CN100348726C - H9N2 type poultry influenze NS1 protein expression plasmid pET-NS1 - Google Patents
H9N2 type poultry influenze NS1 protein expression plasmid pET-NS1 Download PDFInfo
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Abstract
The present invention relates to an H9N2 type avian influenza NS1 protein expression plasmid pET-NS1 which is an expression plasmid obtained by carrying out enzyme cut and connection to pET-32a(+) and an avian influenza NS1 gene containing a restriction enzyme cutting site after PCR amplification. The expression plasmid pET-NS1 is transformed into a colibacillus BL21 seed and then is inducted by IPTG to express a nucleotide sequence in the protein translating region of the H9N2 type avian influenza NS1 protein expression plasmid pET-NS1, and the sequence contains the nucleotide sequence of the H9N2 type avian influenza virus NS1 protein; moreover, an amino acid sequence in the protein translating region of the avian influenza NS1 protein expression plasmid pET-NS1 can be also expressed, and the sequence contains the amino acid sequence of the H9N2 type avian influenza NS1 protein. The present invention has the advantages of wide applicable temperature range of inducing expression, convenient identification and purification of samples and no destruction of protein structure, overcomes the disadvantage of easy degradation of purified protein and can be stored for a long time at-40 DEG C. The expressed NS1 protein can be also used for the detection of the wild virus infection of avian influenza viruses.
Description
Technical field
What the present invention relates to is a kind of protein expressing plasmid of technical field of biological genetic engineering, particularly a kind of H9N2 type bird flu NS1 protein expressing plasmid pET-NS1.
Background technology
Bird flu be OIE regulation a kind of category-A transmissible disease.Its virulence is strong, propagates rapidly, brings great loss to Production of Livestock and Poultry.Avian influenza virus belongs to orthomyxoviridae family, and influenza A virus belongs to.The genome of influenza A virus is the sub-thread strand RNA, contains 8 sections.Wherein the 8th sections is a nonstructural protein gene, coding non-structural protein NS 1 and NS2.Shearing and the translation of NS1 control mRNA play an important role to replying of Interferon, rabbit in virus infection.NS1 albumen can also be used for the detection of avian influenza.Influenza A virus is divided into 15 H types and 9 N types according to hemagglutinin (H) and neuraminidase (N) again.
In the analysis to the prior art document, existing report (Sun Ming, Zhao Tiezhu, Wang Chuanbin etc. to bird flu NS1 protein expression.The expression [J] of bird flue virus H 5 N 1 subtype NS1 gene in intestinal bacteria.Chinese experimental animal journal .2004.12 (4): be that to express NS1 proteic by the reorganization of H5N1 type avian influenza virus NS1 gene and pGEX-4T-1 expression vector being obtained expression plasmid 208-211).GST label of this fusion rotein band can only carry out purifying with commercial resin affinity chromatography, and our experiments show that when purifying, very easily degraded.The fusion rotein of expressing with expression plasmid pET-NS1 has histidine-tagged, can within 40 minutes, obtain purifying protein with commercial MagExtractor-His-tag-kit (TOYOBO company), and the albumen of this purifying can be preserved more than three months in-40 ℃ of refrigerators.The fusion rotein that the pET-NS1 expression plasmid is expressed also all can be induced in 25-30 ℃ of temperature range, and does not produce inclusion body.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of H9N2 type avian influenza virus NS1 protein expressing plasmid pET-NS1 is provided.Make the Applicable temperature scope of its abduction delivering wide, all can express for 25-30 ℃; In the expression process, can not produce inclusion body; Fusion rotein has histidine-tagged, has made things convenient for the evaluation and the purifying of sample, has more avoided the sex change and the renaturation of sample, and protein structure is not damaged; The NS1 albumen that gives expression to has overcome the shortcoming that purifying protein is degraded easily in the prokaryotic expression, can be-40 ℃ of long-time preservations; Expressed NS1 albumen also can be used for the detection of avian influenza virus wild virus infection.
