CN102321651A - Fusion gene containing influenza A virus nucleoprotein, its construction and its expression method - Google Patents
Fusion gene containing influenza A virus nucleoprotein, its construction and its expression method Download PDFInfo
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- CN102321651A CN102321651A CN201110266325A CN201110266325A CN102321651A CN 102321651 A CN102321651 A CN 102321651A CN 201110266325 A CN201110266325 A CN 201110266325A CN 201110266325 A CN201110266325 A CN 201110266325A CN 102321651 A CN102321651 A CN 102321651A
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Abstract
The invention discloses a fusion gene pEGFP-N1-NP containing influenza A virus nucleoprotein, a construction and an expression method, a nucleotide sequence of the fusion gene is shown in SEQIDNO.1. Compared with the prior art, the invention enables the fusion of influenza A virus nucleoprotein and green fluorescent protein to form the fusion gene, the fusion gene is capable of expressing the green fluorescent protein and influenza A virus nucleoprotein after transfecting an eukaryotic cell expression, The successful construction and expression of the fusion gene pEGFP-N1-NP enables a RNA interference to inhibit an influenza virus NP gene, and have a functional identification in cells or virus.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to a kind of fusion gene pEGFP-N1-NP and structure and expression method that contains influenza A virus nucleoprotein.
Background technology
Influenza A virus is the important pathogenic agent of respiratory tract infection, repeatedly causes worldwide flu outbreak: highly pathogenic H5N1 bird flu epidemic situation was broken out in Hong Kong in 1997, confirmed that first bird flu can infect the mankind, and 562 examples have been infected in the whole world so far, dead 329 examples; Influenza A H1N1 have swept the globe in 2009, the virus strain of following influenza pandemic may appear at any time, the world Anywhere and the influenza virus reassortant of any animal-origin.Influenza A virus nucleoprotein (Nucleoprotein; NP) by influenza virus gene group the 5th segment encoding; Its opening code-reading frame contains 1494 Nucleotide; In the transcribing, duplicate and transport of influenza virus, play an important role, in each different subtype, have high conservative property and can produce immune cross protection.Therefore,, will help further to understand the mechanism of causing a disease of influenza A virus, for the prevention and control flu outbreak provides theoretical foundation to the research of nucleoprotein function.
In order to obtain influenza A virus nucleoprotein in a large number,, need to make up influenza A virus NP gene eukaryotic expression vector so that carry out next step research.Eukaryon expression plasmid pEGFP-N1 expresses enhanced green fluorescence protein (EGFP), makes up the fusion gene of NP and EGFP, can make NP genetic expression green fluorescent protein.Constructed fusion gene can carry out eukaryotic expression, and important experiment basis has been established in the further research of the Function Identification of influenza A virus nucleoprotein, drug target screening and pathogenesis thereof.
Summary of the invention
Technical problem to be solved by this invention provides a kind of fusion gene pEGFP-N1-NP and structure and expression method that contains influenza A virus nucleoprotein; The successful structure of this fusion gene pEGFP-N1-NP and expression can be carried out RNA and disturb inhibition influenza virus NP gene, and in cell and virus, carry out Function Identification.
In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:
The present invention contains nucleotide sequence such as the SEQ ID NO.1 of the fusion gene pEGFP-N1-NP of influenza A virus nucleoprotein.
The construction process of above-mentioned fusion gene comprises the steps:
(1) be template with the influenza A virus nucleoprotein gene, design contains the primer of restriction enzyme EcoR I and Sma I restriction enzyme site, and pcr amplification must have the nucleoprotein gene of double enzyme site;
(2) nucleoprotein gene is mixed with T carrier pMD19-T Simple Vector carry out T clone, acquisition pMD19-T-NP plasmid after 0.8% agarose gel electrophoresis is identified;
(3) with the destination carrier pEGFP-N1 of pMD19-T-NP plasmid subclones to enhanced green fluorescent tracing albumen EGFP; Transformed into escherichia coli DH-5 α; Cultivate the back and extract plasmid, Sma I, EcoR I double digestion are identified positive colony, promptly get fusion gene pEGFP-N1-NP.
In the preparation method of above-mentioned fusion gene; Subclone is meant in the step (3): adopt restriction endonuclease sma I and EcoR I double digestion pEGFP-N1 plasmid and pMD19-T-NP plasmid to obtain the pEGFP-N1 and the purpose nucleoprotein fragment of purifying respectively, add to connect in the ligation obtaining ligation liquid to.The said back extraction plasmid of cultivating is the intestinal bacteria DH-5 α after transforming to be inoculated on the LB culture plate that contains kantlex cultivate, and picking mono-clonal bacterium colony is cultivated to the S.O.C liquid nutrient medium that contains kantlex.
