CN109468281A - A BHK-21 cell line capable of stably expressing duck Tembusu virus NS1 protein and its construction method - Google Patents
A BHK-21 cell line capable of stably expressing duck Tembusu virus NS1 protein and its construction method Download PDFInfo
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Abstract
本发明公开了一种能够稳定表达鸭坦布苏病毒NS1蛋白的BHK‑21细胞系建立方法，该鸭坦布苏病毒株保藏在中国典型培养物保藏中心，其保藏名称为鸭黄病毒AH‑F10株，保藏编号为：CCTCC V201213；构建方法如下：1)重组质粒pLOV‑eGFP‑NS1的构建；2)慢病毒样颗粒的制备；3)表达NS1细胞系的构建与筛选；4)NS1基因转录和核酸检测；5)NS1蛋白表达水平的检测。本发明可以获得在细胞外表达的具有生物活性构象的分泌表达的可溶性的鸭坦布苏病毒NS1蛋白。表达NS1细胞系筛选中我们以10μg/mL的嘌呤霉素培养液进行筛选，传代5次后即可检测到NS1基因的表达，第十代NS1细胞系就可以稳定表达，能够稳定表达鸭坦布苏病毒NS1蛋白的BHK‑21细胞系方法比传统细胞系方法的时间缩短2/3。The invention discloses a method for establishing a BHK-21 cell line capable of stably expressing duck Tembusu virus NS1 protein. The duck Tembusu virus strain is preserved in the China Center for Type Culture Collection, and its preservation name is duck flavivirus AH- F10 strain, deposit number: CCTCC V201213; construction method is as follows: 1) Construction of recombinant plasmid pLOV-eGFP-NS1; 2) Preparation of lentivirus-like particles; 3) Construction and screening of cell lines expressing NS1; 4) NS1 gene Transcription and nucleic acid detection; 5) Detection of NS1 protein expression level. The present invention can obtain the secreted and expressed soluble duck Tembusu virus NS1 protein with a biologically active conformation expressed outside the cell. In the screening of cell lines expressing NS1, we screened with 10 μg/mL puromycin culture medium. After 5 passages, the expression of NS1 gene can be detected, and the tenth generation NS1 cell line can be stably expressed, which can stably express duck tembo The BHK‑21 cell line method for the NS1 protein of the Threovirus takes 2/3 less time than the traditional cell line method.
Description
Technical field
The invention belongs to veterinary lemologies and technical field of molecular biology, and in particular to one kind can stablize expression duck
The BHK-21 cells and its construction method of tembusu virus NS1 albumen.
Background technique
Duck tembusu virus disease is that the laying duck caused by tembusu virus (Duck Tembusu Virus, DTMUV) is laid eggs
There is a kind of acute infectious disease that serious nervous symptoms are main feature in rate degradation, duckling.Infect tembusu virus
The common clinical symptoms of laying duck mainly have spiritual depressed, anorexia, arrange green loose stool, general tremor, and laying rate declines 10%-
80%, it is serious even to have no harvest up to 90%.Will appear after duckling infection fall down to the ground, general tremor, last failure is dead.Die of illness duck
The visible ovarian follicle bleeding of dissect, degeneration necrosis, the congestion of blood vessel, meninx oedema.The disease disease incidence is up to 100%, the death rate about 5%-
10%.Duck tembusu virus disease, in Shanghai the first explosion, then traveled to rapidly national most of province in 2010, Anhui,
The disease also occurs in succession for the area such as Shandong, Hunan, Hebei, Guangdong, causes serious economic loss to aviculture.Due to the virus
Vigor is lower, and titre decline is fast, brings great difficulty to viral diagnosis and vaccine development, is not commercialized also on the market
Vaccine and specific treatment medicine.First non-structural protein that duck tembusu virus NS1 albumen is encoded as duck tembusu virus
It is white, it is a highly conserved glycoprotein, can be expressed in the intracellular and cell surface of virus infection, it can also be in post synthesis with can
The form secretion of dissolubility participates in the immune response of virus infection in extracellular, therefore establishes and stablize expression duck Tan Busu disease
The BHK-21 cells of malicious NS1 albumen can be effect of the further investigation duck tembusu virus NS1 albumen in virus infection in the future,
And clinically quick separating duck tembusu virus provides experimental material.
