CN104611343B - The carp antiviral natural immune protein TRIM32 and antiviral activity of separation - Google Patents

Abstract

The invention belongs to aquatic livestock gene engineering technology field, and in particular to the carp antiviral natural immune protein TRIM32 and antiviral activity of a kind of separation.The nucleotide sequence such as SEQ ID NO of described carp antiviral natural immune protein TRIM32 genes：In 1 shown in the 1st 1986bp, its amino acid sequence such as SEQ ID NO encoded：Shown in 2.Experiment shows that the expression for the TRIM32 albumen that the present invention clones can influence SVCV (SVCV) propagation, and TRIM32 albumen plays an important role in SVCV life cycle.The gene or albumen of the present invention provides new target spot to prepare the research of anti-SVCV medicines.

Description

The carp antiviral natural immune protein TRIM32 and antiviral activity of separation

Technical field

The invention belongs to freshwater fish gene engineering technology field, and in particular to a kind of carp antiviral natural of separation is immunized Albumen TRIM32 and antiviral activity.

Background technology

Spring viremia, it is a kind of important aquatic animal disease as caused by SVCV (SVCV), category In International Office of Epizootics (OIE), regulation must notifiable animal epidemic.In China, it is also classified as two class animal epidemics by the Ministry of Agriculture. SVCV is a kind of rhabdovirus.Now it is listed in Rhabdoviridae, the tentative member of bubble stomatitis disease category.The virus is infected with very high The death rate, cause huge economic loss.Fry once infects this disease, shows as moving about slowly, excitement levels step-down, rests on Pond bottom is gathered in water, slow in reacting and until dead.Sick fish is mainly shown as internal bleeding, peritonaeum in appearance Scorching, visceral hemorrhage spot.Meanwhile SVCV can infect black carp, grass carp, chain fish, bighead and other several breams.Therefore, SVC mono- It is directly a kind of fishes virus sexually transmitted disease paid high attention in the world.China's sick report currently without large-scale outbreak.But Never has there is separation in the ornamental fish such as carp, the goldfish of any symptom and has identified SVCV in existing document report China.This is right The outlet of China fancy fishes causes extremely serious influence.Therefore, strengthen SVCV antiviral study, seek effective pre- Anti- and treatment method is the task of top priority.

The innate immunity, i.e. inherent immunity or congenital immunity, which are that body is inherent, can resist microorganism or external Foreign matter invasion and attack physiological function, be body self-defense the first line of defence, have fast reactivity, non-specificity, generality, The features such as diversity.In antiviral natural is immune, ubiquitination plays vital role.

Ubiquitin is a kind of highly conserved small protein being present in eukaryotic, and its expression is quite varied.Entirely With 76 amino acid, molecular weight 8kD.Ubiquitin can be connected to target under a series of catalysis of enzymes in the form of Covalent bonding together On albumen, the ubiquitination to target protein is completed.Modified forms can influence various different albumen point after this protein translation Positioning, expression and activity etc. of son, so as to the various aspects of wide participation body vital movement, such as the cell cycle, point Change, adjust die, shift, signal transmission, inflammatory reaction and genetic transcription etc..Ubiquitination process is mainly by three kinds of ubiquitin GAP-associated protein GAP enzymes Complete, they are E1 ubiquitin kinase, E2 ubiquitin binding enzymes and E3 ubiquitin ligases respectively.And in recent years, it was found that Yi Zhongxin The protein family-TRIM family relevant with ubiquitination.

The title of TRIM (Tripartite motif) family is due to that it has 3 conservative domains and got.This Order of three domains from N-terminal to C-terminal be successively RING domains (RING domain), 1 or 2 B-box domains, One coiled-coil domain (Coiled-coil domain), in addition the family also there is a variable C-terminal, therefore TRIM families are also referred to as RBCC families.RING domains are Zinc finger domain, have E3 ubiquitin ligase activities, mediating proteins are certainly The ubiquitination of body or different substrates.Protein-protein interaction is otherwise engaged in, and it is related to antiviral activity.B-box domains Combined with zinc atom, the generally existing in TRIM albumen, may be relevant with E3 ubiquitin ligase activities.Coiled-coil domain is joined The homologous or oligomeric effect between albumen；And the SPRY domains of c-terminus are in TRIM protein exhibits various biological functions Play an important role.After the first member XNF7 of TRIM (RBCC) protein family in 1991 is cloned identification, the mankind at present Genome has found and identified nearly 70 TRIM protein members, just there is kind more than 100 in fish.And the work(of its family member Can increasingly it be taken seriously.It is many heavy that research shows that the member of TRIM families take part in cell differentiation, propagation, development, apoptosis etc. The biological process wanted.Recent studies have shown that part TRIM albumen has antiviral functions, especially have to retrovirus Restricted effect, and take part in a series of bioprocess related to the innate immunity.

