CN105175554B - One type elastin polypeptide and 90 alpha fusion protein of heat stress proteins and its preparation method and application - Google Patents

One type elastin polypeptide and 90 alpha fusion protein of heat stress proteins and its preparation method and application Download PDF

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CN105175554B
CN105175554B CN201510700114.1A CN201510700114A CN105175554B CN 105175554 B CN105175554 B CN 105175554B CN 201510700114 A CN201510700114 A CN 201510700114A CN 105175554 B CN105175554 B CN 105175554B
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elp
fusion protein
ibdv
disease virus
bursal disease
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孙怀昌
徐碧
邵聪聪
张鑫宇
夏晓莉
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Yangzhou University
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Yangzhou University
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Abstract

The invention belongs to biotechnology research fields, and in particular to a type elastin polypeptide and 90 alpha fusion protein of heat stress proteins and preparation method thereof and the application in infectious bursal disease virus concentrates and purifies.The class elastin polypeptide (ELP) is made of with 90 α of heat stress proteins (Hsp90 α) fusion proteins infectious bursal disease virus (IBDV) the binding fragment M167a of ELP and Hsp90 α.Preparation method includes the structure of fusion expression vector, the expression and purification of fusion protein, the determination of IBDV binding fragments and the concentration of IBDV and elution.Compared with other methods, is concentrated with the fusion protein of the present invention and purifying infectious bursal disease virus has many advantages, such as simple, quick and economical, the infectious bursal disease virus of preparation can be used for experimental study and vaccine preparation.

Description

One type elastin polypeptide and 90 alpha fusion protein of heat stress proteins and its preparation side Method and application
Technical field
The present invention relates to biotechnology research fields, and in particular to a type elastin polypeptide and 90 α of heat stress proteins The preparation method of fusion protein and its application in infectious bursal disease virus concentrates and purifies.
Background technology
Gumboro disease is that a kind of high degree in contact of the chicken caused by infectious bursal disease virus (IBDV) infects Sexually transmitted disease causes huge economic loss to world's aviculture.Viral concentration and purifying be viral molecular biology research and The important link of vaccine preparation.Existing IBDV method for concentration mainly has chloroform and polyethylene glycol precipitation etc., purification process Mainly there are differential centrifugation, density gradient centrifugation and affinity chromatography etc., these methods have that time-consuming, the laborious, rate of recovery is low and expense With it is high the shortcomings of.In addition, lacking quick, sensitive, economic IBDV method of environmental monitoring at present.
Virus receptor integrating capture technology is the viral concentration occurred in recent years and purifying new technology, and basic principle is root According to the specificity that virus is combined with receptor, knitted from infection group and viral infection group using solid phase carriers such as the magnetic beads of virus receptor albumen coupling, Virus is captured in animal foodstuff and environmental sample, is combined with technologies such as PCR, it may also be used for viral environmental monitoring.The technology Have many advantages, such as it is quick, efficient, but magnetic bead used it is expensive, need specialized equipment equipment, practical application to be restricted.
90 α of heat stress proteins (Hsp90 α) is the receptor complex components of IBDV, can be with virion and its VP2 albumen knots It closes, therefore can be as the bait protein for capturing IBDV.Hsp90 α points are N-terminal structural domain, central domain and C-terminal structural domain, But its combined area IBDV is unclear.
Class elastin polypeptide (ELP) is that (Val-Pro-is sweet for the pentapeptide that is synthesized according to elastin laminin correlated series Any amino acids glycine of propylhomoserin -) polymer, there is temperature sensitive reversible transition characteristic, in less than phase transition temperature solution In dissolved state, it is in state of aggregation to be higher than in phase transition temperature solution.This seminar is established with ELP and adenovirus receptor fusion protein Simple, economic recombined adhenovirus concentration and purification process, but after ELP and Hsp90 α or its segment composition can keep reversible Phase-change characteristic, and it is also to be studied for IBDV concentrations and purifying.
The ELP-Hsp90 alpha fusion proteins of expression of recombinant e. coli are used for the concentration and purifying of IBDV by the present invention, although This strategy is feasible in theory, but faces following technical problem in specific design:Can Hsp90 α or its segment In Escherichia coli efficiently and solubility expression, expression ELP merge can be combined with IBDV, which piece the combined areas IBDV are located at What section, best conjugation condition be, how to elute the virus combined with fusion protein and be not inactivated, in conjunction with fusion protein Can IBDV infect permissive cell.
