CN102675469A - Novel duck reovirus recombinant sigma B protein antigen, preparation method and application - Google Patents

Abstract

The invention discloses preparation of novel duck reovirus recombinant sigma B protein antigen, and application of the novel duck reovirus recombinant sigma B protein antigen in detection of a novel duck reovirus antibody. With RNA (ribonucleic acid) of the novel duck reovirus (NDRV) as a template, a nucleotide complete sequence of an NDRV S3 gene coding region can be amplified through a reverse-translation polymerase-chain reaction, is directionally subcloned to a fusion expression vector of the prokaryotic expression vector pET-30a-C(+) of escherichia coli, and transformed into the host cell of the Escherichia coli BL21 (DE3), and is subjected to inducible expression with IPTG (isopropyl-beta-d-thiogalactoside), purification of Ni-NTA Agarose, inclusion body refolding to obtain the expressed NDRV alpha B recombinant protein antigen. The expression mode of the protein is fusion protein (His-alpha B) with molecular weight being about 44kU; and immunoblotting assays prove that the protein has immunoreactivity similar to natural protein..

Description

Novel duck reovirus reorganization σ B (SigmaB) proteantigen, preparation method and application

Technical field

The present invention relates to the preparation and the application in detecting the sick antibody of novel duck reovirus thereof of novel duck reovirus reorganization σ B proteantigen.

Technical background

Duck hemorrhagic necrosis hepatitis be by the novel duck reovirus of Reoviridae Orthoreovirus (Novel duck reovirus, the various kind ducks that NDRV) cause a kind of with irregular necrosis of liver and hemorrhage mixing be the eqpidemic disease of principal character.The equal susceptible of different varieties duck, age in days heal little or during accompanying infection its sickness rate mortality ratio higher, in the majority with 5～10 ages in days, the course of disease 5～7d, sickness rate 5%～20%, mortality ratio 2%～15%.This pain is raised the district China south aquatic bird and is widely current, and there is the trend that enlarges year by year in the epidemic-stricken area, and the feedwater poultry breeding industry causes the certain economic loss.

The representative strains of NDRV is the NP03 strain isolated; The sequence signature of its S3 gene is: according to the Clustal W algorithm of MegAlign gene order comparative analysis software in the DNAStar5.0 software package; Described NDRVNP03 strain isolated S3 gene (GenBank accession number: GQ888710) with kind duck reovirus (Muscovy duck reovirus; MDRV) 89330 strain S3 genes (GenBank accession number: AJ243881), Avianreovirus (Avain reovirus; ARV) (the GenBank accession number: AF059720) coding region nucleotide sequence and deduced amino acid thereof are respectively 68.0%, 66.1% to 176 strain S3 genes; 70.4%, 69.3%.

At present, except serum neutralization test can be used for detecting the sick antibody of novel duck reovirus, still do not have other detection method, though neutralization test is the classical way of antibody test, specificity is good, operates loaded down with trivial detailsly, is inappropriate for veterinary clinic and basic unit and uses.In addition, when utilizing the NDRV totivirus as diagnostic antigen, (Muscovy duck reovirus, serum sample MDRV) produces non-specific cross-reaction with anti-kind duck reovirus in meeting.Utilizing expressing exogenous protein by pronucleus expression system is a kind of easy and high-efficiency method.Some engineered proteins of prokaryotic expression system production can keep necessary antigenicity, can be used in the virus infection detection of antibodies.Research shows; Novel duck reovirus σ B albumen promptly belongs to this proteinoid; Therefore using gene engineering σ B albumen is as antigen; Can set up the serum antibody of detection method in order to differentiate that NDRV, MDRV totivirus infect, this will provide important techniques to support with control NDRV for the using gene engineering vaccine prevention.

Summary of the invention

The object of the present invention is to provide the preparation and the application in detecting the sick antibody of novel duck reovirus thereof of novel duck reovirus reorganization σ B proteantigen;

The present invention realizes through following technical scheme:

Novel duck reovirus reorganization σ B proteantigen, this reorganization prokaryotic expression antigen is His leading peptide and the proteic fusion rotein of S3 genes encoding σ B;

The preparation method of this novel duck reovirus σ B recombinant protein antigen is:

The Auele Specific Primer of a, design amplification NDRV NP03 strain isolated S3 gene:

B, NDRV NP03 strain isolated are cultivated and RNA extracts:

C, NDRV NP03 strain isolated S3 Gene RT-PCR:

The structure of d, prokaryotic expression carrier pET-30a-C (+)-NP03-σ B:

E, NDRV σ B induction expression of protein

The purifying of f, recombinant protein

Renaturation behind g, the reorganization σ B protein purification

The mensuration of reorganization σ B protein content behind h, the purifying

The Western-blot of i, reorganization σ B protein-active detects

Above-mentioned recombinant protein is applied in indirect ELISA detects duck serum anti NDRV antibody.

