CN102352363A - Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as preparation method and application thereof - Google Patents
Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a Scylla paramamosain antiviral type lipopolysaccharide resistant factor as well as a preparation method and application thereof and relates to an antiviral type lipopolysaccharide resistant factor. The invention provides a gene sequence and an amino acid sequence of a Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2, a preparation method of the Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2 and application of the Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2. The preparation method comprises the following steps: constructing a recombinant expression vector of the Scylla paramamosain antiviral type lipopolysaccharide resistant factor; leading the recombinant expression vector into a host cell, carrying out inducing expression on the host cell so as to obtain the expression product; separating and purifying the expression product so as to obtain recombinant protein, namely Sp-ALF2. The Scylla paramamosain antiviral type lipopolysaccharide resistant factor Sp-ALF2 has obvious inhibition effect and killing effect on WSSV (white spot syndrome virus) and multiple pathogenic bacteria, and has wide application value in preparation of disease resistant new medicaments and the Scylla paramamosain antiviral type lipopolysaccharide resistant factor as an animal disease resistant additive also has wide application value.
Description
Technical field
The present invention relates to a kind of anti-virus type coagulogen, especially relate to a kind of Scylla paramamosain anti-virus type coagulogen and preparation method thereof and application.
Background technology
(white spot syndrome virus WSSV) is not only the main diseases toxicity cause of disease of harm prawn, and can infects other many crustaceans decapods, and this is comprising the economic breed variety of many important aquatic products for white spot syndrome virus.The WSSV disease is since 1993; Caused the tremendous economic loss for global culture fishery; Over nearly 20 years; Chinese scholars WSSV infect and the anti-WSSV immune Research of aquatic crustacean aspect obtained some key progress; Regrettably, so far still needleless to this virus the method for effectively preventing and treating.
As everyone knows, compare with vertebrates, no vertebra crustacean such as shrimp, crab lacks the acquired immune response system, and they only rely on non-specific immunity to resist the microbial ingress infection.Existing research shows: can activate host's immune recognition signal conduction path behind virus infection prawn, the crab, like the NF-kappaB signal path
[1,2], TNF (tumor necrosis factor) receptor associated factor 6 (TRAF6) signal path
[3]With the STAT signal path
[4]Deng, thereby cause a series of immune responses, comprise the synthetic of antibacterial peptide and discharge
[3,5], cytophagy
[6]And apoptosis
[7]Deng, these immune responses are resisted in the extraneous cause pathogeny imcrobe infection process Crustacean and are played a significant role.What deserves to be mentioned is have some antiviral active substances to be able to isolation identification in recent years successively, such as: hemocyanin
[8], N,O-Diacetylmuramidase
[9], antibacterial peptide
[5,10-13]Deng, effective control that these researchs are reported as aquaculture virus diseases such as shrimp, crab provides the most important theories foundation.
Coagulogen is found in king crab first
[14,15]In the body, this factor has blood coagulation resisting function, can suppress the agglutination reaction that bacteria lipopolysaccharide (LPS) causes, so the called after coagulogen.Research shows that ALF has high-affinity to LPS, can combine the lipoid aminoacyl site of LPS and its biologic activity that neutralizes, and has vital role for opposing endotoxemia and endotoxin shock; In addition, ALF has the strongly inhibited effect to R-type Gram-negative bacteria growing
[15]ALF also is able to isolation identification in succession in other multiple crustacean bodies, and ALF is at LPS
[16-18], bacterium, fungi
[19-22]And WSSV
[23]Up-regulated expression is in various degree all arranged after the stimulation, and the ALF albumen of in-vitro recombination expression also shows broad spectrum antibiotic activity, comprises inhibition and lethal effect to Gram-negative bacteria and gram-positive microorganism
[16,20,24]Recent research shows that it is active that crayfish Pl-ALF has significant anti-WSSV
[13], and prawn Pm-ALF3
[11]It is active also to demonstrate anti-WSSV.In sum, ALF is the important immune factor of aquatic Crustacean, to its function go deep into identification research not only significant aspect the anti-microbial infection theoretical investigation, and in production practice, also have wide application prospect.
Scylla paramamosain Scylla paramamosain and genetically engineered; Be particularly related to Scylla paramamosain anti-virus type coagulogen (Anti-lipopolysaccharide factor); Be Sp-ALF2 gene clone, recombination expression product and preparation method thereof, especially utilize red claw crayfish hemopoietic tissue stem cell to carry out the antiviral experimental technique that the anti-WSSV of Sp-ALF2 product infects as cell model.
