CN105087607A - Method for prokaryotic expression of third structural domain of DTMUV (duck Tembusu virus) E protein as well as application of method - Google Patents

Abstract

The invention discloses a method for prokaryotic expression of the third structural domain of DTMUV (duck tembusu virus) E protein. The method comprises steps as follows: (1) design and synthesis of a specific primer; (2) clone sequencing of the third structural domain of the DTMUV E protein; (3) clone expression of the third structural domain of the DTMUV E protein; (4) expression and purification of a recombinant plasmid pET-28 alpha-EM of the third structural domain. The prepared and expressed DTMUV E protein has good antigenicity, and a raw material can be provided for establishment of an ELISA method, preparation of immunofluorescence, immunohistochemistry and monoclonal antibodies and the like.

Description

The method of duck tembusu virus E protein the 3rd structural domain prokaryotic expression and application thereof

Technical field:

The present invention relates to a kind of method and application thereof of duck tembusu virus E protein prokaryotic expression, belong to biological gene engineering field.

Background technology:

Duck source tembusu virus virus ( duckTembusuvirus, DTMUV) belong to flaviviridae Flavivirus member, cause the domestic main pathogen taking ovaritis as the aquatic bird of feature and newly send out communicable disease, this disease is popular extensively, propagation is rapid, since reported first in 2010, virus infects on the cultivation aquatic birds such as laying ducks, meat duck and goose, and causes the financial losses such as degradation of laying eggs.

DTMUV encoding viral 3 structural protein and 7 Nonstructural Proteins.Wherein membrane glycoprotein (envelopeprotein, be called for short E protein) be the major structural protein of tembusu virus, be positioned at mature virion surface, form the projection of virion, combine at receptor in target cell, film merges and plays keying action in Virus entry process.Possessing good immunogenicity and reactionogenicity, is the main protection antigen of host's anti-infectious immunity, is also the desirable target antigen detecting this special viral antibody.

Summary of the invention:

Technical problem to be solved by this invention is to provide a kind of method of duck tembusu virus E protein prokaryotic expression.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

The method of duck tembusu virus E protein the 3rd structural domain prokaryotic expression of the present invention, comprises the following steps:

(1) specific primers Design and synthesis:

DTMUV- bamHi-F (upstream primer) P1:

5 '- gGATCC cTAGTGAAGAATCCTACCG-3 ' boldface is what add bamHi restriction enzyme site;

DTMUV- ecoRi-R (downstream primer) P2:

5 '-AAGT gAATTC aCCCCCAACTGAGCC-3 ' boldface is what add ecoRi restriction enzyme site;

(2) cloning and sequencing of duck tembusu virus E protein the 3rd structural domain

(3) clonal expression of duck tembusu virus E protein the 3rd structural domain

(4) expression and purification of recombinant plasmid pET-28a-EM

The cloning and sequencing of the 3rd structural domain of described duck tembusu virus E protein is:

The explanation that the extraction of viral RNA extracts test kit according to virus total RNA operates, and RT-PCR reaction system is according to PrimeScript ^?oneStepRT-PCRKit illustrates and operates, response procedures: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 30 circulations; 72 DEG C of 10min.After reaction terminates, 0.8% agarose gel electrophoresis detects electrophoretic band, and glue reclaims test kit and reclaims purified pcr product.The PCR primer of recovery is sent to the precious biotechnology company limited in Dalian and carries out order-checking qualification.

The clonal expression of described duck tembusu virus E protein the 3rd structural domain is:

With DTMUV-QD strain virus RNA for template, the Auele Specific Primer of E gene the 3rd structural domain carries out RT-PCR amplification, connects to transform to operate according to " Molecular Cloning: A Laboratory guide ", builds recombinant expression plasmid pET-28a-EM, and passes through bamHi He ecoRi carries out double digestion qualification and sequential analysis.

The expression and purification of the 3rd described structural domain recombinant plasmid pET-28a-EM is:

Recombinant plasmid pET-28a-EM is converted into e. coli bl21 (Rosetta) competent cell, 37 DEG C of incubated overnight, select in single bacterium colony to 2 × YT substratum, 37 DEG C of concussion overnight incubation, undertaken in transferred species 2 × YT substratum by 1:100,37 DEG C of joltings are cultivated, and carry out abduction delivering with the IPTG that final concentration is 0.1mM.

After centrifugal for expression bacterium, add the resuspended thalline of PBS, the cracking of ultrasonic wave ice bath, after centrifugal treating, carry out SDS-PAGE qualification, by Ni-NTA affinity column purification of Recombinant E protein.

The duck tembusu virus E protein expressed in method of the present invention can be used for detecting duck tembusu virus and preparation duck tembusu virus antibody.