The present invention is a template with novel duck reovirus (NDRV) RNA; Obtain NDRV S3 gene coding region complete nucleotide sequence through the inverse transcription polymerase chain reaction amplification; And directed subclone to escherichia coli prokaryotic expression carrier pET-30a-C (+) fusion expression vector, use the IPTG abduction delivering after being transformed into e. coli bl21 (DE3) host cell, through the Ni-NTAAgarose purifying; Renaturing inclusion bodies obtains the NDRV σ B recombinant protein antigen of expressing.This proteic expression-form is fusion rotein (His-σ B), and molecular weight is about 44kU; Show that through immunoblotting (Western-blot) detection this albumen has the immune response activity similar with native protein.The present invention does not have when the sick antibody of the novel duck reovirus of a kind of detection is provided that the poison of loosing is dangerous, good stability, and is highly sensitive, can realize suitability for industrialized production, prokaryotic expression antigen that cost is low and preparation method thereof.Use the indirect enzyme-linked immunosorbent assay that this expressing protein is an envelope antigen (ELISA) and can detect the anti-NDRV antibody in the duck serum.

Advantage of the present invention: being the σ B albumen of NDRV reorganization prokaryotic expression owing to this detection antigen 1., is not totivirus, therefore uses and does not have the poison of loosing danger when this antigen detects extremely.2. can be used for differentiating the serum antibody of NDRV, the infection of MDRV totivirus.3. as the sick antibody of the novel duck reovirus of indirect ELISA Detection of antigen, its detection sensitivity is high, high specificity, no cross reaction.4. production technique advanced person, stable performance, production cost are low, and product is paid value added height, are fit to batch production production, and market application foreground is wide.

Description of drawings

Fig. 1 is pET-30a-C (+)-NP03-σ B expression vector establishment figure,

(the NP03-S3 representative is inserted into the NDRV NP03 strain isolated S3 gene among the carrier pET-30a-C (+)).

Fig. 2 is NDRV NP03 strain isolated S3 gene RT-PCR amplified production and recombinant plasmid PCR qualification result figure; (1:NDRV NP03 strain S3 gene RT-PCR amplified production wherein; 2:NDRV NP03 strain S3 dna recombinant expression plasmid PCR qualification result; M:DNA marker DL2000).

Fig. 3 is pET-30a-C (+)-NP03-σ B prokaryotic expression plasmid double digestion qualification result figure;

(wherein 1. pET-30a-C (+)-NP03-σ B prokaryotic expression plasmid double digestion is identified;

2.DNA?MarkerDL10000)。

Fig. 4 is pET-30a-C (+)-NP03-σ B prokaryotic expression plasmid IPTG abduction delivering SDS-PAGE analytical results.(wherein 1 representative does not add the sample that inductor IPTG is got.8 hours institute's sample thiefs behind the 2 representative adding inductor IPTG.M. albumen Marker).

Fig. 5 pET-30a-C (+)-NP03-σ B recombinant protein immunoblotting result.

(Western blot used one anti-be the positive kind duck serum of NDRV polyclone, two anti-ly are the anti-duck IgG of the rabbit polyclonal antibody of HRP mark.The chromogenic substrate of HRP is DAB.)

Embodiment

Below in conjunction with specific embodiment the present invention is further specified.(but not being limitation of the present invention).

1, the Auele Specific Primer of design amplification NDRV NP03 strain isolated S3 gene:

NDRV-NP03-S3F (upstream primer): 5 '-TGCAAGGATCCATGGAGGTGCGTGTGCC-3 '

NDRV-NP03-S3R (downstream primer): 5 '-ACCCAGGTCGACTTACCACCTAC-3 '

2, NDRV NP03 strain isolated is cultivated and the RNA extraction

Get NDRV NP03 strain isolated kind poisons allantoic cavity approach and be inoculated in the non-immunity of 12 ages in days kind duck embryo, 37 ℃ of incubations are collected kind duck embryo allantoic liquid between the 48h-72h, and it is subsequent use to get supernatant after centrifugal.NDR VRNA extracts and is undertaken by Trizol test kit specification sheets; The RNA that extracts is used for RT-PCR.