The present invention is achieved by the following technical solutions, and pET-NS1 expression plasmid provided by the invention is by pET-32a (+) and the bird flu NS1 gene enzyme that contains restriction enzyme site behind pcr amplification being cut, connecting the expression plasmid of acquisition; Among the pET-NS1 expression plasmid transformed into escherichia coli BL21, induce with IPTG, can give expression to the protein translation region nucleotide sequence of H9N2 type bird flu NS1 protein expressing plasmid pET-NS1, in this section sequence, comprise the proteic nucleotide sequence of H9N2 type avian influenza virus NS1; Also can give expression to the protein translation region amino acid sequence of bird flu NS1 protein expressing plasmid pET-NS1, this section sequence contains the proteic aminoacid sequence of H9N2 type bird flu NS1.
The protein translation region nucleotide sequence of described bird flu NS1 protein expressing plasmid pET-NS1, be meant: the nucleotide sequence of coding H9N2 type avian influenza virus NS1 fusion rotein, Trx Tag, the His Tag, S Tag and a series of restriction enzyme site that promptly comprise on pET-32a (+) the carrier sequence add H9N2 type bird flu NS1 protein 69 3bp Nucleotide at interior 504bp Nucleotide, amount to 1197bp.
The protein translation region nucleotide sequence of described bird flu NS1 protein expressing plasmid pET-NS1 is the nucleotide sequence among the SEQ IDNO.1, amounts to 1197bp.It comprises the nucleotide sequence of coding H9N2 type bird flu NS1 gene, and promptly SEQ ID NO.1 505-1197 position Nucleotide amounts to 693bp.It is the aminoacid sequence among the SEQ ID NO.1, about molecular weight 44.2KD.It comprises the proteic aminoacid sequence of H9N2 type bird flu NS1, i.e. the aminoacid sequence of the 169-398 position among the SEQ ID NO.1, molecular weight 26.0KD.
The nucleotide sequence of described coding H9N2 type bird flu NS1 gene is meant the open reading frame of the proteic 693bp of containing of coding H9N2 type avian influenza virus NS1.
The protein translation region amino acid sequence of described bird flu NS1 protein expressing plasmid pET-NS1, be meant: the aminoacid sequence of coding H9N2 type avian influenza virus NS1 fusion rotein, Trx Tag, the His Tag, S Tag and a series of restriction enzyme site that promptly comprise on pET-32a (+) the carrier sequence add 0 amino acid of H9N2 type bird flu NS1 protein 23 at 168 interior amino acid, and molecular weight is 44.2KD.
The aminoacid sequence of described coding H9N2 type avian influenza virus NS1 fusion rotein is meant: proteic 230 aminoacid sequences of H9N2 type avian influenza virus NS1.
The present invention also provides a kind of usefulness above-mentioned carrier transformed host cells, the e. coli bl21 cell.
Described host cell is the dna molecular transformed host cells, is the e. coli bl21 cell.
The present invention extracts genome from spissated H9N2 type avian influenza virus, carry out the sleeve type PCR amplification then, i.e. pcr amplification is taken turns in first round RT-PCR amplification and second, and first round RT-PCR amplified production is connected to order-checking evaluation in the pMD-18T carrier.Cut second with EcoR I and Xho I enzyme and take turns PCR product and pET-32a (+) carrier, connect with the T4 dna ligase.The positive recombinant plasmid of identifying is the pET-NS1 expression plasmid.
Among the pET-NS1 expression plasmid transformed into escherichia coli BL21, induce, can give expression to H9N2 type avian influenza virus NS1 albumen with IPTG.This albumen further identifies it is correct H9N2 type avian influenza virus NS1 albumen through SDS-PAGE electrophoresis and Western blot.
This expressed proteins has histidine-tagged, can carry out purifying with MagExtractor-His-tag-kit (TOYOBO company), can finish within 40 minutes.
The nucleotide sequence in bird flu NS1 protein expressing plasmid pET-NS1 protein translation district and aminoacid sequence are at http: the blast homology highest serial comparative result on the ∥ www.ncbi.nlm.nih.gov website sees Table one and table two.
Table one is listed in http for the nucleotides sequence of H9N2 type bird flu NS1 gene: the blast homology highest serial comparative result on the ∥ www.ncbi.nlm.nih.gov website.