Beneficial effect of the present invention:

Tembusu virus is the novel flavivirus caused a disease to aquatic bird at China's Late Cambrian, propagate rapidly, to the harm of duck goose greatly, cause huge financial loss to waterfowls in China industry, because tembusu virus is short at Chinese explosion time, thus this virus pathogenesis, also there is a difficult problem much in the urgent need to address across in species mechanism of transmission and vaccine development.E protein is the envelope protein of flavivirus, also be main structural protein, result of study shows simultaneously, and E protein is the major target class of external neutralizing effect and the action site of special viral antibody, there is Neutralization effect, body can be stimulated to produce antibody and cause specific immunity.Carry out X-ray Crystal study to E protein to show: the 3rd structural domain possesses immunoglobulin like protein spline structure territory, comprises viral bind receptor.Possess the region of composition major linear epitopes, also comprise the recognition site of viral neutralization monoclonal antibody.

PET-28a-EM recombinant plasmid constructed by the present invention can give expression to through IPTG induction E gene the 3rd structural domain recombinant protein that molecular weight is about 20KD, and Westernblotting analyzes this fusion rotein and DTMUV positive serum and reacts and be positive.Possess good antigenicity, can be the preparation etc. of setting up ELISA method, immunofluorescence, immunohistochemical methods and monoclonal antibody and starting material are provided.

Accompanying drawing illustrates:

Fig. 1 is the CDD analytical results of DTMUVE protein amino acid sequence.

Fig. 2 is the antigenic index analysis of DTMUVE albumen.

Fig. 3 is the qualification of pet28a-EM recombinant plasmid double digestion.M1:λ- HindⅢDNAMarker；M2:DL2000；1：pET28a-EM/ BamHⅠ+ EcoRⅠ；2：DTMUV-Erecombinantplasmid。

Fig. 4 is the SDS-PAGE qualification of expressing protein.M:ProteinMWmarker (Broad); 1: the precipitation not adding IPTG induction; 2: do not add the upper of IPTG induction; 3: the whole protein not adding IPTG induction; The precipitation of 4:IPTG induction; The supernatant of 5:IPTG induction; The whole protein of 6:IPTG induction.

Fig. 5 is that the SDS-PAGE of restructuring 6 protein purification products analyzes.M. protein molecular weight; The bacterium liquid of 1.IPTG induction; 2.Ni-NTA affinity column elution peak (the restructuring EM albumen containing purifying).

Fig. 6 is the SDS-PAGE qualification of expressing protein.M：Protionmarker；1：pET-28-EinducedbyIPTG；2：pET-28-EuninducedbyIPTG；3：ThewesternblottingoftheproteinsinducedbyIPTG；4：ThewesternblottingoftheproteinsuninducedbyIPTG。

Embodiment:

the prokaryotic expression of embodiment 1 duck tembusu virus E gene the 3rd structural domain

1 materials and methods

1.1 strains, bacterial strain and plasmid

DTMUVQD strain is separated by this laboratory and preserves.E.coliDH5 α bacterial strain, Rosetta (DE3) bacterial strain and prokaryotic expression carrier pET28a(+) to be prepared by this room and to preserve; BKH cell, myeloma cell SP2/0 are preserved by this laboratory;

1.2 reagent

M-MLV ThermoScript II, RazoalRNA extract test kit purchased from Promega company; ExTaqDNA amplification enzyme, IPTG, DNAmarker and restriction enzyme bamHi He ecoRi all purchased from the precious biotechnology company limited in Dalian; DNA fragmentation gel reclaims test kit and plasmid extraction kit fast purchased from Tiangen company; 6 × His Protein Purification Resin Ni-NTA is purchased from QIAGEN company; Goat-anti duck HRP-IgG, sheep anti mouse HRP-IgG are purchased from Jackson company; The available from Sigma such as sheep anti mouse FITC-IgG, PEG4000, HAT; BALB/c is purchased from Shandong University's Experimental Animal Center.Many anti-the separation by this laboratory of rehabilitation duck source DTMUV of natural infection are preserved.

primary biological bioinformatics analysis instrument

Application NCBIConservedDomains lookup tool analysis of amino acid sequence conserved domain, application DNAStarProtean software module carries out antigenic index prediction, the possibility of protein surface is positioned at Emini principle prediction specific region, the antigenic index of the Jameson-Wolf method integrated forecasting albumen of application wetting ability, surface property, snappiness and secondary structure 4 kinds of parametric joints, and according to the antigenic index result of each structural domain choose Main Antigenic Region ( mainantigenicdomains, MAD) called after E-M.

the Design and synthesis of primer

According to DTMUVQD pnca gene sequence (GenBank accession number HQ833330.1), design the Auele Specific Primer of a pair E gene the 3rd structural domain, pre-expanding fragment length 345bp, by the precious biosynthesizing in Dalian.