Invention content
Of the existing technology in order to solve the problems, such as, the present invention provides a type elastin polypeptides (ELP) to answer with heat 90 α of shock protein (Hsp90 α) fusion protein and its preparation method and application.
The class elastin polypeptide of the present invention and 90 alpha fusion protein of heat stress proteins, by the infectiousness method of ELP and Hsp90 α The combined area family name's bursal disease virus (IBDV) forms.It is proved through viral concentration and purification experiment, it can be thin from infection using the fusion protein Concentration and purified IBDV in born of the same parents' culture supernatant, lytic cell and viral POLLUTION SIMULATION water sample.
In order to determine that ELP and overall length Hsp90 α or its segment are carried out amalgamation and expression by the combined areas IBDV, the present invention respectively; Fusion protein and IBDV are subjected to total incubation under different condition, to determine the optimum condition of fusion protein combination IBDV;Pass through System optimization to elution requirement enables IBDV effectively to be eluted from fusion protein and is not inactivated.Finally, present invention selection contains The most short fusion protein ELP-M167a of the combined areas IBDV carries out IBDV concentrations and purifying, so as in E. coli With the non-specificity for reducing IBDV combinations.
The technical solution adopted in the present invention is:One type elastin polypeptide and 90 alpha fusion protein of heat stress proteins are ELP-M167a is made of, the amino of the M167a the infectious bursal disease virus binding fragment M167a of ELP and Hsp90 α Acid sequence is as shown in SEQ ID No.1.
Fusion protein ELP-M167a of the present invention is prepared with following method:
(1) with the coded sequence such as SEQ ID No.2 institutes of 167 amino acid M167a of PCR clone's Hsp90 α central domains Show.
(2) above-mentioned coded sequence is inserted into ELP fusion expression vectors, obtains recombinant vector pELP-M167a;
(3) recombinant vector is converted into Escherichia coli, in 20 DEG C of induced fusion protein expressions;
(4) fusion protein is precipitated at 26 DEG C with 2mol/L sodium chloride.
It is specific the invention also discloses application of the ELP-M167a fusion proteins in IBDV is concentrated and is purified Method is as follows:
(1) 320 μ g/ml ELP-M167a fusion proteins are thin with equivalent IBDV infection cells culture supernatant, cracking respectively Born of the same parents and simulated water sample mixing;
(2) use 2mol/L sodium chloride in 26 DEG C of precipitate virus;
(3) pH9.0 buffer solutions elution virus is used;
(4) 2mol/L sodium chloride and 26 DEG C of centrifugation removal ELP-M167a fusion proteins are used.
Further, above-mentioned fusion protein is disclosed to concentrate and the application in purified IBDV.The present invention is used with lower section Method verifies fusion protein:
(1) IBDV infection cells are conventionally used, cells and supernatant and lytic cell are obtained;
(2) by various dose IBDV inoculation tap water and PBS, IBDV POLLUTION SIMULATION water samples are obtained;
(3) 320 μ g/ml ELP-M167a fusion proteins are thin with equivalent IBDV infection cells culture supernatant, cracking respectively Born of the same parents and simulated water sample mixing;
(4) 2mol/L sodium chloride and 26 DEG C of precipitate virus are used;
(5) pH9.0 buffer solutions elution virus is used;
(6) 2mol/L sodium chloride and 26 DEG C of centrifugation removal ELP-M167a fusion proteins are used.
Compared with existing other methods, it can not only be concentrated and purified from infection cell with the fusion protein of the present invention IBDV, and IBDV can be concentrated from viral pollution water sample, the virus of purifying cannot be only used for animal experiment study, and available In vaccine preparation.
Description of the drawings
Fig. 1 Hsp90a structures and its expression strategy.Digital representation amino acid number, arrow indicate Hsp90a structural domains or are used for The segment of clonal expression.
The expression and purification of Fig. 2 ELP-Hsp90a fusion proteins and electrophoretic analysis.M is protein molecular weight standard, and 1 is The recombinant bacterium lysate of IPTG inductions, 2 be the fusion protein after first round phase transformation cycle, and 3 be melting after the second wheel phase transformation cycle Hop protein.