In the construction process of aforementioned fusion gene, the amplimer of pcr amplification is described in the step (1):
Upstream primer: 5'-ccgcgaattcatggcgtctcaaggcaccaaacgat-3', wherein gaattc is the restriction enzyme site of EcoR I;
Downstream primer: 5'-cgcgcccgggcattgtcatactcctctgcatt-3', wherein cccggg is the restriction enzyme site of Sma I.
In aforesaid construction process, said template is an influenza A virus H9N2 nucleoprotein gene.
The expression method of aforementioned influenza A virus nucleoprotein and green fluorescent protein fusion gene pEGFP-N1-NP: with pEGFP-N1-NP fusion gene transfection HEK293T cell, under inverted fluorescence microscope, observe the expression of green fluorescent protein through Lipofectamine 2000; Identify the expression of influenza A virus nucleoprotein with cellular immunization group method.
Compared with prior art; Influenza A virus nucleoprotein and green fluorescent protein are merged in the present invention; Form fusion gene; But this fusion gene is expressing green fluorescent protein and influenza A virus nucleoprotein behind transfection eukaryotic expression cell, and the successful structure of this fusion gene pEGFP-N1-NP and expression can be carried out RNA and disturb inhibition influenza virus NP gene, and in cell and virus, carry out Function Identification.
Description of drawings
Fig. 1 is the structural representation of fusion gene of the present invention;
Fig. 2 is goal gene NP pcr amplification result.M wherein: molecular mass standard; 1:NP-F, NP-R amplification NP gene fragment.
Fig. 3 is a NP gene TA cloned plasmids electrophoresis result.M wherein: molecular mass standard; 1-7: possibly make up successful NP gene TA cloned plasmids; 8,9: do not make up successful NP gene TA cloned plasmids.
Fig. 4 cuts the product electrophoresis result for NP gene TA cloned plasmids enzyme.M wherein: molecular mass standard; 1-7: make up successful NP gene TA cloned plasmids enzyme and cut the product electrophoresis; 8,9: do not make up successful NP gene TA cloned plasmids enzyme and cut the product electrophoresis.
Fig. 5 is NP, EGFP fusion gene plasmid electrophoresis result.M wherein: molecular mass standard; 1-4,6-9,11-18: possibly make up successful NP, EGFP fusion gene plasmid; 5,10: do not make up successful NP fusion gene.
Fig. 6 is NP, EGFP fusion gene plasmid enzyme restriction product electrophoresis result.M wherein: molecular mass standard; 1-4,6-9,11-18: make up successful NP, EGFP fusion gene plasmid enzyme restriction product electrophoresis; 5,10: do not make up successful NP fusion gene plasmid enzyme restriction product electrophoresis.
Fig. 7 is an inverted fluorescence microscope observations (* 400) behind the plasmid transfection HEK293T cell.Wherein A, D:pEGFP-N1 transfection HEK293T cell; B, E:pEGFP-N1-NP transfection HEK293T cell; C, F:HEK293T cell blank transfection contrast.
Fig. 8 is the cellular immunization group evaluation proteic expression observations of NP (* 400).Wherein B:pEGFP-N1-NP transfection HEK293T cell adds the anti-NP one of rabbit and resists the positive coloration result of arrow indication; A, C, D, E, F: do not see the positive staining result.
Embodiment
Embodiment 1: the structure of fusion gene pEGFP-N1-NP
1, design contains the primer of restriction enzyme EcoR I and Sma I restriction enzyme site
Upstream primer: 5'-ccgc
GaattcAtggcgtctcaaggcaccaaacgat-3', wherein gaattc is the restriction enzyme site of EcoR I;
Downstream primer: 5'-cgcg
CccgggCattgtcatactcctctgcatt-3', wherein cccggg is the restriction enzyme site of Sma I.
2, pcr amplification NP fragment: influenza A virus nucleoprotein (NP) gene DNA source virus strain is Influenza A virus (A/swine/Guangxi/S15/2005 (H9N2)) (GenBank:EU086324.1
), be template with the NP gene DNA, step (1) institute designed primer is a primer, introduces restriction enzyme EcoR I and Sma I restriction enzyme site, amplification NP gene fragment total length is 1508bp;
ddH
2O 12.5μl
5×Buffer 5μl
MgCl
2 2μl
dNTPmix 2μl
DNA Taq enzyme 0.5 μ l
NP-F 0.5μl
NP-R 0.5μl
NP?DNA 2μl
Total reaction system 25 μ l.The pcr amplification condition is following: 94 ℃ of 3min; 94 ℃ of 30s, 59 ℃ of 20s, 72 ℃ of 1min30s, totally 42 circulations; 72 ℃ are extended 7min.0.8% agarose gel electrophoresis is observed amplification (as shown in Figure 2), by the U.S. DNA of Omega company purifying and recovering test kit specification sheets cut glue, the purifying size is the NP gene fragment of 1508bp.