Reference is following:
1.Huang XD，Zhao L，Zhang HQ，Xu XP，Jia XT，Chen YH，Wang PH，Weng SP，Yu XQ，YinZX et al.Shrimp NF-kappaB binds to the immediate-early gene ie 1promoter of white spotsyndrome virus and upregulates its activity[J].Virology，2010，406(2)：176-180.
2.Li F，Wang D，Li S，Yan H，Zhang J，Wang B，Xiang J.A Dorsal homolog(FcDorsal)in theChinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge[J].Dev Comp Immunol，2010，34(8)：874-883.
3.Wang PH，Wan DH，Gu ZH，Deng XX，Weng SP，Yu XQ，He JG.Litopenaeus vannameitumor necrosis factor receptor-associated factor 6(TRAF6)responds to Vibrio alginolyticus andwhite spot syndrome virus(WSSV)infection and activates antimicrobial peptide genes[J].DevComp Immunol，2011，35(1)：105-114.
4.Sun C，Shao HL，Zhang XW，Zhao XF，Wang JX.Molecular cloning and expression analysisof signal transducer and activator of transcription(STAT)from the Chinese white shrimpFenneropenaeus chinensis[J].Mol Biol Rep，2011.
5.Antony SP，Singh IS，Sudheer NS，Vrinda S，Priyaja P，Philip R.Molecular characterizationof a crustin-like antimicrobial peptide in the giant tiger shrimp，Penaeus monodon，and itsexpression profile in response to various immunostimulants and challenge with WS SV[J].Immunobiology，2011，216(1-2)：184-194.
6.Wu W，Zong R，Xu J，Zhang X.Antiviral phagocytosis is regulated by a novelRab-dependent complex in shrimp penaeus japonicus[J].J Proteome Res，2008，7(1)：424-431.
7.Wang Z，Hu L，Yi G，Xu H，Qi Y，Yao L.ORF390 of white spot syndrome virus genome isidentified as a novel anti-apoptosis gene[J].Biochem Biophys Res Commun，2004，325(3)：899-907.
8.Zhang X，Huang C，Qin Q.Antiviral properties of hemocyanin isolated from shrimp Penaeusmonodon[J].Antiviral Res，2004，61(2)：93-99.
9.Mai WJ，Wang WN.Protection of blue shrimp(Litopenaeus stylirostris)against the WhiteSpot Syndrome Virus(WSSV)when injected with shrimp lysozyme[J].Fish Shellfish Immunol，2010，28(4)：727-733.
10.Roch P，Yang Y，Toubiana M，Aumelas A.NMR structure of mussel mytilin，andantiviral-antibacterial activities of derived synthetic peptides[J].Dev Comp Immunol，2008，32(3)：227-238.
11.Tharntada S，Ponprateep S，Somboonwiwat K，Liu H，Soderhall I，Soderhall K，Tassanakajon A.Role of anti-lipopolysaccharide factor from the black tiger shrimp，Penaeusmonodon，in protection from white spot syndrome virus infection[J].J Gen Virol，2009，90(Pt6)：1491-1498.
12.Woramongkolchai N，Supungul P，Tassanakajon A.The possible role of penaeidin5 from theblack tiger shrimp，Penaeus monodon，in protection against viral infection[J].Dev Comp Immunol，2011，35(5)：530-536.
13.Liu H，Jiravanichpaisal P，Soderhall I，Cerenius L，Soderhall K.AntilipopolysaccharideFactor Interferes with White Spot Syndrome Virus Replication In Vitro and In Vivo in the CrayfishPacifastacus leniusculus[J].Journal of Virology，2006，80(21)：10365-10371.
14.Tanaka S，Nakamura T，Morita T，Iwanaga S.Limulus anti-LPS factor：an anticoagulantwhich inhibits the endotoxin mediated activation of Limulus coagulation system[J].BiochemBiophys Res Commun，1982，105(2)：717-723.
15.Morita T，Ohtsubo S，Nakamura T，Tanaka S，Iwanaga S，Ohashi K，Niwa M.Isolation andbiological activities of limulus anticoagulant(anti-LPS factor)which interacts withlipopolysaccharide(LPS)[J].J Biochem，1985，97(6)：1611-1620.