The content of the invention

A kind of the defects of it is an object of the invention to overcome prior art, there is provided carp antiviral natural immune protein (TRIM32), while the application of the immune protein (TRIM32) in carp antiviral activity is studied.

A kind of present invention isolated carp antiviral natural immune protein TRIM32 genes in carp by clone technology, its Nucleotide sequence is SEQ ID NO:Sequence in 1 shown in 1-1986bp.

Applicant obtains the carp antiviral natural immune protein TRIM32 of the separation simultaneously, and the amino acid sequence that it is encoded is such as SEQ ID NO:Shown in 2.

Experiment shows that the expression for the TRIM32 albumen that the present invention clones can influence SVCV (SVCV) Propagation, and TRIM32 albumen plays an important role in SVCV life cycle.The gene or albumen of the present invention is anti-to prepare The research of SVCV medicines provides new target spot.

More detailed technical scheme and invention effect referring to《Embodiment》It is described.

Brief description of the drawings

Sequence table SEQ ID NO：1 is the nucleotide sequence (1- of carp antiviral natural immune protein TRIM32 genes 1986bp), sequence 1986bp, wherein 1-1986bp is also the gene coding region (CDS), amino acid corresponding to display Sequence.

Sequence table SEQ ID NO：2 be the protein sequence of carp antiviral natural immune protein TRIM32 gene codes.

The amplification of Fig. 1 .TRIM32 core fragments.Description of reference numerals：Swimming lane M：DNA Marker 5000；Swimming lane 1,2,3：Core fragment.

Fig. 2 .TRIM32 gene magnification results.Description of reference numerals：Swimming lane M：DNA Marker 5000；Swimming lane 1： TRIM32 fragments

Fig. 3 utilize the expression after IIF detection TRIM32 transfections.Description of reference numerals：In Fig. 3 A figures are transfection pCDNA4^TM-/TO/-myc-His A control group：B figures in Fig. 3 are transfection pCDNA4^TM-/TO/-TRIM32- Myc-His A experimental group.

Fig. 4 are overexpressed situation using Diagnosis of Sghistosomiasis notation detection TRIM32.Description of reference numerals：Swimming lane 1：Transfection

pCDNA4^TM-/TO/-TRIM32-myc-His A groups；Swimming lane 2：Transfect pCDNA4^TM-/TO/-myc-His A are unloaded Body group.

Fig. 5：Virus titer measurement result：pCDNA4^TM-/TO/-TRIM32-myc-His A/pCDNA4^TM-/TO/-myc- His A transfected EPC cells after 24 hours, were infected with 0.1MOI SVCV, and collect respectively within 12,24,36,48 hours after infection The supernatant of experimental group and control group, carry out virus titer measure.

Fig. 6：It is the pCDNA4 that the present invention applies^TMThe collection of illustrative plates (5151bp) of/TO/-myc-His A empty carriers.

Fig. 7：Recombinant expression plasmid pCDNA4 prepared by the present invention^TM-/TO/-TRIM32-myc-His A(7134bp)。

Embodiment

Embodiment 1

(1) the RNA extractions of carp

Take size to take the ovary tissue (50-100mg) of mung bean size after dissection for 300g or so female carp, add 1ml TRIzol reagents, homogenized is carried out with Syrup-homogenizing instrument.The ℅ of TRIzol volumes 10 is not to be exceeded in sample volume.

Homogenised sample is placed 5 minutes for (15-30 DEG C) in room temperature, is kept completely separate nucleic acid-protein compound.

Centrifuged 15 minutes in 4 DEG C of 10000 × g.Sample is divided into three layers：Bottom is yellow organic phase, and upper strata is colourless aqueous phase With an intermediate layer.

Aqueous phase is transferred in new pipe, adds the chloroform of 1/5 aqueous phase volume, concussion mixes, and stands 5 minutes, 4 DEG C on ice 10000 × g is centrifuged 15 minutes.

Take supernatant to add isometric isopropanol in a new centrifuge tube, stand 10 minutes on ice, 4 DEG C, 10000 × g Centrifugation 10 minutes.Supernatant is removed, adds 75% ethanol washing precipitation.