3, the TA of NP gene clone:
(1) gets the NP gene fragment of step 2 gained, be connected with pMD19-T Simple Vector mixing by precious biotechnology (Dalian) TA of ltd clone operations specification sheets;
pMD19-T?Simple?Vector 1μl
NP?DNA 4μl
SolutionⅠ 5μl
16 ℃ connect 3h and must connect product.
(2) transformed into escherichia coli: get 100 μ l intestinal bacteria DH-5 α competent cells and in ice bath, melt, avoid competent cell to be exposed to room temperature for a long time; In competent cell, add 10 μ l target DNAs (connection product), the rotating centrifugal pipe makes the liquid mixing gently, must guard against not piping and druming or concussion mixing, ice bath 30min; 42 ℃ of water-bath heat shock 90s transfer in the ice bath fast, and ice bath 3min does not shake centrifuge tube in the process; In centrifuge tube, add 900 μ l LB liquid nutrient mediums, blow and beat mixing gently and be placed on 37 ℃ of shaking table 200rpm concussion cultivation 1h, the plasmid resistant maker gene is expressed, make the thalline recovery; The blue hickie screening and culturing of ultra violet lamp LB (containing penbritin) plate 20min gets the competent cell that 100 μ l have transformed and is added on the plate, evenly is coated with glass stick, and room temperature is placed and after liquid absorbs fully, is inverted in 16h in 37 ℃ of baking ovens.
(3) bacterial strain screening: it is bigger to select the sparkling and crystal-clear transparent volume of cultivation back thalline; And do not have other on every side and merge the sharply marginated white monospecific polyclonal of bacterium colony; Gently be stained with on the fan-shaped blue hickie screening and culturing of LB (the containing penbritin) plate of coating prior lattice in back with toothpick; Label is corresponding one by one, and mark is clear in order to tracing back; 37 ℃ of baking ovens are inverted and are cultivated 16h.
(4) plasmid extraction: still be white bacterium colony to the S.O.C liquid nutrient medium that contains penbritin after selecting the mono-clonal amplification; 37 ℃ of shaking tables are cultivated 12-16h for 200 rev/mins; Utilize the little extraction reagent kit of plasmid (centrifugal column type) of sky, Beijing root biotech firm to extract plasmid, carry out according to the following steps:
1. choose on the mono-clonal plate growing way bacterial strain preferably; Get 1.5ml centrifuge tube adding 1mlS.O.C and contain the penbritin liquid nutrient medium; Stir in centrifuge tube with a small amount of bacterium colony of toothpick picking, in the 15ml centrifuge tube, add 5mlS.O.C and contain the penbritin liquid nutrient medium, in the bacterium liquid adding 15ml centrifuge tube with mixing; About 1/4 stroke of lid double helix is convenient to ventilation, and 37 ℃ of 200rpm 16h shaking tables spend the night.
2. column equilibration step: (adsorption column is put into collection tube) adds the balance liquid BL of 500 μ l in adsorption column CP3, and 12, the centrifugal 1min of 000rpm outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector.(using the pillar of handling the same day)
3. get the bacterium liquid of 2 ml incubated overnight, add in the centrifuge tube, use conventional desk centrifuge, 12, the centrifugal 1min of 000rpm absorbs supernatant as far as possible.
4. in the centrifuge tube that leaves bacterial sediment, add 250 μ l solution P1 (having added RNaseA), use the vortex vibrator bacterial precipitation that thoroughly suspends.
5. in centrifuge tube, add 250 μ l solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time.
6. in centrifuge tube, add 350 μ l solution P3, leniently spin upside down 6-8 time immediately, fully mixing white flocks will occur at this moment.The centrifugal 10min of 12,000 rpm, form deposition in the centrifuge tube bottom this moment.
7. will go up the supernatant of step collection and transfer to (adsorption column is put into collection tube) among the adsorption column CP3, the sucking-off deposition of noting trying not with pipettor.The centrifugal 30-60s of 12,000 rpm outwells the waste liquid in the collection tube, and CP3 puts into collection tube with adsorption column.
8. in adsorption column CP3, add 600 μ l rinsing liquid PW (having added absolute ethyl alcohol before the use), the centrifugal 1min of 12000rpm outwells the waste liquid in the collection tube, and CP3 puts into collection tube with adsorption column; Repeating step 8..
9. adsorption column CP3 is put into collection tube, the centrifugal 2min of 12000rpm removes the remaining rinsing liquid in the adsorption column; The follow-up enzyme reaction of the remaining meeting influence of alcoholic acid (cut by enzyme in the rinsing liquid; PCR etc.) experiment does not receive the influence of residual ethanol for guaranteeing the downstream experiment, and CP3 uncaps with adsorption column; Place 10min as for room temperature, treat to carry out subsequent step again after rinsing liquid remaining in the adsorption column thoroughly dries.