Summary of the invention
The purpose of the present invention is to solve the above problems, and expression duck tembusu virus can be stablized by proposing one kind
The BHK-21 cells of NS1 albumen can stablize the BHK-21 cells method of expression duck tembusu virus NS1 albumen than tradition
The time of cell system, method shortens 2/3.
The present invention provides the building sides that one kind can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen
Method, the specific steps are as follows:
1) building of recombinant plasmid: a pair of NS1 PCR specificity is designed according to duck flavivirus AH-F10 complete genome sequence and is drawn
Object goes out the NS1 gene containing restriction enzyme site using PCR amplification, after pcr amplification product carries out glue recycling, and uses restriction enzyme
Enzyme Nhe I and Not I carries out double digestion, and digestion products are subcloned to equally through the slow virus expression system of double digestion after digestion
In pLOV-eGFP carrier, recombinant plasmid pLOV-eGFP-NS1 is obtained；
2) preparation of slow virus sample particle: use kit extraction step 1) in recombinant plasmid pLOV-eGFP-NS1, obtain nothing
Endotoxic pLOV-eGFP-NS1 recombinant plasmid, to endotoxin-free pLOV-eGFP-NS1 recombinant plasmid, packaging plasmid pSPAX2
And outer membrane plasmid pMD2.G plasmid mixes transfection reagent cotransfection into 293T cell with the ratio of 2:1:1 μ g, after transfection for 24 hours
Collect the cell culture supernatant of the particle of sample containing slow virus；
3) building and screening of NS1 cell line are expressed: by the cell culture supernatant sense of slow virus sample particle in step 2)
BHK-21 cell is contaminated, is screened after infection with puromycin culture medium, every in the culture that 1d discards infection BHK-21 cell
Clear liquid, replaces puromycin culture solution, then continuous passage carries out pressurization screening；
4) detection of NS1 subgenomic mRNA transcription level and DNA level: certain generation BHK-21-NS1 cell is extracted respectively
Genome total serum IgE and total DNA obtain cDNA with M-MLV reverse transcription RNA, and using cDNA and total DNA as template, carry out PCR expansion
Increase, detects PCR product with 1% agarose gel electrophoresis；
5) detection of NS1 protein expression level: take the 10th generation cell carry out Western blotting detection and indirectly
Immunofluorescence, to identify the expression of NS1 albumen.
As preferred means, the sequence of NS1 PCR specific primer is as follows:
NS1-F Nhe I:5 '-CTAGCTAGCATGGACACGGGGTGCTCAATC-3 '；
NS1-R Not I:5 '-ATAAGAATGCGGCCGCTCAAGCCATGACCTTTG-3 '.
As further preferred means, infected in the step 3) with the cell culture supernatant of slow virus sample particle
Twice, every subinfection is for 24 hours for BHK-21 cell.
As further preferred means, the puromycin concentration of puromycin culture medium is 10 μ g/ in the step 3)
mL。
As further preferred means, the NS1 gene order is as shown in SEQ ID No.1.
As further preferred means, the amino acid sequence of the NS1 albumen is as shown in SEQ ID No.2.
It is a kind of it is stable expression duck tembusu virus NS1 albumen BHK-21 cells preventing, treating duck tembusu virus
Application of the NS1 albumen in duck virus infection.
Duck tembusu virus strain is deposited in China typical culture collection center, the entitled duck flavivirus AH-F10 of preservation
Strain, deposit number are as follows: CCTCC V201213.
Beneficial effects of the present invention: it can obtain in the biologically active duck tembusu virus NS1 extracellularly expressed
Albumen.We are screened in expression NS1 cell line selection with the puromycin culture solution of 10 μ g/mL, can be examined after passage 5 times
The expression of NS1 gene is measured, the 10th generation NS1 cell line can stablize expression, can stablize expression duck tembusu virus NS1 egg
White BHK-21 cells method shortens 2/3 than the time of conventional cell system, method.
Detailed description of the invention
Fig. 1 is pLOV-eGFP-NS1 double digestion qualification result.
Fig. 2 is the horizontal testing result of NS1 gene DNA.
Fig. 3 is NS1 gene mRNA transcriptional level testing result.
Fig. 4 is NS1 gene indirect immunofluorescene assay figure.
Fig. 5 is NS1 gene Western blotting detection figure.
Preservation explanation
Use AH-F10 plants of DTMUV of the present invention, which is deposited in China typical culture collection center, and address exists
China, Wuhan, Wuhan University；Deposit number CCTCC NO:V201213；The culture is sent to preservation on April 18th, 2012
Center preservation, and register on the books.Preservation 30 years from the day, the viability of the culture are detected on April 23rd, 2012
It finishes, result is survival.