98％?identity?in?689nt?overlap
Query：509?attccaacactatgtcaagctttcaggtagactgctttctttggcatgtccgcaaacgat?568
Sbjct：5 attccaacactgtgtcaagctttcaggtagactgctttctttggcatgtccgcaaacgat?64
Query：569?ttgcagaccaagaactgggtgatgccccattccttgaccagcttcgccgagatcagaagt?628
Sbjct：65 ttgcagaccaagaactgggtgatgccccattccttgaccggcttcgccgagatcagaagt?124
Query：629?ccctaagaggaagaggcagcactcttggtctggacatcgaaacagctactcgtgcgggaa?688
Sbjct：125?ccctaagaggaagaggcagcactcttggtctggacatcgaaacagctactcgtgcgggaa?184
Query：689?agcagatagtggagcggattctggaggaagagtctgatgaggcacttaaaatgactattg?748
Sbjct：185?agcagatagtggagcggattctggaggaagagtctgatgaggcacttaaaatgactattg?244
Query：749?cttctgtgccggcttcacgctacctaactgacatgactcttgaagaaatgtcaagggact?808
Sbjct：245?cttctgtgccggcttcacgctacctaactgacatgactcttgaagaaatgtcaagggact?304
Query：809?ggttcatgctcatgcccaagcagaaagtggcaggttccctttgcatcaaaatggaccagg?868
Sbjct：305?ggttcatgctcatgcccaagcagaaagtggcaggctccctttgcatcaaaatggaccagg?364
Query：?869 caataatggataaaaacatcatattgaaagcaaatttcagtgtgatttttggccggttag 928
Sbjct：?365 caataatggataaaaacatcatattgaaggcaaacttcagtgtgatttttgaccggttgg 424
Query：?929 aaaccctaatactacttagagccttcacagaagaaggagcaatcgtgggagaaatctcac 988
Sbjct：?425 aaaccctaatactacttagagctttcacagaagaaggagcaatcgtgggagaaatctcac 484
Query：?989 cattaccttcccttccaggacatactgatgaggatgtcaaaaatgcaattggggtcctca?1048
Sbjct：?485 cgttaccttctcttccaggacatactgatgaggatgtcaaaaatgcaattggggtcctca 544
Query：?1049?tcggaggatttgaatggaatgataacacagttcgagtctctaaaactctacagagattcg?1108
Sbjct：?545 tcggaggacttgaatggaatgataacacagttcgagtctctgaaactctacagagattcg 604
Query：?1109?cttggagaagcagtaatgagaatgggagacctccactccctccaaagcagaaacggaaaa?1168
Sbjct：?605 cttggagaagcagtaatgaggatgggagacctccactccctccaaagcagaaacggaaaa 664
Query：?1169?tggcgagaacaattgagtcagaagtttga 1197
Sbjct：665?tggcgagaacaattgagtcagaagtttga 693
Query: the partial nucleotide sequence of coding H9N2 type bird flu NS1 gene
Sbjct: influenza A virus (A/aquatic bird/Hong Kong/399/99 (H3N8)) non-structural protein NS 1 gene (INA427300)
Table two is that the proteic aminoacid sequence of H9N2 type bird flu NS1 exists
Http: the blast homology highest serial comparative result on the ∥ www.ncbi.nlm.nih.gov website.