10. adsorption column CP3 is placed a clean 1.5ml centrifuge tube, drip 100 μ l to the middle part of adsorption film and take off wash buffer EB, room temperature is placed 2min, and the centrifugal 1min of 12000rpm collects plasmid solution in the centrifuge tube, and-20 ℃ of preservations are subsequent use.
(5) the TA clone identifies: 0.8% agarose gel electrophoresis, select target band are the plasmid of 4200bp, the positive clone result of preliminary judgement.Restriction endonuclease sma I, EcoR I are carried out plasmid enzyme restriction and are identified that the endonuclease reaction system is following:
10×Buffer 2μl
SmaⅠ 1μl
ddH
2O 11μl
Add following reagent behind 30 ℃ of connection 1h:
EcoRⅠ 1μl
10×Buffer 2.5μl
37 ℃ connect 1h.
0.8% agarose gel electrophoresis checking (as shown in Figure 3) if having target stripe at 2700bp and 1500bp position, is then signed positive clone, called after pMD19-T-NP.
4, NP gene subclone is to eukaryon expression plasmid pEGFP-N1, gene fusion construct expression vector pEGFP-N1-NP:
(1) restriction endonuclease sma I, EcoR I double digestion pEGFP-N1 and pMD19-T-NP:
10×Buffer 2μl
SmaⅠ 1μl
ddH
2O 11μl
Plasmid (pEGFP-N1/pMD19-T-NP) 6 μ l (1 μ g)
Add following reagent behind 30 ℃ of connection 1h:
EcoRⅠ 1μl
10×Buffer 2.5μl
37 ℃ connect 1h.
0.8% agarose gel electrophoresis (as shown in Figure 4) is cut glue purification and is got big or small be 1500bp purpose fragment NP and the big fragment of 4700bp plasmid pEGFP-N1.
(2) respectively get fragment NP and pEGFP-N1 behind the 2 μ l purifying, 0.8% agarose gel electrophoresis is quantitative roughly; Purpose fragment NP and plasmid pEGFP-N1 5:1 in molar ratio add in the ligation:
pEGFP-N1 4μl
NP 10μl
10×Buffer 2μl
ddH
2O 3μl
16 ℃ of reaction 12-16h.The electrophoresis result of fusion gene plasmid is as shown in Figure 5.
(3) ligation liquid transformed into escherichia coli DH-5 α, evenly coating contains on the LB culture plate of kantlex, cultivates 12-16h for 37 ℃; Picking mono-clonal bacterium colony is cultivated 12-16h for 37 ℃ to the LB culture plate that contains kantlex; The mono-clonal bacterium colony is to the S.O.C liquid nutrient medium that contains kantlex after increasing for picking, and 37 ℃ of shaking tables are cultivated 12-16h for 200 rev/mins, utilize the little extraction reagent kit of plasmid (centrifugal column type) of sky, Beijing root biotech firm to extract plasmid.Sma I, EcoR I double digestion are identified positive colony (as shown in Figure 6), promptly get fusion gene pEGFP-N1-NP, serve extra large Invitrogen Life Technologies, Inc. gene sequencing and identify that further its nucleotide sequence is shown in SEQ ID NO.1.
Embodiment 2: the expression of fusion gene pEGFP-N1-NP
1, plasmid transfection HEK293T cell: well-grown degrees of fusion is reached HEK293T cell inoculation more than 80% in 6 orifice plates; Transfection is carried out in Lipofectamine 2000 explanations by American I nvitrogen company when the culturing cell degrees of fusion reaches 70% left and right sides; Wherein two holes are plasmid pEGFP-N1 transfection HEK293T cell; Two holes are pEGFP-N1-NP transfection HEK293T cell, and two holes are the contrast of HEK293T cell blank, so that the immunohistochemical methods in step detects down.
2, inverted fluorescence microscope is observed the expression of green fluorescent protein: behind the plasmid transfection HEK293T cell 24h, observe down at inverted fluorescence microscope (Olympus IX71-A12FL/PH), the result is as shown in Figure 7.