At 4 DEG C, 10000 × g is centrifuged 5 minutes, the water dissolving RNA without RNase in right amount is added into centrifuge tube, -80 DEG C standby With.

(2) reverse transcription obtains the total cDNA of carp

Using (TAKARA) reverse transcription reagent box of precious bioengineering Dalian Co., Ltd, grasped according to the specification of kit Make, comprise the following steps that：

5 × gDNA Eraser Buffer 2ul, gDNA Eraser 1ul are taken, above-mentioned carried RNA 1ug, are added to RNA In the special EP pipes of reverse transcription, add in right amount without RNase water, supply system to 10ul.42 DEG C, 2 minutes, it is placed at once on ice.

Into above-mentioned 10ul reaction solutions, 5 × PrimeScript Buffer24ul, PrimeScript RT are added Enzyme Mix I 1ul, RT Primer Mix 1ul, and appropriate system is supplied to 20ul without RNase water.37 DEG C, 15 points Clock；85 DEG C, 5 seconds；4 DEG C of incubations.Obtain the total cDNA of carp.

(3) TRIM32 core fragments expand

With reference to the TRIM32 genes design degenerate primer of the species such as zebra fish, Tilapia mossambica, swordfish, latimeria chalumnae, carp is expanded TRIM32 core fragment sequences.Sense primer is：5'-TCCCAGCTGGCTGACaayytnacngt-3', anti-sense primer are：5'- AGGCTTGATCAGCTGGTTCTtrtgrcangcna-3'.Amplification obtains core fragment (as shown in Figure 1), by described core Fragment is held up after the sequencing of Kechuang neoformation Technology Co., Ltd. justifies through Wuhan, public with Invitrogen using RACE technologies Department 5 ' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 kit obtain 5 ' terminal sequences of the gene.Using the SMARTer of Clontech companies^TMRACE cDNA Amplification Kit kits Obtain 3 ' terminal sequences of the gene.Carp TRIM32 full length genes are obtained after splicing.

(4) carp TRIM32 gene magnifications

Using the carp TRIM32 gene orders total length obtained as reference, specific primer is designed, is carried out by masterplate of cDNA Amplification.And BamHI, XhoI restriction enzyme site are added in the primer of upstream and downstream respectively, anti-sense primer removes terminator codon, upstream Primer is：5'-AAAGGATCCATGGCCACACCAACATCTTTAG-3', anti-sense primer are：5'- AAACTCGAGACATGTAGAGGAACGTCTCCTT-3'(underscores part is restriction enzyme site).PCR amplification programs are：95 DEG C pre- It is denatured 5min；95 DEG C of denaturation 30S, 60 DEG C of annealing 30S, 72 DEG C of extension 2min, totally 35 circulate；Then 72 DEG C of fully extensions 10min。

PCR reaction systems (50 μ L) are as follows：CDNA templates (2.5ng/ μ L) 2 μ l, dNTPs (2.5mM) 4 μ l, 10 × Taq enzyme Buffer solution 5 μ L, above-mentioned sense primer (10 μM) 1 μ L, the above-mentioned μ L of anti-sense primer (10 μM) 1, Taq enzyme (5U/ μ L) 1 μ L, in right amount ddH₂O supplies system to 50 μ L.Amplification is as shown in Figure 2.

(5) the purifying recovery of carp TRIM32 fragments

(Beijing DingGuo ChangSheng Biology Technology Co., Ltd, grasped with DNA QIAquick Gel Extraction Kits according to the specification of kit Make) recovery purifying TRIM32 PCR primer or digestion products.Step is as follows：

1. the Ago-Gel blob of viscose containing DNA fragmentation is cut, by weight 1:3 (i.e. 100mg glue adds 300ul solution As Ratio adds solution A (solution A is that the kit carries).

2. 55 DEG C to 60 DEG C water-baths 10 minutes, dissolve, during which can shake hydrotropy 2-3 times completely to blob of viscose.

3. addition 50ul solution Bs (kit carries), concussion, which mixes, (if solution is changed into pink after mixing, to be needed to add The dosage of big solution B is to being changed into orange-yellow or colourless).

4. the solution of step 3. gained is placed in centrifugal column, 2 minutes are stood, 12000 leave the heart 30 seconds, if once plus not It is complete,

Can be at twice.

5. outwell liquid, add 500ul solution Cs (kit carries, and solution C adds 52ml absolute ethyl alcohols before using) in

12000 leave the heart 30 seconds in centrifugal column, abandon liquid.

6. repeat step is 5..