Query：169MNSNTMSSFQVDCFLWHVRKRFADQELGDAPFLDQLRRDQKSLRGRGSTLGLDIETATRA 228
M+SNT+SSFQVDCFLWHVRKRFADQELGDAPFLD+LRRDQKSLRGRGSTLGLDIETATRA
Sbjct：?1?MDSNTVSSFQVDCFLWHVRKRFADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRA 60
Query：?229?GKQIVERILEEESDEALKMTIASVPASRYLTDMTLEEMSRDWFMLMPKQKVAGSLCIKMD?288
GKQIVERILEEESDEALKMTIASVPASRYLTDMTLEEMSRDWFMLMPKQKVAGSLCI+MD
Sbjct：?61?GKQIVERILEEESDEALKMTIASVPASRYLTDMTLEEMSRDWFMLMPKQKVAGSLCIRMD 120
Query：?289?QAIMDKNIILKANFSVIFGRLETLILLRAFTEEGAIVGEISPLPSLPGHTDEDVKNAIGV 348
QAIMDKNIILKANFSVIFGRLETLILLRAFTEEGAIVGEISPLPSLPGHTDEDVKNAIGV
Sbjct：?121?QAIMDKNIILKANFSVIFGRLETLILLRAFTEEGAIVGEISPLPSLPGHTDEDVKNAIGV 180
Query：?349?LIGGFEWNDNTVRVSKTLQRFAWRSSNENGRPPLPPKQKRKMARTIESEV?398
LIGG?EWNDNTVRVS+TLQRFAWRSSNE+GRPPLPPKQKRBARTIESEV
Sbjct：?181LIGGLEWNDNTVRVSETLQRFAWRSSNEDGRPPLPPKQKRKMARTIESEV?230
The proteic aminoacid sequence of Query:H9N2 type bird flu NS1
Sbjct: first type bird flu (A/mallard/Alberta/211/98 (H1N1)) Nonstructural Protein (AAT65469.1)
H9N2 type bird flu NS1 protein expressing plasmid pET-NS1 provided by the invention, the Applicable temperature scope of abduction delivering is wide, all can express for 25-30 ℃.PET-NS1 plasmid provided by the invention can not produce inclusion body in the expression process; Fusion rotein has histidine-tagged, has made things convenient for the evaluation and the purifying of sample, has more avoided the sex change and the renaturation of sample, and protein structure is not damaged.The NS1 albumen that gives expression to has overcome the shortcoming that purifying protein is degraded easily in the prokaryotic expression, can be-40 ℃ of long-time preservations.Utilize the proteic expression plasmid pET-NS1 of H9N2 type avian influenza virus NS1 of the present invention can give expression to stable H9N2 type avian influenza virus NS1.Use the NS1 albumen of expressing and further to study this proteic function.The NS1 albumen that gives expression to can also be introduced chemical mutation, further studies the influence of amino acid whose change to the avian influenza effect.In addition, NS1 albumen and antibody thereof can be used as a vital signs of virus infection body, and expressed NS1 albumen also can be used for the detection of avian influenza virus wild virus infection.
Description of drawings
Fig. 1 obtains expression plasmid pET-NS1 technological line schematic block diagram
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following fact Example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of coding H9N2 type bird flu NS1 gene
1. virus concentrates
Get the centrifugal 50min of chick embryo allantoic liquid 20ml 8000g that contains avian influenza virus, collect the centrifugal 3h of supernatant liquor 110000g, precipitation is dissolved with 1ml STE, and this promptly is spissated chick embryo allantoic liquid.
2. genomic extraction
With spissated chick embryo allantoic liquid 150 μ l, add 850 μ l Trizol, place 5min under the room temperature.
Add 200 μ l chloroforms, use forced oscillation, make it abundant reaction, hatch 2-3min under the room temperature.
12000g, 4 ℃ of centrifugal 15min.
Get supernatant and transfer in the clean centrifuge tube, add Virahol 500 μ l, the concussion mixing is hatched 10min under the room temperature.
12900g, 4 ℃ of centrifugal 15min.
Abandon supernatant, add 75% alcohol 1mL.
12000g, 4 ℃ of centrifugal 2min.
Wash once with alcohol again.
Sop up the alcohol drying, note not with the whole evaporates to dryness of alcohol.
Add 13 μ l DEPC water dissolution.
3. first round RT-PCR amplification
Carry out the RT-PCR amplification according to bird flu NS1 gene conserved sequence design primer.The product fragment is more than the 800bp.Amplified production carries out glue and reclaims.