The RT-PCR of Fig. 3 fusion protein combinations IBDV is detected.M is DNA molecular amount standard, and 1 is virus positive control, and 2 are The virus of ELP-Hsp90a fusion proteins precipitation, 3 be the virus of ELP-N284 fusion proteins precipitation, and 4 be ELP-C444 fusion eggs The virus precipitated in vain, 5 be ELP-M167a fusion proteins precipitation virus, 6 be ELP-M167b fusion proteins precipitation virus, 7 It is the virus of ELP-C132 fusion proteins precipitation, 8 be the virus of ELP precipitations.
Fig. 4 fusion proteins precipitate the condition and efficiency of infectious bursal disease virus.
Fig. 5 elutes the condition and efficiency of infectious bursal disease virus.
Specific implementation mode
Biological material source:
1.pET-30a carriers:It is introduced from Novagen companies of the U.S., this laboratory preserves.
2.pET/EI-GFP carriers:It is introduced from Princeton university, this laboratory preserves.Document:Fong BA, Wood DW.Expression and purification of ELP-intein-tagged target proteins in high cell density E.coli fermentation.Microbial Cell Factories,2010,9:77.
3.DH5a Escherichia coli:It is introduced from BD Biosciences Clontech companies of the U.S., this laboratory preserves.
4.BLR (DE3) Escherichia coli:It is introduced from Novagen companies of the U.S., this laboratory preserves.
5. chicken embryo fibroblasts DF-1 systems:It is introduced from U.S. ATCC, this laboratory preserves;
6.Lukert plants of infectious bursal disease virus:(document is preserved by this laboratory:Lukert PD, Leonard J, Davis RB.Infectious bursal disease virus:antigen production and immunity.Am J Vet Res, 1975,36 (4Pt 2):539-540).
Concrete operation step is as follows:
1. fusion expression vector is built
(1) restriction enzyme NdeI and SacI is used to digest pET/EI-GFP, according to DNA gel QIAquick Gel Extraction Kit after electrophoretic separation (AXYGEN companies) specification recycles ELP genetic fragments, is connect with the pET-30a carriers of same enzyme linearisation, obtains ELP fusions Expression vector pET-ELP.
(2) the good chicken of growth conditions is cultivated 3 hours at fiber DF-1 cells at 42 DEG C, according to RNAisoTMPlus is tried Agent box (TaKaRa companies) specification extracts cell total rna, according to RevertAidTM Reverse Transcriptase (Fermentas companies) illustrates to carry out reverse transcription.
(3) according to chicken Hsp90 α mRNA sequences (GenBank Accession No:X07265 following 1 pair of primer) is designed, It is point that forward primer, which introduces Sal I digestions, and reverse primer introduces Xho I restriction enzyme sites:
Forward primer:5'-TTGTCGACCCGGAAGCTGTGCAAACACAGG-3'(SEQ ID No.3);
Reverse primer:5'-AACTCGAGTTAATCCACCTCCTCCATACGT-3'(SEQ ID No.4)。
Using above-mentioned reverse transcription product as template, according to LA Taq archaeal dna polymerases (Fermentas companies) specification, use PCR amplification overall length Hsp90 α cDNA (Fig. 1) digest PCR product, with the pET- of identical digestion with restriction enzyme Sal I and Xho I ELP carriers connect, and obtain recombinant vector pELP-Hsp90 α.
(4) following 1 pair of primer is designed according to chicken Hsp90 α mRNA sequences, it is point that forward primer, which introduces Sal I digestions, reversely Primer introduces Xho I restriction enzyme sites:
Forward primer:5'-TTGTCGACCCGGAAGCTGTGCAAACACAGG-3'(SEQ ID No.5);
Reverse primer:5'-TTCTCGAGTTCCTCATCAATGTACTTCTCC-3'(SEQ ID No.6).With pELP- Hsp90 α are template, and restriction enzyme is used with PCR amplification N284cDNA segments (Fig. 1) according to LA Taq archaeal dna polymerase specifications Sal I and Xho I digest PCR product, are connected with the pET-ELP carriers of identical digestion, obtain recombinant vector pELP-N284.
(5) following 1 pair of primer is designed according to chicken Hsp90 α mRNA sequences, it is point that forward primer, which introduces Sal I digestions, reversely Primer introduces I restriction enzyme sites of Xho:
Forward primer:5'-TAGTCGACGAGCTCAACAAGACCAAGCC-3'(SEQ ID No.7);
Reverse primer:5'-AACTCGAGTTAATCCACCTCCTCCATACGT-3'(SEQ ID No.8)。
Using pELP-Hsp90 α as template, according to LA Taq archaeal dna polymerase specifications, with PCR amplification MC444cDNA segments (Fig. 1) digests PCR product with restriction enzyme Sal I and Xho I, is connected with the pET-ELP carriers of identical digestion, obtain recombination and carry Body pELP-MC444.