7. 12000 turns centrifuge 1 minute again, remaining liq is dried to remove residual ethanol.

8. centrifugal column is replaced in new centrifuge tube, room temperature opens wide centrifugation lid and places 5-10 minutes, waves ethanol Hair is totally.

9. adding 20ul-60ul solution Ds (the solution D kit carries, or distilled water), (solution D is using being preceding preheated to 50

DEG C), stand 2 minutes.12000 leave the heart 2 minutes, and ttom of pipe solution is required DNA.DNA is stored in -20 DEG C.

(6) recombinant expression plasmid pCDNA4 is built^TM-/TO/-TRIM32-myc-His A

Respectively by pCDNA4^TM/ TO/-myc-His A empty carriers (are purchased from Invitrogen companies), and structure is as shown in Figure 6) It is as follows with the TRIM32 genes BamHI/XhoI double digestions of recovery, reaction system：pCDNA4^TM/ TO/-myc-His A are unloaded Body/TRIM32 recovery fragments, 2 μ g；BamHI, 1 μ L；XhoI, 1 μ L；10 × Buffer3,2 μ L；10 × bovine serum albumin(BSA) (BSA), 2 μ L；With ddH₂O is mended to 20 μ L, and after putting 37 DEG C of reaction 2-3h, recovery is standby.

After double digestion, by the pCDNA4 of recovery^TM/ TO/-myc-His A empty carriers and TRIM32 genes are in T4DNA ligases Under effect, reaction is attached, reaction system (20 μ L) is as follows：TRIM32 genes, 150ng；pCDNA4^TM/TO/-myc-His A Carrier, 50ng；10 × T4DNA Ligase Buffer, 2 μ L；T4DNA Liagse (350U/ μ L), 1 μ L；With ddH₂O is mended to 20 μ L, 16 DEG C of reaction overnights.

(7) conversion of connection product

By connection product conversion E. coli competent DH5 α：The competent cell for taking 50 μ L ice bath melted goes out in 1.5mL In bacterium centrifuge tube, 10 μ L connection products are added, are gently mixed, ice bath 30min；Heat shock 45s, is quickly transferred in 42 DEG C of water-baths 2min in ice bath (process not shake centrifuge tube)；500 μ L sterile LB mediums are added into each centrifuge tube and (are free of antibiosis Element), it is placed in after mixing on 37 DEG C of shaking tables, 180r/min culture 1h, makes bacteria resuscitation；5,000r/min room temperatures centrifuge 3min, abandon Go honest and upright and thrifty 300 μ L, after remaining resuspension, be added on the LB agar mediums containing corresponding antibiotic, it is uniformly spreadable；Flat board is put Absorbed in 37 DEG C to liquid, be inverted flat board, 37 DEG C of overnight incubations

(8) recombinant expression plasmid pCDNA4^TM-/TO/-TRIM32-myc-His A a small amount of preparations

Single bacterium colony on picking flat board, is inoculated into 3mL LB, and adding 3ul ampicillins makes final concentration of 50ug/ mL.It is placed on 37 DEG C of shaking tables, 180r/min overnight incubations；In 1.5mL centrifuge tubes, 10,000g room temperatures centrifuge 1min to collect Strain；Culture medium is poured out, plasmid is extracted with the small extraction reagent kit of plasmid (buying in OMEGA companies), comprises the following steps that：Precipitating 250 μ L Solution I (kit carries) of middle addition, vortex oscillation makes cell suspend completely；250 are added in re-suspension liquid μ L Solution II (kit carries), gently overturn and mix 7-10 times, room temperature places 5-10min；Add 350 μ L Solution III (kit carries), the centrifuge tube that leniently turns upside down mix for several times；10,000g room temperatures centrifuge 10min； By pillar on 2mL collecting pipe, the careful supernatant that shifts is into pillar；10,000g room temperatures centrifuge 1min, make above-mentioned mixing Liquid flows completely through pillar, discards the liquid in collecting pipe；Pillar is mounted in 2mL collecting pipes again, adds 500 μ L HB Buffer is into pillar, 10,000g room temperatures centrifugation 1min washing pillars；The liquid in collecting pipe is discarded, adds 700 μ L DNA Wash Buffer 10,000g room temperatures centrifugation 1min, discard cleaning solution, repeat the step on pillar；10,000g room temperatures centrifuge Void column 2min；Pillar is placed on a clean 1.5mL centrifuge tube, adds 30-50 μ L Elution Buffer, at room temperature Stand 2min；10,000g, room temperature centrifugation 2min, the liquid in centrifuge tube is the recombinant expression plasmid pCDNA4 extracted^TM-/ TO/-TRIM32-myc-His A, are saved backup in -20 DEG C.Recombinant expression plasmid pCDNA4^TM-/TO/-TRIM32-myc-His A structure is as shown in Figure 7.