16.Yedery RD，Reddy KV.Identification，cloning，characterization and recombinant expressionof an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab，Scylla serrata[J].FishShellfish Immunol，2009，27(2)：275-284.
17.Mekata T，Sudhakaran R，Okugawa S，Kono T，Sakai M，Itami T.Molecular cloning andtranscriptional analysis of a newly identified anti-lipopolysaccharide factor gene in kuruma shrimp，Marsupenaeus japonicus[J].Lett Appl Microbiol，2010，50(1)：112-119.
18.Nagoshi H，Inagawa H，Morii K，Harada H，Kohchi C，Nishizawa T，Taniguchi Y，UenobeM，Honda T，Kondoh M et al.Cloning and characterization of a LPS-regulatory gene having anLPS binding domain in kuruma prawn Marsupenaeus japonicus[J].Mol Immunol，2006，43(13)：2061-2069.
19.Tharntada S，Somboonwiwat K，Rimphanitchayakit V，Tassanakajon A.Anti-lipopolysaccharide factors from the black tiger shrimp，Penaeus monodon，are encoded by twogenomic loci[J].Fish Shellfish Immunol，2008，24(1)：46-54.
20.Li C，Zhao J，Song L，Mu C，Zhang H，Gai Y，Qiu L，Yu Y，Ni D，Xing K.Molecularcloning，genomic organization and functional analysis of an anti-lipopolysaccharide factor fromChinese mitten crab Eriocheir sinensis[J].Dev Comp Immunol，2008，32(7)：784-794.
21.Liu F，Liu Y，Li F，Dong B，Xiang J.Molecular cloning and expression profile of putativeantilipopolysaccharide factor in Chinese shrimp(Fenneropenaeus chinensis)[J].Mar Biotechnol(NY)，2005，7(6)：600-608.
22.Supungul P，Klinbunga S，Pichyangkura R，Hirono I，Aoki T，Tassanakajon A.Antimicrobial peptides discovered in the black tiger shrimp Penaeus monodon using the ESTapproach[J].Dis Aquat Organ，2004，61(1-2)：123-135.
23.Liu H，Jiravanichpaisal P，Soderhall I，Cerenius L，Soderhall K.Antilipopolysaccharidefactor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfishPacifastacus leniusculus[J].J Virol，2006，80(21)：10365-10371.
24.Somboonwiwat K，Marcos M，Tassanakajon A，Klinbunga S，Aumelas A，Romestand B，Gueguen Y，Boze H，Moulin G，Bachere E.Recombinant expression and anti-microbial activity ofanti-lipopolysaccharide factor(ALF)from the black tiger shrimp Penaeus monodon[J].Dev CompImmunol，2005，29(10)：841-851.
Summary of the invention
The object of the present invention is to provide the gene order of Scylla paramamosain anti-virus type coagulogen Sp-ALF2.
Second purpose of the present invention is to provide the aminoacid sequence of Scylla paramamosain anti-virus type coagulogen Sp-ALF2.
The 3rd purpose of the present invention is to provide the preparation method of Scylla paramamosain anti-virus type coagulogen Sp-ALF2.
The 4th purpose of the present invention is to provide the application of Scylla paramamosain anti-virus type coagulogen.
Said Scylla paramamosain anti-virus type coagulogen called after Sp-ALF2.
The gene order of said Scylla paramamosain anti-virus type coagulogen Sp-ALF2 is:
cagtatgaag ctctggtagc ttccattctt ggaaagctgt ctggactgtg gcacagcgac 60
acagtggact tcatgggaca cacctgccac atccgccgca ggccgaagtt caggaaattt 120
aagctgtacc acgagggcaa gttttggtgt cctggctgga cacatctcga gggcaattcg 180
aggaccaaga gcaggtcggg gtcagccagg gacgccatca aggacttcgt gtacaaagct 240
ttacaaaaca aactcatcac ggagaataac gcggccgcct ggctgaaggg gtga 294
The aminoacid sequence of said Scylla paramamosain anti-virus type coagulogen Sp-ALF2 is:
Gln Tyr Glu Ala Leu Val Ala Ser Ile Leu Gly Lys Leu Ser Gly Leu
1 5 10 15
Trp His Ser Asp Thr Val Asp Phe Met Gly His Thr Cys His Ile Arg
20 25 30
Arg Arg Pro Lys Phe Arg Lys Phe Lys Leu Tyr His Glu Gly Lys Phe
35 40 45
Trp Cys Pro Gly Trp Thr His Leu Glu Gly Asn Ser Arg Thr Lys Ser
50 55 60
Arg Ser Gly Ser Ala Arg Asp Ala Ile Lys Asp Phe Val Tyr Lys Ala
65 70 75 80
Leu Gln Asn Lys Leu Ile Thr Glu Asn Asn Ala Ala Ala Trp Leu Lys
85 90 95
Gly
The preparation method of said Scylla paramamosain anti-virus type coagulogen Sp-ALF2 may further comprise the steps:
1) makes up Scylla paramamosain anti-virus type coagulogen Sp-ALF2 recombinant expression vector;
2) step 1) gained recombinant expression vector is imported host cell, and host cell is carried out abduction delivering, obtain expression product;
3) purification procedures 2) expression product of gained, obtain recombinant protein, i.e. Sp-ALF2.