3, the evaluation of NP protein expression: observe egfp expression under the inverted fluorescence microscope behind the plasmid transfection cell 24h, but the method for application cell immunohistochemical methods is identified the proteic expression of NP.At first in six orifice plates with PBS buffer solution for cleaning cell 3 times; Add the anti-NP one anti-(1:1000 of rabbit in pEGFP-N1, pEGFP-N1-NP and each hole of blank cell contrast; Contain 0.5% foetal calf serum PBS dilution); Add in 3 holes in addition and contain 0.5% foetal calf serum PBS diluent as contrast, hatch 1h for 37 ℃, 4 ℃ are spent the night; PBS rinsing 3 times, it is anti-to add goat antirabbit two, hatches 1h for 37 ℃; PBS rinsing 3 times; The colour developing of DAB test kit, 5min-10min, adding distil water rinsing color development stopping; Hematorylin is redyed, 3min-5min, zero(ppm) water rinsing; The differentiation of 1% hydrochloride alcohol, the zero(ppm) water rinsing, inverted microscope is observed down, and the result is as shown in Figure 8.
Sequence table
< 110>Affiliated Hospital of Zunyi Medical College
< 120>contain fusion gene and the structure and the expression method of influenza A virus nucleoprotein
<160>?4
<210>?1
<211>?6207
<212>?DNA
< 213>artificial sequence
<400>?1
tagttattaa?tagtaatcaa?ttacggggtc?attagttcat?agcccatata?tggagttccg 60
cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc?cccgcccatt 120
gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc?attgacgtca 180
atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt?atcatatgcc 240
aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt?atgcccagta 300
catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca?tcgctattac 360
catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg?actcacgggg 420
atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc?aaaatcaacg 480
ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg?gtaggcgtgt 540
acggtgggag?gtctatataa?gcagagctgg?tttagtgaac?cgtcagatcc?gctagcgcta 600
ccggactcag?atctcgagct?caagcttcga?attcatggcg?tctcaaggca?ccaaacgatc 660
ttatgaacag?atggaaactg?gtggggaacg?ccagaatgct?actgagatca?gggcatctgt 720
tggaagaatg?gttagtggca?ttgggaggtt?ctacgtacag?atgtgcacag?aactcaaact 780
cagtgactat?gaagggagac?tgatccagaa?cagcataaca?atagagagaa?tggtactctc 840
tgcatttgat?gaaagaagaa?acagatacct?ggaagaacac?cccagtgcag?ggaaggaccc 900
gaagaaaact?ggaggtccaa?tttatcggag?gagagacggg?aaatgggtga?gagagctgat 960
tctgtatgac?aaagaagaga?ttaggaggat?ttggcgtcaa?gcgaacaatg?gagaggacgc 1020
aactgctggt?cttacccacc?tgatgatatg?gcattccaat?ctaaatgatg?ccacatatca 1080
gagaacgaga?gctcttgtgc?gtactggaat?ggatcccagg?atgtgctctc?tgatgcaagg 1140
atcaactctc?ccgaggagat?ctggagctgc?cggtgcagca?gtaaagggag?tagggacaat 1200
ggtgatggag?ctggttcgga?tgataaaacg?agggatcaac?gacaggaatt?tctggagagg 1260
cgaaaatgga?agaagaacaa?ggattgcata?tgagagaatg?tgcaacatcc?tcaaagggaa 1320
attccagaca?gcagcacaaa?gggcaatggt?ggatcaagtg?cgagagagca?gaaatcctgg 1380
gaatgctgaa?attgaagatc?tcatttttct?ggcacggtct?gcactcatcc?tgagaggatc 1440
agtggcccat?aagtcctgct?tgcctgcttg?tgtgtacgga?cttgcagtgg?ccagtggata 1500
tgactttgag?agagaagggt?actctctggt?tggaatagat?cctttccgcc?tgcttcaaaa 1560
cagccaggtc?tttagtctca?ttagaccaaa?tgagaatcca?gcacataaga?gtcaattagt 1620
gtggatggca?tgccattctg?cagcatttga?ggaccttaga?gtctcaagtt?tcatcagagg 1680
gacaagagtg?atcccaaggg?gacagctatc?caccagaggg?gttcaaatag?cttcaaatga 1740
gaacatggaa?gcaatggact?ccaacactct?tgaactgaga?agtagatatt?gggccataag 1800
aaccagaagc?ggaggaaaca?ccaaccagca?gagggcgtct?gcagggcaga?tcagtgtcca 1860