Specific embodiment
Present invention is further described in detail with reference to the accompanying drawing:
Specific embodiment is as follows:
The design of 1.1NS1 gene primer
With reference to DTMUVAH-F10 plants, the complete genome sequence of (GenBank accession number: KM102539) is designed using Oligo7
A pair is for expanding the specific primer of DTMUV NS1 gene, primer sequence are as follows:
NS1-F Nhe I:5 '-CGGGATCCATGGACACGGGGTGCTCAATC-3 '；
NS1-R Not I:5 '-CCCAAGCTTTCAAGCCATGACCTTTGATTTG-3 '；
Target gene size is 1056bp, and Nhe I, Not I restriction enzyme site are added in upstream and downstream primer both ends respectively.
The extraction of 1.2 viral RNAs
The extraction of viral RNA is carried out according to laboratory conventional method, and steps are as follows:
1, the duck tembusu virus liquid that a small amount of freeze thawing is good is taken, 8000r/min is centrifuged 3min, takes supernatant；
2, take 200 μ L supernatants that 1mL TRIzol enzyme, 4 DEG C of cracking 15min are added；
3, it is sufficiently vibrated after 200 μ L chloroforms being added, 12000r/min is centrifuged 10min；
4, it takes supernatant to a sterile EP pipe, the isopropanol of equal volume, 4 DEG C of placement 20min is added；
5,12000r/min is centrifuged 10min, abandons supernatant, mixing of turning upside down manually after addition 750 μ L, 75% ethyl alcohol；
6,12000r/min is centrifuged 10min；
7, supernatant is removed, is upside down on filter paper, dries up, adds 10 μ L ddH2O is placed on -20 DEG C of preservations.
1.3NS1 the RT-PCR of gene is expanded
(1) reverse transcription
The RNA of extraction M-MLV reverse transcription, steps are as follows: RNA template 10 μ L, dNTPs being added into PCR pipe
5 × M-MLV Buffer, 5 μ is added in (10mmol/L) 3 μ L, 1 μ L of random primer, the rapid ice bath 2min after 70 DEG C of water-bath 5min
L, M-MLV reverse transcriptase 1 μ L, ddH25 μ L of O, in 42 DEG C of water-bath 50min, it is standby that the cDNA that reverse transcription is completed is placed in -20 DEG C of preservations
With.
(2) PCR amplification
Using the cDNA after reverse transcription as template, NS1-F Nhe I and NS1-R Not I is that primer carries out PCR amplification, PCR
System is 50 μ L (table 1-1), and PCR condition is 94 DEG C of initial denaturation 3min, and 94 DEG C of denaturation 40s, 55 DEG C of annealing 40s, 72 DEG C extend
1min, 72 DEG C of extensions 10min after 32 circulations, takes the progress gel electrophoresis identification of 10 μ L PCR products, band is big after reaction
Small correct PCR product send company to be sequenced.
Table 1PCR amplification system
1.4 recombinant plasmid pLOV-eGFP-NS1 building
The NS1 gene for containing restriction enzyme site using PCR amplification carries out double digestion to NS1 gene using Nhe I and Not I,
Then double enzyme digestion product is subcloned into the slow virus expression system pLOV-eGFP carrier equally through double digestion, is recombinated
Plasmid pLOV-eGFP-NS1.
The preparation of 1.5 slow virus sample particles
By above-mentioned recombinant plasmid pLOV-eGFP-NS1, with kit, (TIANGEN Biotech (Beijing) Co., Ltd. is without endogenous toxic material
The big extraction reagent kit of quality grain, centrifugal column type) purifying, obtain the recombinant plasmid pLOV-eGFP-NS1 of endotoxin-free.
Packaging plasmid pSPAX2 and outer membrane plasmid pMD2.G plasmid are the present of Chinese Academy of Sciences's Shanghai veterinary institute.
Transfection procedure is as follows:
1, cell is passaged to 10mm Tissue Culture Dish；
2, it configures transfection reagent A liquid: drawing 110 μ L liposomes 2000 (Lipofectamine 2000) in 1.5mL's
In opti-MEM culture medium, 5min is stood；
Transfection reagent B liquid: 20 μ g of endotoxin-free pLOV-eGFP-NS1 recombinant plasmid, 10 μ of packaging plasmid pSPAX2 are drawn
G, and 10 μ g of outer membrane plasmid pMD2.G is in the opti-MEM culture medium of 1.5mL.