4. be connected on the pMD-18 T carrier and check order
Amplified production and pMD-18 T carrier (Takara company) are according to 1: 2-10 is connected.To connect product 2 μ l is transformed in the bacillus coli DH 5 alpha competent cell.Good DH5 α cell inoculation solid ammonia benzyl LB substratum, 37 ℃ of incubated overnight will be transformed.The colony inoculation ammonia liquor benzyl LB substratum of the white that next day, picking grew, 37 ℃ of incubated overnight.Extract plasmid, with restriction enzyme Pst I and 37 ℃ of 2h of EcoR I, enzyme is cut evaluation.To contain the recombinant plasmid order-checking of NSl gene.Sequencing result is to http: with the blast search, know that its sequence and the proteic sequence homology of other bird flu NS1 are very high, so tentatively confirm as bird flu NS1 albumen coded sequence on the ∥ www.ncbi.nlm.nih.gov website.
5. second take turns pcr amplification
Comprise the primer that EcoR I restriction enzyme site and downstream comprise Xho I restriction enzyme site according to sequencing result design upstream, carry out pcr amplification.
Embodiment 2
The structure of H9N2 type bird flu NS1 protein expressing plasmid pET-NS1
1.EcoRI and the XhoI enzyme is cut PCR product and pET-32a (+) carrier
With restriction enzyme EcoR I and Xho I respectively enzyme cut PCR product and pET-32a (+).Behind 37 ℃ of effect 2h, enzyme is cut product and is reclaimed with DNA Fragment Purification Kit (Takara company).
2.T4 the connection of dna ligase
PCR reclaims product and the carrier recovery product presses 1: 3-10 mixes, 10-16 ℃ of connection of spending the night of T4 dna ligase (Takara company).Connect product and change in the bacillus coli DH 5 alpha, 37 ℃ of incubated overnight are extracted plasmid.Cut evaluation with EcoR I and Xho I enzyme.The positive recombinant plasmid order-checking of identifying.Sequencing result is to http: with the blast search, know that its sequence and other bird flu NS1 albumen coded sequence homology are very high, so tentatively confirm as bird flu NS1 albumen coded sequence on the ∥ www.ncbi.nlm.nih.gov website.
Embodiment 3
The proteic expression of H9N2 type avian influenza virus NS1
1. transformed into escherichia coli BL21 kind and picking positive colony
Positive colony upgrading grain changes in the e. coli bl21,1 colony inoculation 2ml of picking Amp LB substratum incubated overnight.Get 200 μ l incubated overnight bacterium liquid inoculation 20ml Amp LB substratum, 37 ℃ 150 rev/mins are shaken bacterium 2h, to OD
600Be 0.4-1.
2.IPTG abduction delivering
Suo Shake bacterium colony add IPTG to final concentration be 1mM, 25 ℃-30 ℃ shake bacterium 4h after, collect bacterium liquid.
Embodiment 4
The proteic evaluation of H9N2 type avian influenza virus NS1
1 SDS-PAGE electrophoresis
The separation gel of preparation 14% and 5% concentrated glue.Bacterium liquid boils 4min with sample-loading buffer boiling water.Concentrate glue voltage 80V, separation gel voltage 136V.Electrophoresis 4-5h.
Coomassie brilliant blue dyeing 2h.
The destainer decolouring.
Observe, have tangible band to occur at the 44KD place.
2 Western Blot identify
When the SDS-PAGE electrophoresis finishes,, wipe dried then with paper with distilled water drip washing graphite cake.
Cut 6 Whatman 3MM filter paper and a nitrocellulose filter.Nitrocellulose filter is immersed on the water surface of deionized water, filter paper is immersed in the transfering buffering liquid, balance 5min.
From the anode to the negative electrode, put correctly by the order of paper, film, glue, paper, according to 0.65mA/cm
2Energising transfer printing 5h.
Nitrocellulose filter is placed in the plastics bag of sealing, in add confining liquid and bird flu positive serum.(the bird flu positive serum is dilution in 1: 100).4 ℃ of night incubation change 37 ℃ again over to and hatch 2h.
Take out nitrocellulose filter PBS rinsing three times, each 10min.
Filter membrane is used TBST rinsing 10min again.
Nitrocellulose filter is placed in the plastics bag of sealing, in add the anti-chicken IgG two of rabbit anti-(Bethyl laboratories.Inc) of confining liquid and 1: 1000 dilution.Hatch 2h for 37 ℃.