gcccactttc?tcggtacaga?gaaatcttcc?cttcgaaaga?gcgaccatta?tggcagcatt 1920
tacaggaaat?actgagggca?gaacgtctga?catgaggact?gaaatcataa?gaatgatgga 1980
gagtgccaga?ccagaagatg?tgtcattcca?ggggcgggga?gtcttcgagc?tctcggacga 2040
aaaggcaacg?aacccgatcg?tgccttcctt?tgacatgaat?aatgaaggat?cttatttctt 2100cggagacaat
gcagaggagt?atgacaatgc?ccgggatcca?ccggtcgcca?ccatggtgag 2160
caagggcgag?gagctgttca?ccggggtggt?gcccatcctg?gtcgagctgg?acggcgacgt 2220
aaacggccac?aagttcagcg?tgtccggcga?gggcgagggc?gatgccacct?acggcaagct 2280
gaccctgaag?ttcatctgca?ccaccggcaa?gctgcccgtg?ccctggccca?ccctcgtgac 2340
caccctgacc?tacggcgtgc?agtgcttcag?ccgctacccc?gaccacatga?agcagcacga 2400
cttcttcaag?tccgccatgc?ccgaaggcta?cgtccaggag?cgcaccatct?tcttcaagga 2460
cgacggcaac?tacaagaccc?gcgccgaggt?gaagttcgag?ggcgacaccc?tggtgaaccg 2520
catcgagctg?aagggcatcg?acttcaagga?ggacggcaac?atcctggggc?acaagctgga 2580
gtacaactac?aacagccaca?acgtctatat?catggccgac?aagcagaaga?acggcatcaa 2640
ggtgaacttc?aagatccgcc?acaacatcga?ggacggcagc?gtgcagctcg?ccgaccacta 2700
ccagcagaac?acccccatcg?gcgacggccc?cgtgctgctg?cccgacaacc?actacctgag 2760
cacccagtcc?gccctgagca?aagaccccaa?cgagaagcgc?gatcacatgg?tcctgctgga 2820
gttcgtgacc?gccgccggga?tcactctcgg?catggacgag?ctgtacaagt?aaagcggccg 2880
cgactctaga?tcataatcag?ccataccaca?tttgtagagg?ttttacttgc?tttaaaaaac 2940
ctcccacacc?tccccctgaa?cctgaaacat?aaaatgaatg?caattgttgt?tgttaacttg 3000
tttattgcag?cttataatgg?ttacaaataa?agcaatagca?tcacaaattt?cacaaataaa 3060
gcattttttt?cactgcattc?tagttgtggt?ttgtccaaac?tcatcaatgt?atcttaaggc 3120
gtaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc 3180
attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga 3240
gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc 3300
caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc 3360
ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag 3420
cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa 3480
agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac 3540
cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtca?ggtggcactt?ttcggggaaa 3600
tgtgcgcgga?acccctattt?gtttattttt?ctaaatacat?tcaaatatgt?atccgctcat 3660
gagacaataa?ccctgataaa?tgcttcaata?atattgaaaa?aggaagagtc?ctgaggcgga 3720
aagaaccagc?tgtggaatgt?gtgtcagtta?gggtgtggaa?agtccccagg?ctccccagca 3780
ggcagaagta?tgcaaagcat?gcatctcaat?tagtcagcaa?ccaggtgtgg?aaagtcccca 3840
ggctccccag?caggcagaag?tatgcaaagc?atgcatctca?attagtcagc?aaccatagtc 3900
ccgcccctaa?ctccgcccat?cccgccccta?actccgccca?gttccgccca?ttctccgccc 3960
catggctgac?taattttttt?tatttatgca?gaggccgagg?ccgcctcggc?ctctgagcta 4020
ttccagaagt?agtgaggagg?cttttttgga?ggcctaggct?tttgcaaaga?tcgatcaaga 4080
gacaggatga?ggatcgtttc?gcatgattga?acaagatgga?ttgcacgcag?gttctccggc 4140
cgcttgggtg?gagaggctat?tcggctatga?ctgggcacaa?cagacaatcg?gctgctctga 4200
tgccgccgtg?ttccggctgt?cagcgcaggg?gcgcccggtt?ctttttgtca?agaccgacct 4260
gtccggtgcc?ctgaatgaac?tgcaagacga?ggcagcgcgg?ctatcgtggc?tggccacgac 4320
gggcgttcct?tgcgcagctg?tgctcgacgt?tgtcactgaa?gcgggaaggg?actggctgct 4380
attgggcgaa?gtgccggggc?aggatctcct?gtcatctcac?cttgctcctg?ccgagaaagt 4440
atccatcatg?gctgatgcaa?tgcggcggct?gcatacgctt?gatccggcta?cctgcccatt 4500
cgaccaccaa?gcgaaacatc?gcatcgagcg?agcacgtact?cggatggaag?ccggtcttgt 4560
cgatcaggat?gatctggacg?aagagcatca?ggggctcgcg?ccagccgaac?tgttcgccag 4620
gctcaaggcg?agcatgcccg?acggcgagga?tctcgtcgtg?acccatggcg?atgcctgctt 4680
gccgaatatc?atggtggaaa?atggccgctt?ttctggattc?atcgactgtg?gccggctggg 4740
tgtggcggac?cgctatcagg?acatagcgtt?ggctacccgt?gatattgctg?aagagcttgg 4800