(6) following 1 pair of primer is designed according to chicken Hsp90 α mRNA sequences, it is point that forward primer, which introduces Sal I digestions, reversely Primer introduces I restriction enzyme sites of Xho:
Forward primer:5'-CTGTCGACGAGGAAGAGCTCAACAAGAC-3'(SEQ ID No.9);
Reverse primer:5'-CGCTCGAGTTAGGAGTCTTCATGTATTCCAA-3'(SEQ ID No.10).
Using pELP-Hsp90 α as template, according to LA Taq archaeal dna polymerase specifications, with PCR amplification M167a cDNA pieces Section (Fig. 1) digests PCR product with restriction enzyme Sal I and Xho I, connects, recombinated with the pET-ELP carriers of identical digestion Carrier pELP-M167a.
(7) following 1 pair of primer is designed according to chicken Hsp90 α mRNA sequences, it is point that forward primer, which introduces Sal I digestions, reversely Primer introduces I restriction enzyme sites of Xho:
Forward primer:5'-ATGTCGACAACATCAAGCTTGGAATACAT-3'(SEQ ID No.11);
Reverse primer:5'-GTCTCGAGTTACATATTGGCAGTCCAGCC-3'(SEQ ID No.12).
Using pELP-Hsp90 α as template, according to LA Taq archaeal dna polymerase specifications, with PCR amplification M167b cDNA pieces Section (Fig. 1) digests PCR product with restriction enzyme Sal I and Xho I, connects, recombinated with the pET-ELP carriers of identical digestion Carrier pELP-M167b.
(8) following 1 pair of primer is designed according to chicken Hsp90 α mRNA sequences, it is point that forward primer, which introduces Sal I digestions, reversely Primer introduces I restriction enzyme sites of Xho:
Forward primer:5'-ATGTCGACACAAGTACATATGGCTGGAC-3'(SEQ ID No.13);
Reverse primer:5'-CTCTCGAGTTAATCCACCTCCTCCATAC-3'(SEQ ID No.14).
Using pELP-Hsp90 α as template, according to LA Taq archaeal dna polymerase specifications, with PCR amplification C132cDNA segments (Fig. 1) digests PCR product with restriction enzyme Sal I and Xho I, is connected with the pET-ELP carriers of identical digestion, obtain recombination and carry Body pELP-C132.
2. expressing fusion protein and purifying
(1) respectively by above-mentioned recombinant vector pELP-Hsp90 α, pELP-N284, pELP-MC444, pELP-M167a, PELP-M167b and pELP-C132 converts BLR (DE3) Escherichia coli, is cultivated on kanamycins (50 μ g/mL) LB tablets Night;Picking single bacterium colony is inoculated with 5mL kanamycins LB, and 37 DEG C of shaking table cultures are stayed overnight, 4 DEG C, 8000g centrifugations 10min;Precipitate thalline with Isometric LB is resuspended, with 1:100 ratios are inoculated with 500mL 2 × YT of kanamycins culture solutions (Tryptone 16g, Yeast Extract 10g, Nacl 5g, 1L), 37 DEG C, 220rpm cultivates to OD600=0.8;Final concentration of 1mmol/L IPTG are added, 20 DEG C of induced expression 20h;4 DEG C, 4000g centrifugation 10min, precipitation thalline is resuspended with 50mL PBS, according to cell crushing instrument (Guangzhou Cumulative bio tech ltd) 2 cracking recombinant bacteriums (4 DEG C, 1300bar) of specification;13000rpm, 4 DEG C of centrifugation 20min, Supernatant is collected respectively and carries out electrophoretic analysis with precipitation, is as a result shown:Recombinant bacterium can express ELP-Hsp90a, ELP- of expected size N284, ELP-MC444, ELP-M167a, ELP-M167b and ELP-C132 fusion protein (Fig. 2).