3, NDRV NP03 strain isolated S3 Gene RT-PCR

(1) reverse transcription of NDRV NP03

In the PCR reaction tubes, add the RNA that 7 μ L have prepared respectively, add 1 μ LNDRV-NP03-S3 downstream primer (20pmol), 70 ℃ of heat absorption 10min; Ice bath 10min continues to add 5 * First Standing Buffer4 μ L, 10mM dNTPs 2 μ L; 0.5 μ L ThermoScript II (M-MLV) 5u (unit)/μ L; 0.25 μ LRNA enzyme inhibitors (40u) transfers to TV 20uL with no RNA enzyme water, instantaneous centrifugal mixing.Response procedures: 30 ℃ of 10min, 37 ℃ of 60min, 99 ℃ of 5min.

(2) PCR of NDRV NP03 S3 gene

PCR reaction system (50 μ L system): the cDNA 2 μ L to prepare make masterplate, add each 1 μ L of corresponding upstream and downstream primers (20pmol), 2.5mM dNTPs4 μ L, 10 * Buffer, 5 μ L, rTaq1 μ L, aqua sterilisa 36 μ L, reaction system 50 μ L.In the pcr amplification appearance, increase, amplification condition is 95 ℃ of 5min, 94 ℃ of 1min, and 53 ℃ of 1min, 72 ℃ of 2min, 35 circulations, last 72 ℃ are extended the 10min end.

4, the structure of prokaryotic expression carrier pET-30a-C (+)-NP03-σ B

(1) the purified test kit of PCR product reclaims the purpose fragment, utilizes the T-A clone to be directly connected in the pMD18-T carrier, connects product Transformed E .coil JM109 competent cell, and the picking mono-clonal carries out PCR and identifies.

(2) identify that with PCR correct positive colony carries out pcr amplification again; After the PCR product carries out purifying with the DNA purification kit; Through BamHI, SalI double digestion, reclaim the purpose fragment and be subcloned between the BamH I and Sal I restriction enzyme site of expression plasmid pET-30a-C (+) structure recombinant expression plasmid pET-30a-C (+)-NP03-σ B; Transformed competence colibacillus host bacterium JM109 extracts recombinant plasmid and carries out PCR, double digestion evaluation and sequencing.

5, NDRV σ B induction expression of protein

With identifying correct recombinant plasmid transformed BL21 (DE3) host bacterium, the picking mono-clonal is inoculated in the 5mL LB substratum of (containing the 100ug/mL kantlex), 37 ℃ of 170r/min overnight cultures.Get 0.5mL and be inoculated in the 5mL LB substratum, 37 ℃ of 220r/min are cultured to OD ₆₀₀0.4～0.6 o'clock, add IPTG to final concentration 0.1mmoL/L, cultivate 8h down at 37 ℃, collect thalline, with the centrifugal 2min of 6000g; Remove supernatant, will precipitate, with the centrifugal 2min of 6000g, remove supernatant again with PBS washing 1 time; 1mLPBS is resuspended, ultrasonic degradation, and 4 ℃ of centrifugal 30min of 12000g get a certain amount of supernatant respectively and with the resuspended deposition of PBS; Add isopyknic 2 * SDS sample-loading buffer, behind the mixing, in boiling water, boil 5min, as all article on the SDS-PAGE electrophoresis.To induce the BL21 (DE) 3 that contains empty plasmid pET-30a-C (+) to compare.

Adopt 12% polyacrylamide gel electrophoresis (SDS-PAGE) analysis, Coomassie brilliant blue dyeing, observations.The result has expressed the fusion rotein of about 44kU the intestinal bacteria that contain pET-30a-C (+)-NP03-σ B plasmid after inducing, and the contrast bacterium does not have corresponding band.Thin layer scanning is analyzed, and this albumen can account for the 35-40% of tropina total amount.

6, the purifying of recombinant protein

(1) preparation of sample to be purified:

By the expression condition of having optimized, express NDRV NP03 strain isolated reorganization σ B protein fungus liquid 1000ml, 4 ℃ of results bacterium liquid; With 8000rpm/min, centrifugal 20min, bacterial sediment is with TE (10mM Tris-HCl; 1mMEDTA, pH8.0) washing is 3 times, precipitates resuspended with TE solution;

The cracking thalline obtains the inclusion body deposition: thalline is carried out ultrasonic disruption, and supersound process must be carried out under condition of ice bath.Ultrasound condition: 800W, work 10S, 10S repeats certain number of times at interval, and with 4 ℃, the centrifugal 10min of 10000rpm abandons supernatant, and deposition is inclusion body;