A liquid and B liquid are mixed, mixed gently.Stand 20min；
3, the solution after mixing A, B is added dropwise to dropwise in the cell plates completed, and is incubated for 6h, and being incubated for the end time is to pass
After generation for 24 hours；
4, transfection liquid addition cell maintenance medium (cell culture fluid for containing 1% serum) is discarded after being incubated for 6h；
5, i.e. transfection finishes afterwards for 24 hours for culture.
The building and screening of 1.6 expression NS1 cell lines
The 293T cells and supernatant of the particle of sample containing slow virus is infected into BHK-21 cell.After infection for 24 hours, superinfection one
It is secondary.Second subinfection for 24 hours after, sieved with puromycin (be purchased from Amresco company) culture medium containing 10 μ g/mL working concentrations
Choosing, discards cell culture supernatant every 1d, replaces normal culture solution, pass on every other day, and 10 μ g/mL working concentrations are used after passage
Puromycin culture medium culture.It continuously passed for 5 generations with the puromycin culture medium containing working concentration and carries out pressurization screening.
1.7NS1 genetic transcription, detection:
The detection of 1.8NS1 protein expression level
The 10th generation cell is taken to carry out Western blotting detection and indirect immunofluorescence, to identify NS1 albumen
Expression.
Western Blotting test procedure
The 10th generation cell for choosing culture carries out Western blot identification, and primary antibody is the anti-NS1 of mouse mostly anti-(providing for oneself), secondary antibody
For the sheep anti-mouse igg of HRP label, it is purchased from Suo Laibao company.Key step is as follows:
(1) preparation of protein sample: cell to be checked is cleaned with 1 × PBS, 2 times, uses cell pyrolysis liquid lytic cell, ice
Upper placement 10min, 6000g are centrifuged 5min, take supernatant, are added 5 × SDS-PAGE albumen sample-loading buffer, and 100 DEG C of water-bath
10min；
(2) PAGE gel electrophoresis: electrophoresis time 80V, 30min, 120V, 100min, until bromophenol blue electrophoresis is extremely
Gel bottommost；
(3) it transfers: transfer time 90V, 50min,；
(4) it closes: preparing 5% skimmed milk power, 37 DEG C of closing 2h using PBS；
(5) primary antibody is incubated for: corresponding primary antibody is diluted according to 1:200 using PBS, 4 DEG C are incubated overnight, and TBST is cleaned 3 times,
10min/ times；
(6) secondary antibody is incubated for: dilute corresponding secondary antibody according to 1:2000 using PBS, 37 DEG C incubations 1h, TBST cleaning 3 times,
10min/ times；
(7) develop the color: under the conditions of being protected from light, it is clear to band that DAB develops the color,
Indirect immunofluorescence assay step:
By the 10th generation cell culture 36h after passage.
Indirect immunofluorescence experiment is carried out according to the following steps:
It is cleaned cell 3 times with PBS, the 500 μ L of paraformaldehyde that every hole is added 4% fixes, 4 DEG C of fixed 15min, and PBS cleans 3
It is secondary, 5min/ times；
Preparing for primary antibody source of mouse NS1 polyvalent antibody is as follows:
6 week old BALB/c female mices are immunized using the NS1 albumen of preparation as antigen.Immune programme are as follows: when first immunisation,
NS1 albumen and isometric Freund's complete adjuvant are mixed, subcutaneous injection, 100 μ g/ are only；Secondary exempt from is carried out after first immunisation 2 weeks
Epidemic disease mixes NS1 albumen and isometric Freund Freund's incomplete adjuvant, subcutaneous injection, and 100 μ g/ are only；After secondary immunity 2 weeks and 4 weeks
Be immunized three times and four times immune, the same secondary immunity of method.After four times 1 week immune, polyvalent antibody is collected.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
The present invention is not limited to above to the description of embodiment, the content that those skilled in the art disclose according to the present invention,
The improvement and modification that need not be carried out by creative work on the basis of the present invention, all should protection scope of the present invention it
It is interior.