(2) ELP fusion protein purifications bibliography (Kang HJ, Kim JH, Chang WJ.Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system.Microbiology and Biotechnology,2007,17(11):1751- 1757) it carries out.Cracking thalline is centrifuged into 20min in 4 DEG C, 16000g, collects supernatant;With spectrophotometric determination albumen concentration, Albumen concentration is adjusted to 10mg/mL with PBS;Isometric 4mol/L NaCl, 26 DEG C of incubation 10min is added;14000g is centrifuged 5min collects centrifugation;It is repeated 1 times phase transformation cycle under the same conditions, centrifugation is dissolved with PBS;Partial purification is taken to produce Object carries out 12%SDS-PAGE analyses, as a result shows:It is essentially single band by the fusion protein of two-wheeled phase transformation circulatory purification (Fig. 2).
3. the measurement of fusion protein combination IBDV
Different fusion proteins are diluted to 50 μ g/mL with PBS, 200 μ L is respectively taken to be mixed, 4 with 30 μ L IBDV (Lukert plants) DEG C be incubated 1h;Isometric 4mol/L NaCl are added, 26 DEG C of incubations 10min, 14000g centrifuge 5min;100 μ L PBS weights of precipitation It is outstanding, according to RNAisoTMPlus (TaKaRa companies) specification extracts RNA, according to RevertAidTM Reverse Transcriptase (Fermentas companies) operation instructions carry out reverse transcription.
According to IBDV VP2 gene orders (GenBank Accession No:KR028197 following 1 pair of primer) is designed:
Forward primer:5'-AGCTCGAAGTTGCTCACC-3'(SEQ ID No.15);
Reverse primer:5'-CAACAGCCAACATCAACG-3'(SEQ ID No.16).
PCR amplification is carried out by template and primer of above-mentioned reverse transcription product, is as a result shown:In ELP-Hsp90a, ELP- Expected 679bp IBDV VP2 genetic fragments can be amplified in the virus of MC444, ELP-M167a fusion protein precipitation, Corresponding genetic fragment (Fig. 3) cannot be amplified in other fusion protein precipitate virus.
4. fusion protein precipitates the condition optimizing of IBDV
(1) ELP-M167a fusion proteins are diluted to 10,20,40,80,160,320,640 μ g/mL with PBS (pH7.0), Respectively take 100 μ L and equivalent IBDV (10-6.5/0.1mL TCID50) mixing, 4 DEG C of incubation 1h;It is separately added into 200 μ L 4mol/L chlorinations Sodium, 26 DEG C of incubation 10min;Room temperature 14000g centrifuges 5min, and precipitation uses PBS centrifuge washings 2 times, with 30 μ L PBS dissolvings;With reference to Document (Wang Y, Sun H, Shen P, Zhang X, Xia X.Efficient inhibition of replication of infectious bursal disease virus by miRNAs delivered by vectors and targeting the VP2gene.Journal of Virological Methods,2010,165:127-132) carried out on DF-1 cells IBDV is titrated, if 3 holes repeat, is as a result shown:ELP-M167a fusion proteins precipitate the 160 μ g/mL of optimum concentration of IBDV, virus The rate of recovery is up to 82%.
(2) ELP-M167a fusion proteins are diluted to 320 μ g/mL with the PBS of different pH, respectively take 100 μ L and equivalent IBDV Mixing, 4 DEG C of incubation 1h;Such as preceding progress viral pellet and titration, as a result show:ELP-M167a fusion proteins precipitate IBDV most Suitable pH is 7.0, and viral recovery is up to 80%.
(3) ELP-M167a fusion proteins are diluted to 320 μ g/mL with the PBS of pH7.0, respectively take 100 μ L and equivalent IBDV Mixing is incubated 1h in different temperatures;Such as preceding progress viral pellet and titration, as a result show:ELP-M167a fusion proteins precipitate The optimum temperature of IBDV is 16 DEG C, and viral recovery is up to 82%.
(4) ELP-M167a fusion proteins are diluted to 320 μ g/mL with the PBS of pH7.0, respectively take 100 μ L and same volume IBDV is mixed, and different time is incubated at 16 DEG C;Such as preceding progress viral pellet and titration, as a result show:ELP-M167a fusion proteins The optimum time for precipitating IBDV is 1h, and viral recovery is up to 81% (Fig. 4).
(5) ELP-M167a fusion proteins are diluted to 320 μ g/mL with the PBS of pH7.0, respectively take 100 μ L and equivalent IBDV Infection cell culture supernatant or lytic cell mixing, 16 DEG C of incubation 1h;Such as preceding progress viral pellet and titration, as a result show:With The rate of recovery that ELP-M167a fusion proteins precipitate IBDV from infection cell culture supernatant and lytic cell distinguishes 75.4% He 71.7%.