cggcgaatgg?gctgaccgct?tcctcgtgct?ttacggtatc?gccgctcccg?attcgcagcg 4860
catcgccttc?tatcgccttc?ttgacgagtt?cttctgagcg?ggactctggg?gttcgaaatg 4920
accgaccaag?cgacgcccaa?cctgccatca?cgagatttcg?attccaccgc?cgccttctat 4980
gaaaggttgg?gcttcggaat?cgttttccgg?gacgccggct?ggatgatcct?ccagcgcggg 5040
gatctcatgc?tggagttctt?cgcccaccct?agggggaggc?taactgaaac?acggaaggag 5100
acaataccgg?aaggaacccg?cgctatgacg?gcaataaaaa?gacagaataa?aacgcacggt 5160
gttgggtcgt?ttgttcataa?acgcggggtt?cggtcccagg?gctggcactc?tgtcgatacc 5220
ccaccgagac?cccattgggg?ccaatacgcc?cgcgtttctt?ccttttcccc?accccacccc 5280
ccaagttcgg?gtgaaggccc?agggctcgca?gccaacgtcg?gggcggcagg?ccctgccata 5340
gcctcaggtt?actcatatat?actttagatt?gatttaaaac?ttcattttta?atttaaaagg 5400
atctaggtga?agatcctttt?tgataatctc?atgaccaaaa?tcccttaacg?tgagttttcg 5460
ttccactgag?cgtcagaccc?cgtagaaaag?atcaaaggat?cttcttgaga?tccttttttt 5520
ctgcgcgtaa?tctgctgctt?gcaaacaaaa?aaaccaccgc?taccagcggt?ggtttgtttg 5580
ccggatcaag?agctaccaac?tctttttccg?aaggtaactg?gcttcagcag?agcgcagata 5640
ccaaatactg?tccttctagt?gtagccgtag?ttaggccacc?acttcaagaa?ctctgtagca 5700
ccgcctacat?acctcgctct?gctaatcctg?ttaccagtgg?ctgctgccag?tggcgataag 5760
tcgtgtctta?ccgggttgga?ctcaagacga?tagttaccgg?ataaggcgca?gcggtcgggc 5820
tgaacggggg?gttcgtgcac?acagcccagc?ttggagcgaa?cgacctacac?cgaactgaga 5880
tacctacagc?gtgagctatg?agaaagcgcc?acgcttcccg?aagggagaaa?ggcggacagg 5940
tatccggtaa?gcggcagggt?cggaacagga?gagcgcacga?gggagcttcc?agggggaaac 6000
gcctggtatc?tttatagtcc?tgtcgggtt?tcgccacctct?gacttgagcg?tcgatttttg 6060
tgatgctcgt?caggggggcg?gagcctatgg?aaaaacgcca?gcaacgcggc?ctttttacgg 6120
ttcctggcct?tttgctggcc?ttttgctcac?atgttctttc?ctgcgttatc?ccctgattct 6180
gtggataacc?gtattaccgc?catgcat 6207
<210>?2
<211>?1565
<212>?DNA
< 213>H9N2 nucleoprotein gene
<400>?2
agcgaaagca?gggttgataa?tcactcaccg?agtgacatca?acaccatggc?gtctcaaggc 60
accaaacgat?cttatgaaca?gatggaaact?ggtggggaac?gccagaatgc?tactgagatc 120
agggcatctg?ttggaagaat?ggttagtggc?attgggaggt?tctacgtaca?gatgtgcaca 180
gaactcaaac?tcagtgacta?tgaagggaga?ctgatccaga?acagcataac?aatagagaga 240
atggtactct?ctgcatttga?tgaaagaaga?aacagatacc?tggaagaaca?ccccagtgca 300
gggaaggacc?cgaagaaaac?tggaggtcca?atttatcgga?ggagagacgg?gaaatgggtg 360
agagagctga?ttctgtatga?caaagaagag?attaggagga?tttggcgtca?agcgaacaat 420
ggagaggacg?caactgctgg?tcttacccac?ctgatgatat?ggcattccaa?tctaaatgat 480
gccacatatc?agagaacgag?agctcttgtg?cgtactggaa?tggatcccag?gatgtgctct 540
ctgatgcaag?gatcaactct?cccgaggaga?tctggagctg?ccggtgcagc?agtaaaggga 600
gtagggacaa?tggtgatgga?gctggttcgg?atgataaaac?gagggatcaa?cgacaggaat 660
ttctggagag?gcgaaaatgg?aagaagaaca?aggattgcat?atgagagaat?gtgcaacatc 720
ctcaaaggga?aattccagac?agcagcacaa?agggcaatgg?tggatcaagt?gcgagagagc 780
agaaatcctg?ggaatgctga?aattgaagat?ctcatttttc?tggcacggtc?tgcactcatc 840
ctgagaggat?cagtggccca?taagtcctgc?ttgcctgctt?gtgtgtacgg?acttgcagtg 900
gccagtggat?atgactttga?gagagaaggg?tactctctgg?ttggaataga?tcctttccgc 960
ctgcttcaaa?acagccaggt?ctttagtctc?attagaccaa?atgagaatcc?agcacataag 1020
agtcaattag?tgtggatggc?atgccattct?gcagcatttg?aggaccttag?agtctcaagt 1080
ttcatcagag?ggacaagagt?gatcccaagg?ggacagctat?ccaccagagg?ggttcaaata 1140
gcttcaaatg?agaacatgga?agcaatggac?tccaacactc?ttgaactgag?aagtagatat 1200
tgggccataa?gaaccagaag?cggaggaaac?accaaccagc?agagggcgtc?tgcagggcag 1260
atcagtgtcc?agcccacttt?ctcggtacag?agaaatcttc?ccttcgaaag?agcgaccatt 1320
atggcagcat?ttacaggaaa?tactgagggc?agaacgtctg?acatgaggac?tgaaatcata 1380
agaatgatgg?agagtgccag?accagaagat?gtgtcattcc?aggggcgggg?agtcttcgag 1440
ctctcggacg?aaaaggcaac?gaacccgatc?gtgccttcct?ttgacatgaa?taatgaagga 1500