(9) recombinant expression plasmid pCDNA4 is utilized^TM-/TO/-TRIM32-myc-His A/ empty carriers pCDNA4^TM-/TO/- Myc-His A transfect EPC cells

Comprise the following steps that：

1. 1 day before transfection, in 12 porocyte culture plates inoculating cells, treat that EPC cell monolayers length to 80% or so, prepares to turn Dye.

2. take the sterile EP pipes of two 1.5ml.2 μ l liposomes 2000 and 100 μ l serum free mediums Opti- are added in pipe A MEM (is bought in Gibco companies), and 5min is stored at room temperature after mixing.Therebetween, 100 μ l serum free mediums are added in pipe B Opti-MEM and 1-2ug recombinant expression plasmids pCDNA4^TM-/TO/-TRIM32-myc-His A。

3. the liposome 2000 in pipe A and Opti-MEM mixed liquors are added dropwise in pipe B, mix after being stored at room temperature 20min.Therebetween, the culture medium for abandoning cell hole to be transfected is inhaled, changes serum free medium (Opti-MEM or corresponding basis cultures Base).

4. inhaling the serum free medium for abandoning cell hole to be transfected, 200 μ l mixed liquors in pipe B are added drop-wise to cell monolayer On, 800 μ l Opti-MEM (buying in Gibco companies) are added, are gently shaken up.Cultivated in 28 DEG C of incubators.

5. after cultivating 4-6h, transfection cocktail is abandoned in suction, adds complete medium (MEM adds 10% hyclone) to continue to cultivate.

Control group, by pCDNA4^TM-/TO/-TRIM32-myc-His A are changed to empty carrier pCDNA4^TM-/TO/-myc-His A (is bought in Invitrogen companies), and remaining step is operated by above-mentioned the step of 1. arriving 5..

After transfection 24 hours, indirect immunofluorescence and immunoblotting analysis experiment detection TRIM32 protein expression situations are utilized.

Concrete operations are as follows：

(10) indirect immunofluorescence experiment (IFA)

1. transfectional cell EPC to be detected (abbreviation PBS, is bought in the prosperous biology of Beijing ancient cooking vessel state with phosphate buffer Technology Co., Ltd) wash 3 times, each 5min.

2. 100% methanol room temperature fixes 10min, PBS is washed 3 times, each 5min.

3. with the confining liquid PBS of 1% bovine serum albumin(BSA) (BSA) (contain) closing 30min, 3 times are washed with PBS, every time 5min。

4. the mouse source primary antibody for adding confining liquid suitable (seeing below) dilution (is bought in Abclonal companies；Anti- His labels；With Volume ratio is 1：1000 dilution antibody are primary antibody), 1-2h is incubated, is washed 3 times with PBS, each 5min.

5. the anti-mouse fluorescence secondary antibody of donkey for adding confining liquid suitable (seeing below) dilution (is bought in Invitrogen companies；With body Product is than being 1：500 dilution antibody are secondary antibody), 45min is incubated, is washed 3 times with PBS, each 5min.

6. adding appropriate PBS, dry sample is prevented.Fluorescence and the photograph of transfectional cell are observed under inverted fluorescence microscope.Knot Fruit is as shown in figure 3, show that TRIM32 expressions of results are good.

(11) immunoblotting analysis experiment (Western Blot)：

1. match somebody with somebody glue：The preparation of separation gel and concentration glue is referring to table 1.

Table 1SDS--PAGE separation gel formula tables

Table 2SDS--PAGE concentrates glue (5%Acrylamide) formula table

2. glue：The glass plate for recording polyacrylamide gel is clipped, the SDS acrylamide separating gels about 5ml that will be made Pour into the gap of two glass plates rapidly, reserve space needed for perfusion spacer gel, one layer of isopropanol of addition, puts room temperature on separation gel It polymerize about 45min, after separation gel polymerize, pours out coating liquid, is washed with deionized water at the top of gel for several times, is inhaled with blotting paper Net residual liquid, then the spacer gel just prepared is poured on separation gel, comb is immediately inserted into, polymerized at room temperature about 45min is put, treats glue Comb all is removed after polymerization, the residual liquid of loading slot is washed away with electrophoretic buffer (1 × Tris- glycine), is dialled with syringe needle The glue for falling loading slot straightens tooth, is fixed on electrophoretic apparatus.