The cracking of inclusion body and the recovery of soluble proteins: the insoluble inclusion body of acquisition is with inclusion body washings (20mM Tris-HCl [pH8.0], 5mM EDTA, 2M urea) washing 2 times; This inclusion body resolution of precipitate in containing 8M urea Tris-HCl damping fluid, is placed 2h on ice, and 4 ℃ are spent the night then; With 12000rpm, 4 ℃ of centrifugal 20min remove infusible precipitate; Get supernatant and be the solubility inclusion body protein, filter through 0.45 μ m aperture small offence filter ,-80 ℃ frozen subsequent use;

(2) the proteic affinity chromatography purification of reorganization σ B:

1. use the freshly prepared precooling binding buffer of 25ml liquid (20mM Tris-HCl [pH7.9], 10mM imidazoles, 0.5M NaCl, 8M urea) flushing and balance His nickel post;

2. the His protein purification medium after the resuspended balance of 10ml solubility inclusion body protein solution (again through 0.22 μ m pore size filter small offence filter filter) places the clean centrifuge tube of 50ml, and room temperature is fully felt and made 30min; Again upper prop then; Discard effluent, flow velocity is 10 times of column volumes/hour (column volume, i.e. volume of resin purification post; 2ml, below all with);

3. use binding buffer liquid (containing 8M urea) the flushing pillar of 15 times of column volumes, collect stream and wear the peak;

4. use freshly prepared elution buffer (20mM Tris-HCl [pH7.9], 0.5M imidazoles, 0.5M NaCl, the 8M urea) wash-out of 3 times of column volumes, collect elution peak, be sub-packed in the clean Eppendorf pipe by 500 μ l amount.

5. the regeneration of purification column and preservation: use successively 8 times of volumes binding buffer liquid and 5 times of column volumes provide deionized water wash for oneself after, provide 20% ethanol balance (ethanol will soak medium), 4 ℃ of preservations for oneself with 3 times of column volumes.

7, the renaturation behind the reorganization σ B protein purification

Dialysis tubing (the molecular weight cut-off: 14000Da), place TGE dialyzate (50mmol/L Tris-HCl, 0.5mmol/LEDTA that the expressing protein that purifying is good (containing 8M urea) is packed into and handled well by ordinary method; 50mmol/LNacl, 10% glycerine, 1% glycocoll; PH 8.0) in; Dialysis was repeatedly changed dialyzate one time in 8 hours under 4 ℃ of conditions, changed liquid three times.The dialysis back is covered in the dialysis tubing surface with the sucrose powder protein solution is concentrated, and is concentrated into 1/4 of original volume.Gained renaturation purifying protein-80 is ℃ frozen subsequent use.

8, the mensuration of reorganization σ B protein content behind the purifying

Adopt the uItraviolet absorption methods quantitative protein.After the suitable dilution of sample to be checked; After measuring this protein and Coomassie brilliant blue G250 association reaction with BioPhotometer spectrophotometer (Eppendorf) Bradford method simultaneously; The OD value at the colored compound absorption peak 595nm place that produces; Use Coomassie brilliant blue G250 as blank simultaneously, show protein content.The mass concentration of NDRV-σ B/His purifying protein is >=1.0mg/ml.

9, the Western-blot of reorganization σ B protein-active detects

The reorganization σ B albumen that the renaturation purifying is good is through being transferred to behind the SDS-PAGE electrophoresis on the nitrocellulose filter (NC), with 4 ℃ of incubated overnight sealings of the TBS (pH=8.0) that contains 3% (w/v) BSA NC film; Wash 3 times in the gentle concussion of shaking table with TTBS, each 10min at interval adds with the NDRV polyclone kind duck serum that contains 1% (w/v) BSA dilution (1: 100), room temperature shaking table mild action 90min again; TTBS washes 3 times in the concussion of shaking table gentleness, adds with the anti-duck IgG two of the rabbit of the HRP mark that contains 1% (w/v) BSA dilution (1: 1500) again and resists room temperature shaking table mild action 60min; TTBS washes 3 times in the gentle concussion of shaking table, inhales quiet surplus liquid, places DAB colour developing liquid to develop the color, and the result has observed specific reaction band at about 44kU place.