In step 1), said expression vector can be selected pPIC9k etc. for use.
In step 2) in, said host cell can be pichia spp etc.
In step 3), said purification procedures 2) method of the expression product of gained can be dialysed expression product earlier, carries out affinity chromatography again.
Said Scylla paramamosain anti-virus type coagulogen Sp-ALF2 has obvious restraining effect and lethal effect to WSSV and multiple pathogenic bacteria, has widespread use value at the antiviral kind new medicine of preparation with in as the animal disease resistant fodder additives.
The present invention is on the basis of isolating Scylla paramamosain anti-virus type coagulogen Sp-ALF2; Successfully make up recombinant expression vector and in Bichi yeast system, express also purifying acquisition Sp-ALF2 albumen according to the Sp-ALF2 gene sequence characteristic, this recombinant protein has stronger anti-WSSV activity and broad spectrum antibiotic activity.Result of study shows; Sp-ALF2 maybe wide participation the reaction of Scylla paramamosain anti-microbial infection; It is a kind of important congenital immunity factor; Therefore; Recombination engineering product Sp-ALF2 albumen demonstrates very tempting application prospect at anti-microbial infection in the particularly antiviral kind new medicine Application and Development.
Description of drawings
Fig. 1 is an electrophoretogram before and after the pPIC9k carrier double digestion.1 is the pPIC9k carrier of enzyme before cutting; 2 cut back pPIC9k carrier for enzyme; M is DS2000 DNA Marker.
Fig. 2 is an electrophoretogram before and after expression vector pPIC9k and the pPIC9k/Sp-ALF2 Sal I linearizing.1 is pPIC9k, and 2 cut back pPIC9k for enzyme, and 3 is pPIC9k/Sp-ALF2, and 4 are the pPIC9k/Sp-ALF2 of enzyme after cutting, and M is DS2000 DNAMarker.
Fig. 3 clones sub-methanol induction detection of expression SDS-PAGE electrophoretogram for the pPIC9k/Sp-ALF2 recombinant yeast pichia pastoris.1 for inducing preceding expression; 2～4 expression products that are respectively methanol induction 12h, 24h, 48h; M is SDS-PAGE standard protein Marker, and the size of recombinant protein Sp-ALF2 is 13kDa, with the 14kDa band position consistency of Marker.
Fig. 4 is a pPIC9k/Sp-ALF2 recombinant yeast pichia pastoris methanol induction expression product purification assays electrophoretogram.1～2 is deposition and the supernatant of dialysis sample after centrifugal; 3 for passing the peak; 4 is the solution C elution peak; 5～9 is the solution D elution peak; M is SDS-PAGE standard protein Marker.
Fig. 5 detects electrophoretogram for the antiviral experiment of recombinant protein rSp-ALF2: detect the expression of confidential reference items phosphoglyceraldehy-de dehydrogenase (GAPDH) gene (1～6) and WSSV utmost point early gene IE1 (7～12) respectively.1,7 are the normal cell contrast; 2,8 is that 5 μ M rSp-ALF2 are hatched group; 3,9 is that 10 μ M rSp-ALF2 are hatched group; 4,10 is that 5 μ M r Sp-ALF are hatched group; 5,11 is that 10 μ M r Sp-ALF are hatched group; 6,12 are WSSV infection control group.
Embodiment
Below be described with reference to the accompanying drawings technical scheme of the present invention through embodiment.