Nitrocellulose filter is put among the TBST rinsing 3 times, each 1h.
Add substrate colour developing 2-3min.Observations has black stripe to occur at the 44KD place.
Embodiment 5
Utilize MagExtractor-His-tag-kit (TOYOBO company) purified fusion protein
1, bacterial cell disruption
The thalline multigelation of expressing adds 10mg/mL N,O-Diacetylmuramidase and 10U/ μ l DNase I effect 30min.The centrifugal 1min of 12000rpm.
2, purification process
Bacterial cell disruption liquid supernatant adds 40 μ l magnetic beads and at room temperature stirs 10-30min with the micro test tube agitator.The centrifugal supernatant that removes.Add 500 μ l absorption ablution, stir 10sec with eddy mixer, the centrifugal supernatant that removes.Add 500 μ l absorption ablution again, stir 10sec with eddy mixer, the centrifugal supernatant that removes.Add 100-200 μ l dissolution fluid and at room temperature stir 1-10min with the micro test tube agitator.Instantaneous high speed centrifugation reclaims supernatant liquor and is purifying protein.
In concrete enforcement, H9N2 type bird flu NS1 protein expressing plasmid pET-NS1 provided by the invention, the Applicable temperature scope of abduction delivering is wide, all can express for 25-30 ℃.PET-NS1 plasmid provided by the invention can not produce inclusion body in the expression process, fusion rotein has histidine-tagged, can use the MagExtractor-His-tag-kit purifying.Made things convenient for the evaluation and the purifying of sample like this, more avoided the sex change and the renaturation of sample, protein structure is not damaged.The NS1 albumen that gives expression to has overcome the shortcoming that purifying protein is degraded easily in the prokaryotic expression, can be-40 ℃ of long-time preservations.
Utilize the pET-NS1 expression plasmid can great expression H9N2 type bird flu NS1 albumen, can study the proteic basic function of H9N2 type avian influenza virus NS1.The NS1 albumen that gives expression to can also be introduced chemical mutation, further studies the influence of amino acid whose change to the avian influenza effect.In addition, expressed NS1 albumen and antibody thereof a vital signs can be used as virus infection chicken body.Utilize the bird flu NS1 albumen can differential diagnosis natural infection chicken group and vaccine immune chicken group.
The sequence and the mark apportion of the present invention's design are as follows:
(1) information of SEQ ID NO.1
＜110〉Shanghai Communications University
＜120〉the protein translation district encoding sequence of bird flu NS1 protein expressing plasmid pET-NS1
<160>2
<170>PatentIn?version?3.3
<210>1
<211>1197
<212>DNA
＜213〉artificial sequence
<400>1
atgagggata?aaattattca?cctgactgac?gacagttttg?acacggatgt?actcaaagcg 60
gacggggcga?tcctcgtcga?tttctgggca?gagtggtgcg?gtccgtgcaa?aatgatcgcc 120
ccgattctgg?atgaaatcgc?tgacgaatat?cagggcaaac?tgaccgttgc?aaaactgaac 180
atcgatcaaa?accctggcac?tgcgccgaaa?tatggcatcc?gtggtatccc?gactctgctg 240
ctgttcaaaa?acggtgaagt?ggcggcaacc?aaagtgggtg?cactgtctaa?aggtcagttg 300
aaagagttcc?tcgacgctaa?cctggccggt?tctggttctg?gccatatgca?ccatcatcat 360