DTMUV- bamHi-F (upstream primer) P1:

5 '- gGATCC cTAGTGAAGAATCCTACCG-3 ' boldface is what add bamHi restriction enzyme site

DTMUV- ecoRi-R (downstream primer) P2:5 '-

AAGT gAATTC aCCCCCAACTGAGCC-3 ' boldface is what add ecoRi restriction enzyme site

the cloning and sequencing of the 3rd structural domain major antigen section of 1.5DTMUV membrane glycoprotein

The explanation that the extraction of viral RNA extracts test kit according to virus total RNA operates, and RT-PCR reaction system is according to PrimeScript ^?oneStepRT-PCRKit illustrates and operates, response procedures: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 30 circulations; 72 DEG C of 10min.After reaction terminates, 0.8% agarose gel electrophoresis detects electrophoretic band, and glue reclaims test kit and reclaims purified pcr product.The PCR primer of recovery is sent to Shanghai Sheng Gong biotechnology company limited and carries out order-checking qualification.

the clonal expression of 1.6DTMUV membrane glycoprotein E gene

With DTMUV-BZ strain virus RNA for template, the Auele Specific Primer of E gene the 3rd structural domain carries out RT-PCR amplification, connects to transform to operate according to " Molecular Cloning: A Laboratory guide ", builds recombinant expression plasmid, called after pET-28a-EM, and passes through bamHi He ecoRi carries out double digestion qualification and sequential analysis.

the expression and purification of recombinant plasmid pET-28a-EM

Recombinant plasmid pET-28a-EM is converted into e. coli bl21 (Rosetta) competent cell, 37 DEG C of incubated overnight, select in single bacterium colony to 2 × YT substratum, 37 DEG C of concussion overnight incubation, undertaken in transferred species 2 × YT substratum by 1:100,37 DEG C of joltings are cultivated, and carry out abduction delivering with the IPTG that final concentration is 0.1mM.

After centrifugal for expression bacterium, add the resuspended thalline of PBS, the cracking of ultrasonic wave ice bath, after centrifugal treating, carry out SDS-PAGE qualification, by Ni-NTA affinity column purification of Recombinant E protein.

result

2.1DTMUVQD strain E gene primary structure domain analysis and Epitope prediction

The analysis of NCBIConservedDomains lookup tool finds that this gene amino acid sequence contains 3 conserved domains, possesses similar function (Fig. 1) to other flavivirus capsid protein.The display of X-ray Crystal study is carried out to E protein: the 1st structural domain is positioned at active site of protein and is rich in glycosylation site.What its side was connected is the 2nd structural domain, comprises fusogenic peptide section.3rd structural domain is positioned at the opposite side of the 1st structural domain, possesses immunoglobulin like protein spline structure territory, comprises viral bind receptor.In addition the 3rd structural domain is also considered to the region possessing composition major linear epitopes, possesses the recognition site of viral neutralization monoclonal antibody.

Jameson-Wolf method is adopted to predict the outcome display, the antigenic index of the 3rd structural region of this strain E protein is all higher, although these regions are distributed in hydrophilic region and plasticity-region more, it is generally acknowledged that epitope also must be present in solvent accessibility region.Therefore the antigenic index of the section such as Pro315-Thr322, Ala330-Ile340, Asp345-ILe356, Glu376-Phe383, Gly387-Phe408 is higher, also may be the dominant area (> 0, Fig. 2) of B cell epi-position.

Comprehensive above-mentioned predictive analysis results, chooses and contains the stretches of amino acids that Pro315-Gly429 contains the 3rd structural domain of Dominant Epitopes and carry out cloning, expressing.Nucleotide sequence is the 943-1287bp section of E gene, called after EM, altogether 345bp.

the qualification of recombinant plasmid pET-28a-EM, Expression and purification

Utilize RT-PCR method to increase and comprise the cDNA fragment of the E gene Main Antigenic Region of DTMUVQD strain, PCR primer is cloned into pMD-18T, obtain recombinant vectors (pMD-18T-EM).Further order-checking qualification, proves that the E gene fragment of cloning conforms to expanded sequence.By object fragment EM subclone to pET28a's bamHi He ecoRi site, obtains recombinant expression vector pET28a-EM.There are two bands through double digestion qualification result in recombinant plasmid, size conforms to expection (Fig. 3).

expression of recombinant plasmid

E.coliRosetta (DE3) genetic engineering bacterium containing RT-PCR expression plasmid has been carried out IPTG abduction delivering.And respectively SDS-PAGE is carried out to expression bacterium supernatant, precipitation and whole protein.Electrophoresis detection result is presented at 20ku a specific band (containing 6 × His label protein), consistent with expected results, the PET28a-E recorded with thin layer chromatography scanner merges expression of recombinant proteins amount and accounts for 38.4% of bacterial protein, shows that goal gene obtains high expression with the form of fusion rotein in colon bacillus.Empty vector control and do not induce bacterium then without this protein band (Fig. 4).And precipitation capacity is apparently higher than supernatant, is indicated as inclusion body and expresses.