The structure of embodiment 1 Scylla paramamosain anti-virus type coagulogen Sp-ALF2 recombinant eukaryotic expression plasmid
According to pPIC9k carrier multiple clone site, specificity upstream primer F1 and the downstream primer R1 of design amplification coding Scylla paramamosain Sp-ALF2 (cDNA) gene ORF.Add the base of SnaB I restriction enzyme site and coding His-tag at 5 of upstream primer F1 ' end; Add terminator codon and Avr II restriction enzyme site at 5 of downstream primer R1 ' end.Upstream primer F1:5 '-CGTACGTACACCATCATCATCATCATCAGTATGAAGCTCTGGTAGC-3 ', downstream primer R1:5 '-AACCTAGGTC A CCCCTTCAGCCAGGCGGCC-3 ', the coding region fragment of amplification Sp-ALF2.The PCR reaction conditions is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and repeated 30 circulations; 72 ℃ are extended 10min.
The PCR product is carried out the sepharose purification kit to be reclaimed; The PCR product that reclaims is purifying and recovering after SnaB I and Avr II enzyme are cut; Be connected with Avr II double digestion linearizing pPIC9k carrier with SnaB I; Build yeast expression recombinant vectors pPIC9k/Sp-ALF1, order-checking identifies that reading frame is correct.
Electrophoretogram is referring to Fig. 1 before and after the pPIC9k carrier double digestion.
The abduction delivering of embodiment 2 pPIC9k/Sp-ALF2 recombinant plasmids in pichia spp GS115
Order-checking correct plasmid pPIC9k/Sp-ALF2 is converted in the pichia spp GS115 competent cell with electric shocking method through Sal I linearization for enzyme restriction (referring to Fig. 2), and abduction delivering.
The pichia spp GS115 that transforms with the pPIC9k empty plasmid is contrast, is experimental group with the pichia spp GS115 of pPIC9k/Sp-ALF2 recombinant plasmid transformed, utilizes the proteic expression of polyacrylamide gel electrophoresis (SDS-PAGE) testing goal.
The result shows; With the pichia spp GS115 of pPIC9k/Sp-ALF2 recombinant plasmid transformed induce the back and induce before compare and have tangible recombinant protein abduction delivering; Protein band is (referring to Fig. 3) about 14kDa; Similar with the theoretical molecular that calculates; After the mass spectrum evaluation is errorless, confirm as Sp-ALF2.
The purifying of embodiment 3 pPIC9k/Sp-ALF2 recombinant plasmids abduction delivering product in pichia spp GS115, antiviral activity and anti-microbial activity are identified
1, affinity chromatography purifying target protein
To recombinate behind a large amount of abduction deliverings of positive pichia spp GS115 bacterial strain, centrifugal removal microorganism collection supernatant of culture medium 1L, after the PBS dialysis twice (12h changes once new dialyzate), 4 ℃, the centrifugal 35min of 12000rpm gets supernatant, the upper prop sample.Adopt the metal chelate affinity chromatography post that the back albumen of dialysing is carried out affinity chromatography subsequently.Collect the solution D elution peak, be accredited as Sp-ALF2 recombinant protein (referring to Fig. 4) through SDS-PAGE electrophoretic analysis and mass spectrum.
2, the anti-WSSV activity identification of Sp-ALF2 recombinant protein
Application of B radford method is measured the BSA standard substance and is obtained the protein concentration typical curve.Can calculate Sp-ALF2 recombinant protein concentration according to formula.
The anti-WSSV activity identification of Sp-ALF2 recombinant protein: target protein is diluted to 5 μ M and 10 μ M, with the WSSV mixing; After hatching 30min, add in crayfish hemopoietic tissue stem cell (Hpt) cultured cell in vitro mixing; Hatched 1 hour, and removed whole substratum subsequently, add fresh culture, mixing; After continuing to hatch 3 hours, collect cell in the culture hole, extract the Hpt cell total rna, get the total RNA reverse transcription in 1 μ g DNaseI processing back and become cDNA.
With the HS of Dong Sheng company
TMTaq Mix test kit is RT-PCR, detects WSSV duplicating in the Hpt cell: detect virus replication utmost point early gene IE1, as confidential reference items, the GAPDH primer is with the GAPDH gene:
F：AATGCTTCTTGCACCACCAAC；
R：AGGTCTTGCTCAGCTGGATACC；
The IE1 primer is:
F：GGTATTGAGGTGATGAAGAGGCG；
R：TGACATGGGAACCACTGTTGAG。
The PCR reaction system is as shown in table 1:
Table 1
Mix, on thermal cycler, carry out the PCR reaction by following program:
The PCR program is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 30sec, GAPDH gene cycle number 33, IE1 gene cycle number 37; 72 ℃ are extended 7min.Get 5 μ 1 afterwards and carry out agarose gel electrophoresis.