catcattctt?ctggtctggt?gccacgcggt?tctggtatga?aagaaaccgc?tgctgctaaa 420
ttcgaacgcc?agcacatgga?cagcccagat?ctgggtaccg?acgacgacga?caaggccatg 480
gctgatatcg?gatccgaatt?cataatgaat?tccaacacta?tgtcaagctt?tcaggtagac 540
tgctttcttt?ggcatgtccg?caaacgattt?gcagaccaag?aactgggtga?tgccccattc 600
cttgaccagc?ttcgccgaga?tcagaagtcc?ctaagaggaa?gaggcagcac?tcttggtctg 660
gacatcgaaa?cagctactcg?tgcgggaaag?cagatagtgg?agcggattct?ggaggaagag 720
tctgatgagg?cacttaaaat?gactattgct?tctgtgccgg?cttcacgcta?cctaactgac 780
atgactcttg?aagaaatgtc?aagggactgg?ttcatgctca?tgcccaagca?gaaagtggca 840
ggttcccttt?gcatcaaaat?ggaccaggca?ataatggata?aaaacatcat?attgaaagca 900
aatttcagtg?tgatttttgg?ccggttagaa?accctaatac?tacttagagc?cttcacagaa 960
gaaggagcaa?tcgtgggaga?aatctcacca?ttaccttccc?ttccaggaca?tactgatgag 1020
gatgtcaaaa?atgcaattgg?ggtcctcatc?ggaggatttg?aatggaatga?taacacagtt 1080
cgagtctcta?aaactctaca?gagattcgct?tggagaagca?gtaatgagaa?tgggagacct 1140
ccactccctc?caaagcagaa?acggaaaatg?gcgagaacaa?ttgagtcaga?agtttga 1197
<210>2
<211>398
<212>PRT
＜213〉artificial sequence
<400>2
Met?Arg?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr?Asp
15?10?15
Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala?Glu?Trp
20?25?30
Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu?Ile?Ala?Asp
35?40?45
Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn?Ile?Asp?Gln?Asn
50?55?60
Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly?Ile?Pro?Thr?Leu?Leu
65?70?75?80
Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr?Lys?Val?Gly?Ala?Leu?Ser
85?90?95
Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp?Ala?Asn?Leu?Ala?Gly?Ser?Gly
100?105?110
Ser?Gly?His?Met?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
115?120?125
Arg?Gly?Ser?Gly?Met?Lys?Glu?Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln
130?135?140
His?Met?Asp?Ser?Pro?Asp?Leu?Gly?Thr?Asp?Asp?Asp?Asp?Lys?Ala?Met
145?150?155?160
Ala?Asp?Ile?Gly?Ser?Glu?Phe?Ile?Met?Asn?Ser?Asn?Thr?Met?Ser?Ser
165?170?175
Phe?Gln?Val?Asp?Cys?Phe?Leu?Trp?His?Val?Arg?Lys?Arg?Phe?Ala?Asp
180?185?190
Gln?Glu?Leu?Gly?Asp?Ala?Pro?Phe?Leu?Asp?Gln?Leu?Arg?Arg?Asp?Gln
195?200?205
Lys?Ser?Leu?Arg?Gly?Arg?Gly?Ser?Thr?Leu?Gly?Leu?Asp?Ile?Glu?Thr
210?215?220
Ala?Thr?Arg?Ala?Gly?Lys?Gln?Ile?Val?Glu?Arg?Ile?Leu?Glu?Glu?Glu
225?230?235?240
Ser?Asp?Glu?Ala?Leu?Lys?Met?Thr?Ile?Ala?Ser?Val?Pro?Ala?Ser?Arg
245?250?255
Tyr?Leu?Thr?Asp?Met?Thr?Leu?Glu?Glu?Met?Ser?Arg?Asp?Trp?Phe?Met
260?265?270
Leu?Met?Pro?Lys?Gln?Lys?Val?Ala?Gly?Ser?Leu?Cys?Ile?Lys?Met?Asp
275?280?285
Gln?Ala?Ile?Met?Asp?Lys?Asn?Ile?Ile?Leu?Lys?Ala?Asn?Phe?Ser?Val
290?295?300
Ile?Phe?Gly?Arg?Leu?Glu?Thr?Leu?Ile?Leu?Leu?Arg?Ala?Phe?Thr?Glu
305?310?315
Glu?Gly?Ala?Ile?Val?Gly?Glu?Ile?Ser?Pro?Leu?Pro?Ser?Leu?Pro?Gly
320?325?330?335
His?Thr?Asp?Glu?Asp?Val?Lys?Asn?Ala?Ile?Gly?Val?Leu?Ile?Gly?Gly
340?345?350
Phe?Glu?Trp?Asn?Asp?Asn?Thr?Val?Arg?Val?Ser?Lys?Thr?Leu?Gln?Arg
355?360?365
Phe?Ala?Trp?Arg?Ser?Ser?Asn?Glu?Asn?Gly?Arg?Pro?Pro?Leu?Pro?Pro
370?375?380
Lys?Gln?Lys?Arg?Lys?Met?Ala?Arg?Thr?Ile?Glu?Ser?Glu?Val
385?390?395?398
Claims (5)