10, the application of reorganization σ B albumen in indirect ELISA method

The reorganization σ B albumen that the renaturation purifying is good encapsulates enzyme reaction plate after encapsulating the damping fluid dilution with the carbonate of pH9.6, concentration 1 μ g/mL, and every hole 50 μ L, 4 ℃ are spent the night; Take out the ELISA Sptting plate next day, with washing lotion (PBS that contains the pH7.4 of 0.5%TWEEN-20) washing three times, each 3min dries; With the PBST sealing that contains 1%BSA, every hole 180 μ L place 37 ℃ to hatch 1h; It is inferior to give a baby a bath on the third day after its birth with method; Serum to be checked (NDRV positive serum, MDRV positive serum and non-immune health kind duck negative serum) is done to add in the hand-hole after the dilution in 1: 40 with the PBST that contains 1%BSA respectively, place 37 ℃ to hatch 1h after, with method washing three times; Add the anti-duck IgG two of horseradish peroxidase (HRP) labelled goat and resist, with dilution in 1: 500, every hole 50 μ L added each hole, place 37 ℃ to hatch 1h; With method flushing three times; Add the FAST OPD colour developing with the 20ml deionized water dissolving then, every hole 50 μ L place 37 ℃ of 10-15min; Last every hole adds the 2M sulfuric acid termination reaction of 25 μ L; Enzyme mark detector reading reads the OD value in each hole under the 490nm wavelength, P/N >=2 o'clock are judged to be the NDRV antibody positive.The recombinant expressed σ B albumen of result only reacts with NDRV positive serum to be checked, and with MDRV positive serum, non-immunity kind duck negative serum cross reaction does not take place all.

The information of SEQ ID NO.1:

(a) NDRV S3 sequence signature:

Full mrna length: 1202 bases

Type: nucleic acid

Chain: strand

Topological framework: linearity

(b) molecule type: cDNA

(c) initial source: novel duck reovirus

(d) sequence description: SEQ ID NO 1:

SEQ?ID?NO.1

GCTTTTTGAGTCCTCAGCGTGCAAGCCGCAATGGAGGTGCGTGTGCCAAACTTTCACTCCTTTATTGAGGGTATTACTACTAGTTACTTGCGGTCTCCTGCTTGCTGGAATTCGAAGACGTTATGGGATATTGAAGAGTTTCACACACCTGACGTTATCAGGGTCGGCAACGCTTACTGTTGCACTCAGTGCTGTGGTGTTCTGTACTATGGTGCCCCTCCCTCTGATGGTAACTGTTTTCCACATCACAAGTGTCATCAACAGCAATATCGTACTGAGACTCCGCTCATGAGATATATTAAGGTGGGTCGCACTACAGAGCAACTACTTGATCAATATGCCGTTGCTCTGCATGTCATTGCAGATTACTATGATGAGGCGAGTAAGCGACCTCAGGATATCGCTGAAACTGAGTCAATCGCACCATTTGATATCGTAACCAGGACTGAATCTATTCGCAGTGACCGTGCCGTTGACCCGGAGTTCTGGACTTATCCGTTGGAGAGACGAGGATACGACGCGCGACATGAGATTGCTAGAGCGGGTTGGAAGATGATAGATGCTTCATCGCGAAGTCACACTCTTCCTGACTGTCTGGTGTCAAATATGCTCCATACTAGGCATGTCTTCAGCCAAATGTTAACCACGACAACCATCTATGATGTCGCTGTCACGGGTAAAGCTGTTAAATTCAGCCCGATGTTAGTAACCATGCCAACTCGAGGAGATGGTGCTGTGGCTCTGTCAAGAGGTAACTTGGATCATGATGTCGAGGACTGTTGGATGAATGGTTTTGCATTCTCCCCTATCATCGGCGGTGTTGGCATCACTGGTCAATTTGAGCGTGGTTCCTACCATAATTTTGGACACCCCATGGTTGGGAGTGGTAAGAAAGCTTCTCACTACCGCAATTTGTTCATGGAGTCCTGGCGTGGATGGTCAAAGTCG?TGCTTTACATGTGCTGCAGGATGGAGCCCGCCGAGTGCGAATCTAGGCTGCGAGGCCATGCCAGAACTATGTTCGGACGTTCTCTTCCGGATATCTGTGACTTCGAGGAGACTACCCACGTTGGCCAGTCGTCCGCGCCATTAAAGAAGGCCACGAAATTGTCCTTCCTGGAGTGTAGGTGGTAAGCACCTCTGGGTCAAAATGCACATAGGCTCCCACCTATGTGACGGTTAGCGGGACTCGCCTATTCATC

The information of SEQ ID NO.2:

(a) NDRV S3 coding region sequence characteristic:

