference to the specification sheets of Invitrogen company, Version F), and then the clone who selects 6 multiple copieies carries out methanol induction expression experiment, sample by abduction delivering after 4 days, respectively get the centrifugal (8000r/min of 1mL, 10min), respectively get 100uL and then add the expression that carries out 15%SDS-PAGE check FcALF recombinant protein after 20uL6 × SDS sample-loading buffer, as seen from Figure 5, in selected 6 clones, No. 2-4 clone's expression amount is relatively high, and No. 3 expression amounts are relatively the highest under similarity condition, selection is used for carrying out amplification test, this recombinant bacterial strain called after FcALF-60 simultaneously.
3) cation-exchange chromatography purifying target protein restructuring FcALF
(1) engineering strain (FcALF-60) the highest the above-mentioned expression amount screening is carried out after a large amount of abduction deliverings, 4 ℃, the centrifugal 15min of 10000r/min, gets supernatant liquor.
(2) utilize 5M NaOH solution that the supernatant liquor of acquisition is adjusted to pH7.0.
(3) utilize 5 column volumes of Sodium phosphate dibasic-sodium dihydrogen phosphate buffer balance cation post (HITRAP Q FF, 5mL) of the pH7.0 of 50mM.
(4) by the supernatant liquor upper prop of above-mentioned acquisition.
(5) utilize 5 column volumes of Sodium phosphate dibasic-sodium dihydrogen phosphate buffer balance of the pH7.0 of 50mM.
(6) utilize NaCl solution (NaCl concentration is 0-1.0M) to carry out gradient elution, be collected in the component of 400mMNaCl wash-out, the Protein reconstitution FcALF(being after purifying is rFcALF) (referring to Fig. 2), wherein the detection of albumen wash-out adopts the UV-detector under 280nM to carry out.
By above-mentioned collection elution peak, through 16.5% Tricine-SDS-PAGE electrophoretic analysis (referring to Fig. 3), as shown in Figure 3, the molecular weight of recombinant protein, in 11.5kD left and right, cuts the protein spots of separation and purification shown in Fig. 3, utilizes LC-ESI-MS/MS to carry out Identification of Fusion Protein, authentication method reference (Zhang Jiquan etc., Journal of Biotechnology, 2006,125 (2): 173-184).After utilizing Bioworks software to compare with SEQUEST database, find 1 peptide section (TAEDFVR) with restructuring Crustin ALF albumen mate (table 1 and Fig. 4) completely.
Table 1 Mass Spectrometric Identification peptide segment information
| Scan(s | Sequence | MH+ | Charg | XC | Delta | Sp | RS | Ions |
| ALF | 20.06 | 2(2000) | ||||||
| 2411 | -.TAEDFVR | 837.9 | 2 | 1.2 | 0.00 | 441 | 1 | 2/3 |
| 2447 | -.TAEDFVR. | 837.9 | 2 | 1.2 | 0.00 | 299 | 1 | 2/3 |
4) recombinant protein desalination
By the component that contains restructuring FcALF target protein of cation-exchange chromatography purifying, utilize HiPrep26/10 desalting column on AKTA avant25, to carry out desalination, as shown in Figure 6, as seen from Figure 6, target protein obtains fully separate with salt desalination result.
Restructuring FcALF albumen after purifying, through freeze drier freeze-drying, is preserved.Get in Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution that 1mg lyophilized protein is dissolved in 1mL20mM pH7.2 the recombinant protein mother liquor of preparation 1g/L.Then utilize Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 20mM pH7.2 to be diluted to the recombinant protein liquid that concentration is 50mg/L.
(1) Oxford agar diffusion method detects inhibition zone
By cell concentration, be 10
8the intestinal bacteria (E.coli) of cell/mL and streptococcus aureus (Staphylococcus aureus) are uniformly coated on respectively on LB solid plate, 37 ℃ of incubators are placed 30-60min, treat planar surface complete drying, place Oxford cup, then add respectively wherein the recombinant protein liquid of the above-mentioned purifying 50mg/L of 200 μ L, simultaneously using Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of same volume 20mM pH7.2 as negative control.Then be placed in 37 ℃ of incubators and cultivate 16-18 hour observations, then use vernier caliper measurement inhibition zone size (referring to Fig. 7), result shows that the restructuring FcALF of 50mg/L can effectively suppress the growth of Gram-negative bacteria E.coli.