tcttatttct?tcggagacaa?tgcagaggag?tatgacaatt?aaagaaaaat?acccttgttt 1560
ctact 1565
<210>?3
<211>?35
<212>?DNA
< 213>artificial sequence
<400>?3
ccgcgaattc?atggcgtctc?aaggcaccaa?acgat 35
<210>?4
<211>?32
<212>?DNA
< 213>artificial sequence
<400>?4
cgcgcccggg?cattgtcata?ctcctctgca?tt 32
Claims (7)
1. a nucleotide sequence such as SEQ ID NO.1 who contains the fusion gene pEGFP-N1-NP of influenza A virus nucleoprotein.
2. the said construction process that contains the fusion gene of influenza A virus nucleoprotein of claim 1 is characterized in that comprising the steps:
(1) be template with the influenza A virus nucleoprotein gene, design contains the primer of restriction enzyme EcoR I and Sma I restriction enzyme site, and pcr amplification must have the nucleoprotein gene of double enzyme site;
(2) nucleoprotein gene is mixed with T carrier pMD19-T Simple Vector carry out T clone, acquisition pMD19-T-NP plasmid after 0.8% agarose gel electrophoresis is identified;
(3) with the destination carrier pEGFP-N1 of pMD19-T-NP plasmid subclones to enhanced green fluorescent tracing albumen EGFP; Transformed into escherichia coli DH-5 α; Cultivate the back and extract plasmid, Sma I, EcoR I double digestion are identified positive colony, promptly get fusion gene pEGFP-N1-NP.
3. according to the said construction process that contains the fusion gene of influenza A virus nucleoprotein of claim 2; It is characterized in that: subclone is meant in the step (3): adopt restriction endonuclease sma I and EcoR I double digestion pEGFP-N1 plasmid and pMD19-T-NP plasmid to obtain the pEGFP-N1 and the purpose nucleoprotein fragment of purifying respectively, add to connect in the ligation obtaining ligation liquid to.
4. according to the said construction process that contains the fusion gene of influenza A virus nucleoprotein of claim 2; It is characterized in that: cultivating back extraction plasmid described in the step (3) is the intestinal bacteria DH-5 α after transforming to be inoculated on the LB culture plate that contains kantlex cultivate, and picking mono-clonal bacterium colony is cultivated to the S.O.C liquid nutrient medium that contains kantlex.
5. according to the said construction process that contains the fusion gene of influenza A virus nucleoprotein of claim 2, it is characterized in that: the amplimer of pcr amplification is described in the step (1):
Upstream primer: 5'-ccgcgaattcatggcgtctcaaggcaccaaacgat-3';
Downstream primer: 5'-cgcgcccgggcattgtcatactcctctgcatt-3'.
6. state the construction process of the fusion gene that contains influenza A virus nucleoprotein according to claim 2, it is characterized in that: influenza A virus is H9N2 described in the step (1).
7. the said expression method that contains the fusion gene of influenza A virus nucleoprotein of claim 1; It is characterized in that: with pEGFP-N1-NP fusion gene transfection HEK293T cell, under inverted fluorescence microscope, observe the expression of green fluorescent protein through Lipofectamine 2000; Identify the expression of influenza A virus nucleoprotein with cellular immunization group method.
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Cited By (1)
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| CN105505848A (en) * | 2016-01-08 | 2016-04-20 | 中国科学院武汉病毒研究所 | Lassa fever virus nucleoprotein, preparation method and application |
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