The result shows: compare with rSp-ALF; RSp-ALF2 has stronger restraining effect to the infection duplication of WSSV in the Hpt cell; And this restraining effect strengthens with the protein concentration raising; Therefore; It is active that rSp-ALF2 has stronger anti-WSSV, can be used as a potential antiviral agent preparation and give Application and Development.
2, Sp-ALF2 recombinant protein anti-microbial activity is identified
The mensuration of Sp-ALF2 recombinant protein anti-microbial activity: according to MIC (Minimum Inhibitory Concentration; Minimum inhibitory concentration) sets concentration and dilute target protein; With μ M is that unit dilutes proportioning; As adopting 25,12.5,6.25,3.12 and 1.6 μ M respectively, on 96 hole microtest plates, carry out for the dilution gradient.Protein sample dilutes proportioning again after 0.22 μ m membrane filtration degerming, each concentration is provided with 2 parallel holes.Get tested bacterium, on MH or 2216 flat boards, rule, be inverted and cultivate 12～16h; The picking mono-clonal is inoculated in MH or 2216 inclined-planes, continues overnight incubation and reaches the logarithmic growth stage in mid-term; With DPBS (1.58mM NaH
2PO
4, 8.42mMNa
2HPO
4, pH 7.2～7.4) and clean slant culture, adjustment is diluted to OD
600=0.1, get 18 μ l OD
600It is that this bacteria concentration then is the MIC working concentration in the MH diluted medium (DPBS:600 μ l, MH:400 μ l) of 1ml that=0.1 dilution bacterium adds final volume.Wherein intestinal bacteria type strain, Corynebacterium glutamicum, streptococcus aureus, bacillus cereus and shigella flexneri culture temperature are 37 ℃, and Vibrio parahemolyticus, to separate alginic acid vibrios, Pseudomonas fluorescens and Pseudomonas stutzeri culture temperature be 28 ℃.
MIC carries out on 96 porocyte culture plates, and every kind of tested bacterium is according to following operation setting blank group, negative control group and testing sample experimental group, and every group is provided with 2 parallel appearance, and the application of sample system is respectively:
1. blank group: add 50 μ l protein samples and 50 μ l DPBS;
2. negative control group: add 50 μ l bacteria suspensions and 50 μ l DPBS;
3. sample experimental group: add 50 μ l each concentration protein sample to be measured and 50 μ l bacteria suspensions.
28 ℃ cultivate 24h after, judge the MIC of Sp-ALF2 recombinant protein to bacterium, criterion is less than for this concentration bacterial count or equals half in the contrast (not adding antibacterial peptide).
Utilize and measure above each the hole culture of MIC, draw 5 μ l respectively, culture transferring is on nutrient agar; Through incubated overnight; Judge the MBC (Minimum Bactericidal Concentration, minimum bactericidal concentration) of Sp-ALF2 recombinant protein, the lowest concentration of drug of promptly killing certain bacterium to bacterium.
The result shows; Eukaryotic expression Sp-ALF2 protein product is to being tried bacterium: intestinal bacteria type strain, shigella flexneri, Vibrio parahemolyticus, separate alginic acid vibrios, Pseudomonas stutzeri, Pseudomonas fluorescens, Corynebacterium glutamicum, streptococcus aureus and bacillus cereus (all available from DSMZ of Institute of Microorganism, Academia Sinica) all have strong lethal effect (referring to table 2).
Table 2
A:MIC (Minimum Inhibitory Concentration, minimum inhibitory concentration)
B::MBC (Minimum Bactericidal Concentration, minimum bactericidal concentration)
The antiviral experiment that Fig. 5 provides recombinant protein rSp-ALF2 detects electrophoretogram: detect the expression of confidential reference items phosphoglyceraldehy-de dehydrogenase (GAPDH) gene (1～6) and WSSV utmost point early gene IE1 (7～12) respectively.
