CN115074389A - Construction method and application of cell line for stably expressing duck tembusu virus NS1 protein - Google Patents

Abstract

The invention discloses a construction method and application of a cell line for stably expressing duck tembusu virus NS1 protein, and relates to the technical field of molecular biology. The construction method comprises the following steps: (1) constructing recombinant plasmids; (2) transfecting cells by using the recombinant plasmid to obtain recombinant cells; (3) co-culturing the recombinant cells and the lentivirus packaging plasmids to obtain a culture solution of lentivirus-like particles; (4) infecting host cells with the culture solution of the lentivirus-like particles, screening to obtain stable strains, and culturing to obtain a cell line. By implementing the invention, the expression level of NS1mRNA and protein can be stably improved.

Description

Construction method and application of cell line for stably expressing duck tembusu virus NS1 protein

Technical Field

The invention relates to the technical field of biological engineering, in particular to a construction method and application of a cell line for stably expressing duck tembusu virus NS1 protein.

Background

Duck tembusu virus (DTMUV) is a causative agent that causes infected birds to exhibit egg drop syndrome as a major symptom, and its host of infection includes chickens, ducks, geese, sparrows and pigeons. In the last ten years of 2010, DTMUV disease firstly erupts in the southeast region of China and rapidly spreads to the middle part and all parts of the country in a short time. After the meat duck is infected, the meat duck is mainly characterized by ataxia, slow growth, listlessness and inappetence; infected laying ducks are mainly characterized in that the laying rate is low for a long time, and the fertility rate and the hatching rate of the laying eggs after recovery are also affected; the clinical symptoms of the infected ducks are blue and thin stool accompanied by nerve symptoms such as quadriplegia, head swing and the like, and the death rate is 5-25%. After the diseased duck is subjected to cesarean examination, the liver has hemorrhage and slight swelling; the spleen has a uniform needle-like white dead center; ovaries can show bleeding, atrophy, necrosis and chapping due to different infection durations; pathological section observation can diagnose hemorrhagic oophoritis. At present, DTMUV is mainly popular in most duck and goose raising areas in China, and causes huge economic loss to the water poultry industry in China.

NS1 has a molecular weight of about 40kDa and consists of 352 amino acids. Mainly exists in infected cells, but can be positioned on the cell surface and slowly secreted from the cells. Under the action of a host signal peptidase, NS1 is translocated into the endoplasmic reticulum and separated from the E protein, and in the process, a host protease is also involved. It was considered that NS1 functions early in viral replication, and NS1 and NS4A participate in RNA replication through interaction. The NS1 protein is also the major antigen of the virus and induces the production of non-neutralizing antibodies by cells accompanied by a complement response. NS1 can inhibit induction of host cell type I IFNs to antagonize natural immune response, and provide favorable conditions for virus replication. The cell line over-expresses the NS1 protein and can be used for the subsequent research on the functional interaction of the NS1 protein.

Disclosure of Invention

The invention aims to solve the technical problem of providing a construction method and application of a cell line for stably expressing duck tembusu virus NS1 protein, wherein the cell line can stably express NS1 protein.

In order to solve the technical problems, the invention provides a primer combination for NS1 gene in the Canine duck Tembusu virus, which is characterized by comprising a primer I and a primer II, wherein the primer I has a nucleotide sequence shown in SEQ ID NO: 1; the second primer has a nucleotide sequence shown as SEQ ID NO: 2.

Correspondingly, the invention also discloses a construction method of a cell line for stably expressing the duck tembusu virus NS1 protein, which comprises the following steps:

(1) constructing recombinant plasmids by adopting the primer combination;

(2) transfecting cells by using the recombinant plasmid to obtain recombinant cells;

(3) co-culturing the recombinant cells and the lentivirus packaging plasmids to obtain a culture solution of lentivirus-like particles;

(4) infecting host cells with the culture solution of the lentivirus-like particles, screening to obtain stable strains, and culturing to obtain a cell line.

As an improvement of the technical scheme, the step (1) comprises the following steps:

(1.1) carrying out PCR amplification on the NS1 gene by adopting the primer combination;

(1.2) carrying out homologous recombination on the PCR amplification product and a PSE5519 vector obtained by enzyme digestion, and transforming the PCR amplification product into DH5 alpha competent bacteria;

(1.3) carrying out monoclonal bacterial amplification culture on DH5 alpha competent bacteria, and isolating to obtain a PSE5519-NS1 recombinant plasmid.

As an improvement of the technical scheme, in the step (1.2), the PMT406 vector is subjected to enzyme digestion by using BamHI-HF and XhoI, and a 8537bp vector fragment is recovered to obtain a PSE5519 vector.

As an improvement of the above technical scheme, in the step (2), the cell is a BHK-21 cell.

As an improvement of the above technical scheme, in the step (3), the lentivirus packaging plasmids are pCMV-dR8.9 plasmid and pCMV-VSV-G plasmid.

As an improvement of the above technical scheme, in the step (4), the host cell is BHK-21 cell.

Correspondingly, the invention also discloses a plasmid which is the PSE5519-NS1 recombinant plasmid.

Correspondingly, the invention also discloses a plasmid set which comprises the PSE5519-NS1 recombinant plasmid, the pCMV-dR8.9 plasmid and the pCMV-VSV-G plasmid.

Correspondingly, the invention also discloses an application of the cell subline constructed by the construction method in any one of the following (1) to (3):

(1) preparing and/or screening a medicine for treating duck tembusu virus;

(2) preparing a vaccine for preventing duck tembusu virus;

(3) a kit for detecting duck tembusu virus.