(2) utilize microplate reader to measure Mlc
1. the monoclonal intestinal bacteria of picking, are seeded in LB liquid nutrient medium, in 37 ℃ of shaking tables, cultivate 10 minutes;
2. the recombinant protein liquid of getting above-mentioned 50mg/L carries out Mlc mensuration;
3. by step, the intestinal bacteria of the cultivation in are 1. divided and are filled in 96 orifice plates, add 140 μ L in the each hole of first row, in all the other every row holes, add 75 μ L;
4. rFcALF albumen is diluted at double: get the recombinant protein liquid that 10 μ L concentration are 50mg/L and add in the hole of first row, mix rear absorption 75 μ L and add in secondary series, get so according to this liquid and mix, reach the effect to ALF gradient dilution; Control group is the same, only rFcALF albumen is changed to Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of isopyknic 20mM pH7.2;
5., after gradient dilution completes, the initial absorbancy while measuring 600nm in microplate reader is then cultivated in 37 ℃ of shaking tables; After incubated overnight, statistical experiment the data obtained, analyzes its fungistatic effect (referring to Fig. 8).
The concentration that suppresses bacteria growing with recombinant protein rFcALF Cmin defines the minimal inhibitory concentration of bacterium for this reason, result as shown in Figure 8, along with the reduction of recombinant protein rFcALF concentration, bacteriostatic activity weakens gradually, recombinant protein rFcALF has inhibition growth to intestinal bacteria, and minimum inhibition concentration is 3.12 μ g/mL.
Claims (6)
1. a Fenneropenaeus chinensis Anti-LPS factor FcALF, is characterized in that: Fenneropenaeus chinensis Anti-LPS factor FcALF is
(a) by the protein shown in aminoacid sequence in SEQ ID NO.1, or
(b) (a) limit aminoacid sequence in through replacement, lack or add one or several amino acid and have coagulogen activity by (a) derivative protein.
2. a preparation method of Fenneropenaeus chinensis Anti-LPS factor FcALF claimed in claim 1, is characterized in that:
(a) build Fenneropenaeus chinensis Anti-LPS factor recombinant expression vector pPIC9k-FcALF;
(b) step (a) gained recombinant expression vector is imported to host cell after linearizing, the method for combination antibiotic G418 and PCR is carried out the screening of positive colony, and the positive colony obtaining is carried out to abduction delivering, obtains expression product;
(c) expression product of purification procedures (b) gained, obtain shown in the SEQ ID NO.1 sequence of restructuring in the amino acid shown in amino acid whose albumen (being rFcALF) or SEQ ID NO.1 sequence through replacement, lack or add one or several amino acid and have coagulogen activity by the derivative protein shown in aminoacid sequence in SEQID NO.1.
3. by the preparation method of Fenneropenaeus chinensis Anti-LPS factor FcALF claimed in claim 2, it is characterized in that: in described step (b), host cell is pichia spp.
4. an application of Fenneropenaeus chinensis Anti-LPS factor FcALF claimed in claim 1, is characterized in that: described Fenneropenaeus chinensis Anti-LPS factor FcALF can be used for preparing broad-spectrum antimicrobial class medicine.
5. by the application of Fenneropenaeus chinensis Anti-LPS factor FcALF claimed in claim 4, it is characterized in that: described Fenneropenaeus chinensis Anti-LPS factor FcALF can be used for preparing the anti-microbial type medicine of Gram-negative bacteria.
6. an application of Fenneropenaeus chinensis Anti-LPS factor FcALF claimed in claim 1, is characterized in that: described Fenneropenaeus chinensis Anti-LPS factor FcALF can be used for preparing immunostimulant, healthcare products or fodder additives.
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