Coding region length: 1104 bases

Type: nucleic acid

Chain: strand

Topological framework: linearity

(b) molecule type: cDNA

(c) initial source: novel duck reovirus

(d) sequence description: SEQ ID NO.2:

SEQ?ID?NO.2

ATGGAGGTGCGTGTGCCAAACTTTCACTCCTTTATTGAGGGTATTACTACTAGTTACTTGCGGTCTCCTGCTTGCTGGAATTCGAAGACGTTATGGGATATTGAAGAGTTTCACACACCTGACGTTATCAGGGTCGGCAACGCTTACTGTTGCACTCAGTGCTGTGGTGTTCTGTACTATGGTGCCCCTCCCTCTGATGGTAACTGTTTTCCACATCACAAGTGTCATCAACAGCAATATCGTACTGAGACTCCGCTCATGAGATATATTAAGGTGGGTCGCACTACAGAGCAACTACTTGATCAATATGCCGTTGCTCTGCATGTCATTGCAGATTACTATGATGAGGCGAGTAAGCGACCTCAGGATATCGCTGAAACTGAGTCAATCGCACCATTTGATATCGTAACCAGGACTGAATCTATTCGCAGTGACCGTGCCGTTGACCCGGAGTTCTGGACTTATCCGTTGGAGAGACGAGGATACGACGCGCGACATGAGATTGCTAGAGCGGGTTGGAAGATGATAGATGCTTCATCGCGAAGTCACACTCTTCCTGACTGTCTGGTGTCAAATATGCTCCATACTAGGCATGTCTTCAGCCAAATGTTAACCACGACAACCATCTATGATGTCGCTGTCACGGGTAAAGCTGTTAAATTCAGCCCGATGTTAGTAACCATGCCAACTCGAGGAGATGGTGCTGTGGCTCTGTCAAGAGGTAACTTGGATCATGATGTCGAGGACTGTTGGATGAATGGTTTTGCATTCTCCCCTATCATCGGCGGTGTTGGCATCACTGGTCAATTTGAGCGTGGTTCCTACCATAATTTTGGACACCCCATGGTTGGGAGTGGTAAGAAAGCTTCTCACTACCGCAATTTGTTCATGGAGTCCTGGCGTGGATGGTCAAAGTCGTGCTTTACATGTGCTGCAGGGATGGAGCCCGCGGAGTGCGAATCTAGGCTGCGAGGCCATGCCAGAACTATGTTCGGACGTTCTCTTCCGGATATCTGTGACTTCGAGGAGACTACCCACGTTGGCCAGTCGTCCGCGCCATTAAAGAAGGCCACGAAATTGTCCTTCCTGGAGTGTAGGTGGTAA

The information of SEQ ID NO.3:

(a) NDRV σ B protein sequence characteristic:

Length: 367 amino acid

Type: amino acid

Topological framework: linearity

(b) molecule type: polypeptide

(c) sequence description: SEQ ID NO.3:

SEQ?ID?NO.3

MEVRVPNFHSFIEGITTSYLRSPACWNSRTLWDIEEFHTPDVIRVGNAYCCTQCCGVLYYGAPPSDGNCFPHHKCHQQQYRTETPLMRYIKVGRTTEQLLDQYAVALHVIADYYDEASKRPQDIAETESIAPFDIVTRTESIRSDRAVDPEFWTYPLERRGYDARHEIARAGWKMIDASSRSHTLPDCLVSNMLHTRHVFSQMLTTTTIYDVAVTGKAVKFSPMLVTMPTRGDGAVALSRGNLDHDVEDCWMNGFAFSPIIGGVGITGQFERGSYHNFGHPMVGSGKKASHYRNLFMESWRGWSKSCFTCAAGMEPAECESRLRGHARTMFGRSLPDICDFEETTHVGQSSAPLKKATKLSFLECRW?。

Claims (3)

1. novel duck reovirus reorganization σ B proteantigen, it is characterized in that: this reorganization prokaryotic expression antigen is His leading peptide and the proteic fusion rotein of S3 genes encoding σ B.