SEQUENCE LISTING
<110>Agricultural University Of Anhui
<120>a kind of BHK-21 cells and its construction method that can stablize expression duck tembusu virus NS1 albumen
<130> 2018/10/26
<160> 1
<170> PatentIn version 3.5
SEQ ID No.1
<210> 1
<211> 1056
<212> DNA
<213> Pabellonia incrassata
<400> 1
gacacggggt gctcaatcga cttggctagg aaagaattga aatgtggaca aggcatgttc 60
gtcttcaacg atgttgaggc ttggaaagat aattataagt actatccatc cacaccaagg 120
agacttgcca aagtcgtggc agaagctcat gaggctggaa tttgtggcat acgatcagtc 180
agcaggctcg agcacaacat gtgggtaagc atcaaacatg agttgaacgc gatcttggaa 240
gacaacgcca ttgacttgac tgtggtggtt gaagaaaatc ctggaagata caggaaaact 300
aatcagaggc tgccgaacgt tgatggagag ctcatgtacg gatggaagaa atgggggaaa 360
agtattttta gcagcccgaa gatgtcaaat aatacatttg tcatcgatgg accaaaaact 420
aaagagtgcc cagatgagag aagagcatgg aatagtatga agattgaaga ctttgggttt 480
ggagtgttgt ccacaaaggt atggatggaa atgcggacag aaaatacaac tgattgtgac 540
accgcagtaa tgggcacagc aattaaagga aatagagctg tgcacagtga cctgagctat 600
tggatagaga gcaagaataa tggaagctgg aaactggaga gagctgtgct gggcgaggtg 660
aagtcatgca catggccgga aacccacaca ctgtggagtg acagcgttgt ggagagtgaa 720
ctcatcatac ctaaaacatt gggaggaccg aagagtcatc acaacacgag gacaggatac 780
aaggttcaga gttccggacc gtgggatgag aaagagattg tagtagactt cgactactgc 840
cctggaacaa ctgtcacagt aacgagctcg tgccgcgaca gagggccttc agctaggaca 900
acaacagcga gtgggaaact gataacagat tggtgttgta ggtcttgcac caccccacca 960
ctgagatttg ttacaaaaag tggatgctgg tatgggatgg aaattcggcc aactgctcac 1020
ggagacgaca tgttgatcaa atcaaaggtc atggct 1056
SEQ ID NO.2
<210> 2
<211> 352
<212> PRT
<213> Pabellonia incrassata
<400> 2
Asp Thr Gly Cys Ser Ile Asp Leu Ala Arg Lys Glu Leu Lys Cys Gly
1 5 10 15
Gln Gly Met Phe Val Phe Asn Asp Val Glu Ala Trp Lys Asp Asn Tyr
20 25 30
Lys Tyr Tyr Pro Ser Thr Pro Arg Arg Leu Ala Lys Val Val Ala Glu
35 40 45
Ala His Glu Ala Gly Ile Cys Gly Ile Arg Ser Val Ser Arg Leu Glu
50 55 60
His Asn Met Trp Val Ser Ile Lys His Glu Leu Asn Ala Ile Leu Glu
65 70 75 80
Asp Asn Ala Ile Asp Leu Thr Val Val Val Glu Glu Asn Pro Gly Arg
85 90 95
Tyr Arg Lys Thr Asn Gln Arg Leu Pro Asn Val Asp Gly Glu Leu Met
100 105 110
Tyr Gly Trp Lys Lys Trp Gly Lys Ser Ile Phe Ser Ser Pro Lys Met
115 120 125
Ser Asn Asn Thr Phe Val Ile Asp Gly Pro Lys Thr Lys Glu Cys Pro
130 135 140
Asp Glu Arg Arg Ala Trp Asn Ser Met Lys Ile Glu Asp Phe Gly Phe
145 150 155 160
Gly Val Leu Ser Thr Lys Val Trp Met Glu Met Arg Thr Glu Asn Thr
165 170 175
Thr Asp Cys Asp Thr Ala Val Met Gly Thr Ala Ile Lys Gly Asn Arg
180 185 190
Ala Val His Ser Asp Leu Ser Tyr Trp Ile Glu Ser Lys Asn Asn Gly
195 200 205
Ser Trp Lys Leu Glu Arg Ala Val Leu Gly Glu Val Lys Ser Cys Thr
210 215 220
Trp Pro Glu Thr His Thr Leu Trp Ser Asp Ser Val Val Glu Ser Glu
225 230 235 240
Leu Ile Ile Pro Lys Thr Leu Gly Gly Pro Lys Ser His His Asn Thr
245 250 255
Arg Thr Gly Tyr Lys Val Gln Ser Ser Gly Pro Trp Asp Glu Lys Glu
260 265 270
Ile Val Val Asp Phe Asp Tyr Cys Pro Gly Thr Thr Val Thr Val Thr
275 280 285
Ser Ser Cys Arg Asp Arg Gly Pro Ser Ala Arg Thr Thr Thr Ala Ser
290 295 300
Gly Lys Leu Ile Thr Asp Trp Cys Cys Arg Ser Cys Thr Thr Pro Pro
305 310 315 320
Leu Arg Phe Val Thr Lys Ser Gly Cys Trp Tyr Gly Met Glu Ile Arg
325 330 335
Pro Thr Ala His Gly Asp Asp Met Leu Ile Lys Ser Lys Val Met Ala
340 345 350
Claims (7)
1. the construction method that one kind can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen, it is characterised in that:
Specific step is as follows:
1) pair for amplification NS1 gene PCR specificity the building of recombinant plasmid: is designed according to duck flavivirus AH-F10 complete genome sequence
Primer carries out PCR amplification, double digestion, digestion products to NS1 gene and is subcloned to equally through the slow virus expression system of double digestion