1, a kind of H9N2 type bird flu NS1 protein expressing plasmid pET-NS1, it is characterized in that, the pET-NS1 expression plasmid that is provided is by pET-32a (+) and the bird flu NS1 gene enzyme that contains restriction enzyme site behind pcr amplification being cut, connecting the expression plasmid of acquisition; Among the pET-NS1 expression plasmid transformed into escherichia coli BL21, induce with IPTG, can give expression to the protein translation region nucleotide sequence of H9N2 type bird flu NS1 protein expressing plasmid pET-NS1, it is the sequence of SEQ ID NO.1, amount to 1197bp, SEQ ID NO.1 505-1197 position Nucleotide is the nucleotide sequence of H9N2 type avian influenza virus NS1 gene, total 693bp; Also can give expression to the protein translation region amino acid sequence of bird flu NS1 protein expressing plasmid pET-NS1, it is the aminoacid sequence of SEQID NO.2, amount to 398 amino acid, the amino acid of SEQ ID NO.2 169-398 position is the proteic aminoacid sequence of H9N2 type bird flu NS1, amounts to 230 amino acid.
2, H9N2 type bird flu NS1 protein expressing plasmid pET-NS1 according to claim 1, it is characterized in that, the protein translation region nucleotide sequence of described H9N2 type bird flu NS1 protein expressing plasmid pET-NS1, be meant: the nucleotide sequence of coding H9N2 type avian influenza virus NS1 fusion rotein, promptly add that at interior 504bp Nucleotide the 693bp Nucleotide of H9N2 type avian influenza virus NS1 gene order forms, amount to 1197bp by Trx Tag, His Tag, S Tag and a series of restriction enzyme site on pET-32a (+) the carrier sequence.
3, according to claim 1 or 2 described H9N2 type bird flu NS1 protein expressing plasmid pET-NS1, it is characterized in that, the nucleotide sequence of described H9N2 type bird flu NS1 gene is meant the open reading frame of the coding H9N2 type avian influenza virus NS1 proteic 693bp of containing.
4, H9N2 type bird flu NS1 protein expressing plasmid pET-NS1 according to claim 1, it is characterized in that, the protein translation region amino acid sequence of described bird flu NS1 protein expressing plasmid pET-NS1, be meant: the aminoacid sequence of H9N2 type avian influenza virus NS1 fusion rotein promptly adds that at 168 interior amino acid 0 amino acid of H9N2 type bird flu NS1 protein 23 forms by TrxTag, His Tag, S Tag and a series of restriction enzyme site on pET-32a (+) the carrier sequence.
5, according to claim 1 or 4 described H9N2 type bird flu NS1 protein expressing plasmid pET-NS1, it is characterized in that the proteic aminoacid sequence of described H9N2 type bird flu NS1 is meant: proteic 230 aminoacid sequences of H9N2 type avian influenza virus NS1.
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| CN108130340B (en) * | 2018-02-06 | 2021-02-23 | 福建省农业科学院畜牧兽医研究所 | Method for expressing duck source avian influenza virus NS1 protein and application of method |
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Non-Patent Citations (4)
| Title |
|---|
| H9N2亚型禽流感病毒非结构蛋白(NS1)基因的克隆与表达 刘金华，吴清民.中国病毒学，第15卷第8期 2003 * |
| pET System Manual. 第1页，第19页，第35页,Novagen Corp.. 2003 * |
| 大肠杆菌中外源基因表达的研究进展 李太华.微生物免疫学进展，第28卷第2期 2000 * |
| 真核基因在pet系统中表达出现的问题与拟解决的方案 张锐，孙美榕.生物技术，第14卷第2期 2004 * |
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