The present invention is intended to obtain the Sp-ALF2 gene engineering expression product of the anti-WSSV of Scylla paramamosain, and, antimicrobial acivity antiviral to it identify, in the hope of the novel drugs of exploitation efficient compound resisting pathogenic microbes.The present invention has obtained Scylla paramamosain Sp-ALF2 gene engineering expression recombinant plasmid pPIC9k/Sp-ALF2; And at the recombinant expressed acquisition Sp-ALF2 gene engineering product of pichia spp success; Anti-WSSV activity and the broad spectrum antibiotic activity of Sp-ALF2 have been confirmed, for good basis in early stage has been established in its exploitation as animal feedstuff additive and resisting pathogenic microbes novel drugs.
The present invention is according to Scylla paramamosain anti-virus type coagulogen Sp-ALF2 gene sequence characteristic; Make up recombinant eukaryotic expression plasmid, transform pichia spp abduction delivering and purifying acquisition Sp-ALF2 recombinant protein; Identify the recombinant protein antimicrobial acivity, discover that the Sp-ALF2 recombinant protein has efficient antimicrobial acivity: white spot syndrome virus and multiple pathogenic bacteria are all had obvious restraining effect and lethal effect.
Claims (8)
1. the gene order of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 is:
cagtatgaag ctctggtagc ttccattctt ggaaagctgt ctggactgtg gcacagcgac 60
acagtggact tcatgggaca cacctgccac atccgccgca ggccgaagtt caggaaattt 120
aagctgtacc acgagggcaa gttttggtgt cctggctgga cacatctcga gggcaattcg 180
aggaccaaga gcaggtcggg gtcagccagg gacgccatca aggacttcgt gtacaaagct 240
ttacaaaaca aactcatcac ggagaataac gcggccgcct ggctgaaggg gtga 294。
2. the aminoacid sequence of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 is:
Gln Tyr Glu Ala Leu Val Ala Ser Ile Leu Gly Lys Leu Ser Gly Leu
1 5 10 15
Trp His Ser Asp Thr Val Asp Phe Met Gly His Thr Cys His Ile Arg
20 25 30
Arg Arg Pro Lys Phe Arg Lys Phe Lys Leu Tyr His Glu Gly Lys Phe
35 40 45
Trp Cys Pro Gly Trp Thr His Leu Glu Gly Asn Ser Arg Thr Lys Ser
50 55 60
Arg Ser Gly Ser Ala Arg Asp Ala Ile Lys Asp Phe Val Tyr Lys Ala
65 70 75 80
Leu Gln Asn Lys Leu Ile Thr Glu Asn Asn Ala Ala Ala Trp Leu Lys
85 90 95
Gly。
3. the preparation method of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 is characterized in that may further comprise the steps:
1) makes up Scylla paramamosain anti-virus type coagulogen Sp-ALF2 recombinant expression vector;
2) step 1) gained recombinant expression vector is imported host cell, and host cell is carried out abduction delivering, obtain expression product;
3) purification procedures 2) expression product of gained, obtain recombinant protein, i.e. Sp-ALF2.
4. the preparation method of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 as claimed in claim 3 is characterized in that in step 1) said expression vector is selected pPIC9k for use.
5. the preparation method of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 as claimed in claim 3 is characterized in that in step 2) in, said host cell is a pichia spp.
6. the preparation method of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 as claimed in claim 3; It is characterized in that in step 3); Said purification procedures 2) method of the expression product of gained is earlier expression product to be dialysed, and carries out affinity chromatography again.
7. the application of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 in the preparation antiviral drug.
8. the application of Scylla paramamosain anti-virus type coagulogen Sp-ALF2 in preparation animal disease resistant fodder additives.
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| CN105219779B (en) * | 2015-11-12 | 2018-06-15 | 厦门大学 | Red claw crayfish coagulogen and preparation method and application |
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| CN114957430A (en) * | 2022-06-09 | 2022-08-30 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Sparepcin 173-194 And uses thereof |
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| CN105219779B (en) * | 2015-11-12 | 2018-06-15 | 厦门大学 | Red claw crayfish coagulogen and preparation method and application |
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| CN112159460A (en) * | 2020-09-29 | 2021-01-01 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamprin4-23And uses thereof |
| CN112159460B (en) * | 2020-09-29 | 2021-12-17 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamprin4-23And uses thereof |
| CN114957430A (en) * | 2022-06-09 | 2022-08-30 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Sparepcin 173-194 And uses thereof |
| CN114957430B (en) * | 2022-06-09 | 2023-12-12 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Sparepcin 173-194 And applications thereof |
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