In pLOV-eGFP carrier, recombinant plasmid pLOV-eGFP-NS1 is obtained；
2) preparation of slow virus sample particle: to step 1) recombinant plasmid pLOV-eGFP-NS1 with kit purify without in
The recombinant plasmid pLOV-eGFP-NS1 of toxin, using recombinant plasmid pLOV-eGFP-NS1, the packaging plasmid of endotoxin-free
With transfection reagent cotransfection into 293T cell, transfection is collected afterwards for 24 hours containing slow disease for pSPAX2 and outer membrane plasmid pMD2.G plasmid
The cell culture supernatant of malicious sample particle；
3) building and screening of NS1 cell line are expressed: the cell culture supernatant of slow virus sample particle in step 2) is infected
BHK-21 cell is screened with purine-containing mycin culture medium；
4) NS1 genetic transcription and detection of nucleic acids: extraction step 3) certain generation BHK-21-NS1 cell genome total serum IgE, carry out
Reverse transcription distinguishes PCR amplification with the total DNA of extraction, and detects PCR product with 1% agarose gel electrophoresis；
5) detection of NS1 protein expression level: the 10th generation cell is taken to carry out Western blotting detection and be immunized indirectly
Fluorescence, to identify the expression of NS1 albumen.
2. one kind according to claim 2 can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen
Construction method, it is characterised in that: the sequence of the NS1 gene PCR specific primer is as follows:
NS1-F Nhe I:5 '-CTAGCTAGCATGGACACGGGGTGCTCAATC-3 '；
NS1-RNot I:5 '-ATAAGAATGCGGCCGCTCAAGCCATGACCTTTG-3 '.
3. one kind according to claim 1 can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen
Construction method, it is characterised in that: infect BHK-21 cell with the cell culture supernatant of slow virus sample particle in the step 3)
Twice, every subinfection is for 24 hours.
4. one kind according to claim 1 can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen
Construction method, it is characterised in that: the puromycin concentration of puromycin culture medium is 10 μ g/mL in the step 3).
5. one kind according to claim 1 can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen
Construction method, it is characterised in that: the NS1 gene order is as shown in SEQIDNo.1.
6. one kind according to claim 1 can stablize the BHK-21 cells of expression duck tembusu virus NS1 albumen
Construction method, it is characterised in that: the amino acid sequence of the NS1 gene is as shown by seqid no.2.
7. a kind of BHK-21 cells of stable expression duck tembusu virus NS1 albumen are preventing, treating, are detecting duck Tan Busu disease
Poison and the application in duck virus infection.
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| CN115074389A (en) * | 2022-06-13 | 2022-09-20 | 华南农业大学 | Construction method and application of cell line for stably expressing duck tembusu virus NS1 protein |
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| CN114380921A (en) * | 2022-01-19 | 2022-04-22 | 中国农业科学院北京畜牧兽医研究所 | Human ferritin-based duck tembusu virus E protein nano vaccine, antigen and application thereof |
| CN114380921B (en) * | 2022-01-19 | 2023-05-30 | 中国农业科学院北京畜牧兽医研究所 | Nanometer vaccine and antigen of duck tembusu virus E protein based on human ferritin and application thereof |
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| CN117448284A (en) * | 2023-11-02 | 2024-01-26 | 广东省农业科学院动物卫生研究所 | Recombinant tembusu virus expressing EGFP (enhanced green fluorescent protein) through stable passage and construction method |
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