(6) IBDV is diluted to 10 with 10mL tap water and PBS (pH7.0) respectively4TCID50, respectively take 100 μ L and equivalent 320 μ g/mL ELP-M167a fusion proteins mix, 16 DEG C of incubation 1h;Such as preceding progress viral pellet and titration, as a result show:With The rate of recovery difference 73% and 68% of ELP-M167a fusion proteins precipitate virus from two IBDV POLLUTION SIMULATION water samples.
5. the optimization of infectious bursal disease virus elution requirement
(1) with the IBDV of the PBS of different pH dissolving ELP-M167a fusion proteins precipitations, 30min is eluted at 4 DEG C, is then used ITC precipitates fusion protein, collects centrifugation supernatant and carries out titration of virus, as a result shows:The PBS of pH4.5 and 9.0 can be eluted effectively IBDV, viral recovery are respectively 46% and 47%.
(2) IBDV for using the PBS dissolving ELP-M167a fusion proteins precipitations of pH9.0, elutes 30min, so in different temperatures Afterwards such as preceding carry out titration of virus, as a result show:The optimum temperature for eluting IBDV is 22 DEG C, and viral recovery is up to 46%.
(3) IBDV for using the PBS dissolving ELP-M167a fusion proteins precipitations of pH9.0, different time is eluted at 22 DEG C, so Afterwards such as preceding carry out titration of virus, as a result show:The optimum time for eluting IBDV is 30min, and viral recovery is up to 47% (Fig. 5).
(4) it uses the PBS of pH9.0 to dissolve ELP-M167a fusion proteins to precipitate from infection cell culture supernatant and lytic cell IBDV, 30min is eluted at 22 DEG C, then such as preceding carry out titration of virus, is as a result shown:The rate of recovery of IBDV is respectively 39.3% With 42.5%.

Claims (4)

1. the infectious bursal disease virus binding fragment fusion protein of a type elastin polypeptide and 90 α of heat stress proteins ELP-M167a, it is characterised in that:It is made of the infectious bursal disease virus binding fragment M167a of ELP and Hsp90 α, it is described The amino acid sequence of M167a is as shown in SEQ ID No.1;The ELP is the ELP coded by said gene from pET/EI-GFP.
2. the infectious bursal disease virus bonding pad of class elastin polypeptide described in claim 1 and 90 α of heat stress proteins Section fusion protein ELP-M167a, preparation method are as follows:
(1) with the coded sequence SEQ ID No.2 of 167 amino acid M167a of PCR clone's Hsp90 α central domains;
(2) above-mentioned coded sequence is inserted into ELP fusion expression vectors, obtains recombinant vector pELP-M167a;Concrete operations are:
1) restriction enzyme NdeI and SacI is used to digest pET/EI-GFP, according to DNA gel QIAquick Gel Extraction Kit specification after electrophoretic separation ELP genetic fragments are recycled, is connect with the pET-30a carriers of same enzyme linearisation, obtains ELP fusion expression vectors pET-ELP;
2) the good chicken of growth conditions is cultivated 3 hours at fiber DF-1 cells at 42 DEG C, according to RNAisoTMPlus kits Specification extracts cell total rna, according to RevertAidTMReverse Transcriptase illustrate to carry out reverse transcription;
3) PCR product for using restriction enzyme Sal I and Xho I digestion steps (1), connects with the pET-ELP carriers of identical digestion, obtains Obtain recombinant vector pELP-M167a;
(3) recombinant vector is converted into Escherichia coli, in 20 DEG C of induced fusion protein expressions;
(4) fusion protein is precipitated at 26 DEG C with 2mol/L sodium chloride.
3. the infectious bursal disease virus bonding pad of class elastin polypeptide described in claim 1 and 90 α of heat stress proteins Section fusion protein ELP-M167a is concentrated and the application in purifying in infectious bursal disease virus.
4. the application described in claim 3, which is characterized in that the specific method is as follows:
(1) by 160 μ g/ml ELP-M167a fusion proteins as described in claim 1 respectively in IBDV infection cell cultures Clearly, lytic cell and simulated water sample mixing;
(2) use 2mol/L sodium chloride in 26 DEG C of precipitate virus;
(3) pH9.0 buffer solutions elution virus is used;
(4) removal ELP-M167a fusion proteins are centrifuged at 26 DEG C with 2mol/L sodium chloride.
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