2. the preparation method of novel duck reovirus σ B recombinant protein antigen according to claim 1 is characterized in that:

The Auele Specific Primer of a, design amplification NDRV NP03 strain isolated S3 gene:

NDRV-NP03-S3F (upstream primer): 5 '-TGCAAGGATCCATGGAGGTGCGTGTGCC-3 '

NDRV-NP03-S3R (downstream primer): 5 '-ACCCAGGTCGACTTACCACCTAC-3 '

B, NDRV NP03 strain isolated are cultivated and RNA extracts:

Get NDRV NP03 strain isolated kind poisons allantoic cavity approach and be inoculated in the non-immunity of 12 ages in days kind duck embryo, 37 ℃ of incubations are collected kind duck embryo allantoic liquid between the 48h-72h, and it is subsequent use to get supernatant after centrifugal; NDRV RNA extracts and is undertaken by Trizol test kit specification sheets; The RNA that extracts is used for RT-PCR;

C, NDRV NP03 strain isolated S3 Gene RT-PCR:

(1) reverse transcription of NDRV NP03

In the PCR reaction tubes, add the RNA that 7 μ L have prepared respectively, add 1 μ LNDRV-NP03-S3 downstream primer (20pmol), 70 ℃ of heat absorption 10min; Ice bath 10min continues to add 5 * First Standing Buffer4 μ L, 10mM dNTPs 2 μ L; 0.5 μ L ThermoScript II (M-MLV) 5u (unit)/μ L; 0.25 μ LRNA enzyme inhibitors (40u) transfers to TV 20uL with no RNA enzyme 7 water, instantaneous centrifugal mixing; Response procedures: 30 ℃ of 10min, 37 ℃ of 60min, 99 ℃ of 5min;

(2) PCR of NDRV NP03 S3 gene

PCR reaction system (50 μ L system): the cDNA2 μ L to prepare makes masterplate, adds each 1 μ L of corresponding upstream and downstream primers (20pmol), 2.5mM dNTPs4 μ L, 10 * Buffer, 5 μ L, rTaq1 μ L, aqua sterilisa 36 μ L, reaction system 50 μ L; In the pcr amplification appearance, increase, amplification condition is 95 ℃ of 5min, 94 ℃ of 1min, and 53 ℃ of 1min, 72 ℃ of 2min, 35 circulations, last 72 ℃ are extended the 10min end;

The structure of d, prokaryotic expression carrier pET-30a-C (+)-NP03-σ B:

(1) the purified test kit of PCR product reclaims the purpose fragment, utilizes the T-A clone to be directly connected in the pMD18-T carrier, connects product Transformed E .coil JM109 competent cell, and the picking mono-clonal carries out PCR and identifies;

(2) identify that with PCR correct positive colony carries out pcr amplification again; After the PCR product carries out purifying with the DNA purification kit; Through BamH I, Sal I double digestion, reclaim the purpose fragment and be subcloned between the BamH I and Sal I restriction enzyme site of expression plasmid pET-30a-C (+) structure recombinant expression plasmid pET-30a-C (+)-NP03-σ B; Transformed competence colibacillus host bacterium JM109 extracts recombinant plasmid and carries out PCR, double digestion evaluation and sequencing.

E, NDRV σ B induction expression of protein

With identifying correct recombinant plasmid transformed BL21 (DE3) host bacterium, the picking mono-clonal is inoculated in the 5mL LB substratum of (containing the 100ug/mL kantlex), 37 ℃ of 170r/min overnight cultures; Get 0.5mL and be inoculated in the 5mL LB substratum, 37 ℃ of 220r/min are cultured to OD ₆₀₀0.4～0.6 o'clock, add IPTG to final concentration 0.1mmoL/L, cultivate 8h down at 37 ℃, collect thalline, with the centrifugal 2min of 6000g; Remove supernatant, will precipitate, with the centrifugal 2min of 6000g, remove supernatant again with PBS washing 1 time; 1mL PBS is resuspended, ultrasonic degradation, and 4 ℃ of centrifugal 30min of 12000g get a certain amount of supernatant respectively and with the resuspended deposition of PBS; Add isopyknic 2 * SDS sample-loading buffer, behind the mixing, in boiling water, boil 5min, as all article on the SDS-PAGE electrophoresis; To induce the BL21 (DE) 3 that contains empty plasmid pET-30a-C (+) to compare;

The purifying of f, recombinant protein

(1) preparation of sample to be purified:

By the expression condition of having optimized, express NDRV NP03 strain isolated reorganization σ B protein fungus liquid 1000ml, 4 ℃ of results bacterium liquid; With 8000rpm/min, centrifugal 20min, bacterial sediment is with TE (10mM Tris-HCl; 1mMEDTA, pH8.0) washing is 3 times, precipitates resuspended with TE solution;

The cracking thalline obtains the inclusion body deposition: thalline is carried out ultrasonic disruption, and supersound process must be carried out under condition of ice bath; Ultrasound condition: 800W, work 10S, 10S repeats certain number of times at interval, and with 4 ℃, the centrifugal 10min of 10000rpm abandons supernatant, and deposition is inclusion body;