3. electrophoresis：Appropriate 1 × Tris- glycine running buffers (3.03g, Tris are added toward electrophoretic apparatus；18.8g, Glycine；SDS, 1g, it is dissolved in 1L deionized waters), the sample loading handled well is taken, per the μ l of hole about 20.Switch on power, voltage 80V is transferred to, when bromophenol blue indicator goes to separation gel, voltage is transferred to 120V, waits bromophenol blue indicator to go to separation gel bottom (about needing 2h) powers off during portion, terminates electrophoresis.

4. electricity turns：After SDS-PAGE electrophoresis terminates, the gel after SDS-PAGE electrophoresis is taken, it is not dyed, directly use and turn Film device goes to albumen on pvdf membrane.

5. transferring film is stayed overnight under 12V voltages, ice bag is put in its surrounding.

6. close：Pvdf membrane in buffer solution (TBST, sodium chloride, 8.8g；1M Tris-HCL, 20ml；Be dissolved in 1L go from In sub- water) in rinse and, be put into confining liquid (TBST containing 1%BSA), lie against room temperature on shaking table and gently shake and incubate 1- 2h or 4 DEG C overnight.

7. primary antibody (the anti-His labels in mouse source, buy in Abclonal companies) is incubated pvdf membrane：Pvdf membrane is put into new envelope Close in liquid, primary antibody (the anti-His labels in mouse source) is 1 according to volume ratio：1000 dilutions, lie against and incubate 1h on shaking table at room temperature.Will Pvdf membrane is rinsed 4-6 times with TBST, each 5-10min.

8. secondary antibody (sheep anti-mouse igg) is incubated pvdf membrane：Pvdf membrane is put into new confining liquid, adds the two of acceptable diluent Anti- (sheep anti-mouse igg；Volume ratio is 1：1000) incubation at room temperature 1h on shaking table, is lain against.Again by NC with TBST rinse 4-6 times, often Secondary 5-10min.

9. develop the color：Add 1 × TBST to wash 3 times, each 5min.Pvdf membrane is put into A the and B liquid (purchase of chemical luminous substrate Buy in Bio-Rad companies) equivalent mixed liquor in carry out lucifuge colour developing, taken a picture with chemiluminescence imaging system.

Immunoblotting analysis result works well as shown in figure 4, showing that TRIM32 is overexpressed.

(12) SVCV virus titers determine

After transfection 24 hours, above-mentioned experimental group and control group are infected with 0.1MOI SVCV (SVCV) EPC cells, and 12,24,36,48 hours supernatants for collecting experimental group and control group respectively after infection, carry out SVCV viruses Titer determination.Concrete operations are as follows：

1. will be accessed after the EPC cell dissociations for growing up to individual layer in 96 orifice plates, 100 μ L are accessed per hole；

2. second day, in 1.5ml EP pipes, (bought prosperous raw in Beijing ancient cooking vessel state with the MEM culture mediums containing 5% serum Thing technology Co., Ltd) SVCV virus liquids are made to continuous 10 times of dilution, from 10^-1-10^-9；

3. by the SVCV virus inoculations diluted into 96 hole microtest plates, each dilution factor is inoculated with 8 of a tandem Hole, 100 μ L are inoculated with per hole；Other 3 tandem makees blank control；

4. observing cytopathy situation day by day, and make a record, generally require 5 days or so time；

5. result calculates, TCID is calculated according to Karber methods₅₀Value.

Calculation formula：lgTCID₅₀=L-d (s-0.5)

In formula：The logarithm of L=highest dilutions；Difference between D=dilution factor logarithms；S=positive boring ratio rate summations；

The above-mentioned experiment of repetition 3 times, acquired results are as shown in Figure 5：In 12h, 24h, TRIM32 groups compared to control group, TRIM32 group virus titers are relatively low, and display 24h TRIM32 have obvious inhibiting effect to virus multiplication.

Claims (2)

1. a kind of carp antiviral natural immune protein TRIM32 genes of separation are preparing anti-SVCV medicine group Application in compound, it is characterised in that the nucleotide sequence of described gene is SEQ ID NO:In 1 shown in 1-1986bp Sequence.

2. a kind of carp antiviral natural immune protein TRIM32 of separation is preparing anti-SVCV pharmaceutical composition In application, it is characterised in that the amino acid sequence of described albumen such as SEQ ID NO:Shown in 2.