recombinant protein purification

Expression product after a series of pre-treatments such as lysozyme lysis, ultrasonic disruption, urea washes, nickel sepharose affinitive layer purification restructuring EM albumen.Collect imidazoles elution peak simultaneously, carry out SDS-PAGE electrophoresis, inspection purity and concentration, result shows: containing a large amount of highly purified restructuring EM albumen (Fig. 5) in Ni-NTA affinity column elution peak.After the recombinant protein dialysis renaturation of purifying, packing is for subsequent use.

recombinant protein

Westernblotting detected result shows, and occurs a brown reaction zone, show that His-EM can by duck natural immunity DTMUV polyclonal serum identification (Fig. 6) at object band place (20kd).

Nucleotides sequence list

<110> Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine

The method of <120> duck tembusu virus E protein the 3rd structural domain prokaryotic expression and application thereof

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<170>PatentInversion3.5

<210>1

<211>25

<212>DNA

<213> artificial sequence

<220>

<221>primer_bind

<222>(1)..(25)

<223> is for the upstream primer of the duck tembusu virus E protein that increases

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GGATCC CTAGTGAAGAATCCTACCG25

<210>2

<211>25

<212>DNA

<213> artificial sequence

<220>

<221>primer_bind

<222>(1)..(25)

<223> is for the downstream primer of the duck tembusu virus E protein that increases

<400>2

AAGT GAATTC ACCCCCAACTGAGCC25

Claims (6)

1. the method for duck tembusu virus E protein the 3rd structural domain prokaryotic expression, is characterized in that, comprise the following steps:

(1) specific primers Design and synthesis:

DTMUV- bamHi-F (upstream primer) P1:

5 '- gGATCC cTAGTGAAGAATCCTACCG-3 ' boldface is what add bamHi restriction enzyme site;

DTMUV- ecoRi-R (downstream primer) P2:

5 '-AAGT gAATTC aCCCCCAACTGAGCC-3 ' boldface is what add ecoRi restriction enzyme site;

(2) cloning and sequencing of duck tembusu virus E protein the 3rd structural domain;

(3) clonal expression of duck tembusu virus E protein the 3rd structural domain;

The expression and purification of (4) the 3rd structural domain recombinant plasmid pET-28a-EM.

2. the method for duck tembusu virus E protein prokaryotic expression according to claim 1, is characterized in that,

The cloning and sequencing of described duck tembusu virus E protein the 3rd structural domain is:

The explanation that the extraction of viral RNA extracts test kit according to virus total RNA operates, and RT-PCR reaction system is according to PrimeScript ^?oneStepRT-PCRKit illustrates and operates, response procedures: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 30 circulations; 72 DEG C of 10min;

After reaction terminates, 0.8% agarose gel electrophoresis detects electrophoretic band, and glue reclaims test kit and reclaims purified pcr product;

The PCR primer of recovery is sent to the precious biotechnology company limited in Dalian and carries out order-checking qualification.

3. the method for duck tembusu virus E protein the 3rd structural domain prokaryotic expression according to claim 1, is characterized in that,

The clonal expression of described duck tembusu virus E protein is:

With DTMUV-QD strain virus RNA for template, the Auele Specific Primer of E gene carries out RT-PCR amplification, connects to transform to operate according to " Molecular Cloning: A Laboratory guide ", builds the 3rd structural domain recombinant expression plasmid, called after pET-28a-EM, and passes through bamHi He ecoRi carries out double digestion qualification and sequential analysis.

4. the method for duck tembusu virus E protein prokaryotic expression according to claim 1, is characterized in that

The expression and purification of described recombinant plasmid pET-28a-EM is:

Recombinant plasmid pET-28a-EM is converted into e. coli bl21 (Rosetta) competent cell, 37 DEG C of incubated overnight, select in single bacterium colony to 2 × YT substratum, 37 DEG C of concussion overnight incubation, undertaken in transferred species 2 × YT substratum by 1:100,37 DEG C of joltings are cultivated, and carry out abduction delivering with the IPTG that final concentration is 0.1mM;

After centrifugal for expression bacterium, add the resuspended thalline of PBS, the cracking of ultrasonic wave ice bath, after centrifugal treating, carry out SDS-PAGE qualification, by Ni-NTA affinity column purification of Recombinant E protein.

5. the duck tembusu virus E protein described in above-mentioned any one claim is detecting the application on duck tembusu virus.

6. the application of the duck tembusu virus E protein described in above-mentioned any one claim on preparation duck tembusu virus antibody.