The cracking of inclusion body and the recovery of soluble proteins: the insoluble inclusion body of acquisition is with inclusion body washings (20mM Tris-HCl [pH8.0], 5mM EDTA, 2M urea) washing 2 times; This inclusion body resolution of precipitate in containing 8M urea Tris-HCl damping fluid, is placed 2h on ice, and 4 ℃ are spent the night then; With 12000rpm, 4 ℃ of centrifugal 20min remove infusible precipitate; Get supernatant and be the solubility inclusion body protein, filter through 0.45 μ m aperture small offence filter ,-80 ℃ frozen subsequent use;

(2) the proteic affinity chromatography purification of reorganization σ B:

1. use the freshly prepared precooling binding buffer of 25ml liquid (20mM Tris-HCl [pH7.9], 10mM imidazoles, 0.5MNaCl, 8M urea) flushing and balance His nickel post;

2. the His protein purification medium after the resuspended balance of 10ml solubility inclusion body protein solution (again through 0.22 μ m pore size filter small offence filter filter) places the clean centrifuge tube of 50ml, and room temperature is fully felt and made 30min; Again upper prop then; Discard effluent, flow velocity is 10 times of column volumes/hour (column volume, i.e. volume of resin purification post; 2m1, below all with);

3. use binding buffer liquid (containing 8M urea) the flushing pillar of 15 times of column volumes, collect stream and wear the peak;

4. use freshly prepared elution buffer (20mM Tris-HCl [pH7.9], 0.5M imidazoles, 0.5M NaCl, the 8M urea) wash-out of 3 times of column volumes, collect elution peak, be sub-packed in the clean Eppendorf pipe by 500 μ l amount;

5. the regeneration of purification column and preservation: use successively 8 times of volumes binding buffer liquid and 5 times of column volumes provide deionized water wash for oneself after, provide 20% ethanol balance (ethanol will soak medium), 4 ℃ of preservations for oneself with 3 times of column volumes;

Renaturation behind g, the reorganization σ B protein purification

Dialysis tubing (the molecular weight cut-off: 14000Da), place TGE dialyzate (50mmol/L Tris-HCl, 0.5mmol/LEDTA that the expressing protein that purifying is good (containing 8M urea) is packed into and handled well by ordinary method; 50mmol/LNacl, 10% glycerine, 1% glycocoll; PH 8.0) in; Dialysis was repeatedly changed dialyzate one time in 8 hours under 4 ℃ of conditions, changed liquid three times; The dialysis back is covered in the dialysis tubing surface with the sucrose powder protein solution is concentrated, and is concentrated into 1/4 of original volume; Be purified recombinant NDRV σ B albumen ,-80 ℃ frozen subsequent use;

The mensuration of reorganization σ B protein content behind h, the purifying

Adopt the uItraviolet absorption methods quantitative protein.After the suitable dilution of sample to be checked; After measuring this protein and Coomassie brilliant blue G250 association reaction with BioPhotometer spectrophotometer (Eppendorf) Bradford method simultaneously; The OD value at the colored compound absorption peak 595nm place that produces; Use Coomassie brilliant blue G250 as blank simultaneously, show protein content; The mass concentration of NDRV-σ B/His purifying protein is >=1.0mg/ml;

The Western-blot of i, reorganization σ B protein-active detects

The reorganization σ B albumen that the renaturation purifying is good is through being transferred to behind the SDS-PAGE electrophoresis on the nitrocellulose filter (NC), with 4 ℃ of incubated overnight sealings of the TBS (pH=8.0) that contains 3% (w/v) BSA NC film; Wash 3 times in the gentle concussion of shaking table with TTBS, each 10min at interval adds with the NDRV polyclone kind duck serum that contains 1% (w/v) BSA dilution (1: 100), room temperature shaking table mild action 90min again; TTBS washes 3 times in the concussion of shaking table gentleness, adds with the anti-duck IgG two of the rabbit of the HRP mark that contains 1% (w/v) BSA dilution (1: 1500) again and resists room temperature shaking table mild action 60min; TTBS washes 3 times in the gentle concussion of shaking table, inhales quiet surplus liquid, places DAB colour developing liquid to develop the color, and the result has observed specific reaction band at about 44kU place.

3. the novel duck reovirus σ B recombinant protein of preparing method's preparation of novel duck reovirus σ B recombinant protein antigen according to claim 2 detects the application in the sick antibody of anti-novel duck reovirus at indirect ELISA.

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