The implementation of the invention has the following beneficial effects:

according to the invention, a carrier PSE5519-NS1 for stably over-expressing NS1 is constructed through a specific primer combination, and a cell line for stably expressing duck Tembusu virus non-structural protein NS1 is further constructed. The cell line can stably express NS1 protein, is complementary with a proper flavivirus lacking NS1 in a trans-form manner, is used for rescuing the lacking virus, and can realize laboratory operation of low biological safety level on the flavivirus with high biological level safety. The cell line of the invention has the advantages that: the expression quantity of the NS1 protein is strong, and the puro resistance of a stable strain of BHK cells is obvious; the fluorescence efficiency is above 90%, and the detection is effective through WB and QPCR. Compared with CN109468281A, the cell line has high mRNA expression efficiency, and protein expression can be detected without NS1 specific antibody.

Drawings

FIG. 1 is a map of tool vector PMT406 in example 1;

FIG. 2 is a map of the support PSE5519 in example 1;

FIG. 3 is a diagram showing PCR identification of colonies in example 1; wherein M is a DNA molecular weight standard, and 1-4 is a selected positive transformant;

FIG. 4 is a graph showing the results of the q-PCR detection in example 4;

FIG. 5 is a fluorescent picture of the stable strain BHK-PSE5519 in example 4;

FIG. 6 is a fluorescent photograph of the stabilized strain BHK-PMT406 in example 4;

FIG. 7 is a graph showing the results of the western blot test in example 4, wherein 1 is BHK-PMT406, and 2 is BHK-PSE 5519;

FIG. 8 is a graph showing the results of the western blot test in example 4 of example 4, wherein 1 is BHK-PMT406 and 2 is BHK-PSE 5519.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

The apparatus used in the examples of the present invention, the reagents were as follows:

ultra clean bench SW-CJ-2FD (Suzhou Antai air technologies, Inc., Jiangsu, China); CO2 constant temperature incubator Forma 371(Thermo Corp., USA); biosafety cabinet 1300SERIES A2(Thermo corporation, USA); inverted optical microscope (Nikon corporation, japan); pipette Research plus (Eppendorf company, germany); high speed Centrifuge 5804R (Eppendorf corporation, germany); PCR apparatus C1000 Touch (Bio-Rad, USA); vertical electrophoresis tank Mini protein Tetra (Bio-Rad, USA); electrophoresis apparatus Power Pac Basic (Bio-Rad, USA); gel imaging system 2500(R) (Tanon corporation, shanghai, china).

Gel Extraction Kit (D2500-02) was purchased from OMEGA; BCA quantification kit was purchased from Thermo (23227); flag (hrp) antibody purchased from YEASEN (30502ES 60); ACTIN antibodies were purchased from YEASEN (30103ES 60); the TIAN prep Mini plasma Kit was purchased from Promega (A1460); lipofectamine LTX and Plus Reagent (15338-100) from Invitrogen; PBS from GIBCO, (cat. No. 14190); Opti-MEM was obtained from GIBCO (cat. No.31985) 0.25% Trypsin-EDTA (25200-056), DMEM basic (C11995500BT) from Gibco; FBS (10099-141C) was purchased from Gibco; Premix-Taq (RR902A) was purchased from TAKARA; prime STAR GXL (R050) from TAKARA; TRIZOL RNA isolation reagents were purchased from Invitrogen; PrimeScriptRT Master Mix was purchased from taraka; TaKaRa TB Green ^TM Premix Ex Taq ^TM II from takara; RT T4 DNA ligase and other commonly used restriction enzymes were purchased from TAKARA; anti-DTMUV NS1 protein antibodies were prepared by tokensry bio-limited, tokyo.

pCMV-dR8.9 plasmid and pCMV-VSV-G plasmid, purchased from Addgene. Coli competent cell DH5 α was purchased from Dalianbao Nissan Co., Ltd.

The sequencing and primer synthesis related to the embodiment of the invention are completed by the company of Biotechnology engineering (Shanghai).

Example 1 construction of plasmid PSE5519-NS1 for overexpression of NS1

1. Designing a primer for fishing the NS1 gene;

Primer 1(+)：

5’-TGGCAAAGAATTGGATCCGCCACCATGGACACGGGGTGCTCA-3’。

Primer 2(－)：

5’-AGCCATGACCTTTGATTTGATC-3’。

the Primer I (Primer 1, SEQ ID NO: 1) and the Primer II (Primer 2, SEQ ID NO: 2) are used for PCR to obtain the sequence of the target gene NS 1.

Primer 3(+)

5’-CGGCTCTAGAGCCTCTGCTA-3’

Primer 4(－)

5’-CGTGAGTCAAACCGCTATCCAC-3’

The third Primer (Primer 3, SEQ ID NO: 3) and the fourth Primer (Primer 4, SEQ ID NO: 4) are used for colony PCR to identify transformants. The primers were synthesized by bioengineering.

2. Amplifying a target gene fragment by PCR;

specifically, the PCR system is shown in the following table:

reagent	Volume (μ L)
		ddH 2 O	32.5
5×Buffer(with Mg 2+ )	10
		10×T4 Ligation Buffer	1
dNTPs (2.5 mM each)	4
		Primer1(+)(10μM)	1
Primer2(－)(10μM)	1
		Template	1
PrimeSTAR	0.5

The PCR conditions were: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.

3. The instrument vector PMT406 (see FIG. 1) was digested with BamHI-HF and XhoI, and a 8537bp vector fragment was recovered to obtain PSE 5519.

4. The target fragment amplified by PCR is subjected to homologous recombination with linearized PSE5519, and the specific formula is shown in the following table:

reagent	Positive control/. mu.L	Self-ligation control/. mu.L	Ligation group/. mu.L
				Target gene fragment after glue recovery	4	4	4
Linearized expression vector	1	1	1
				Seamless cloning reaction solution	15	0	15
dd H2O	Up to 20	Up to 20	Up to 20

After mixing, incubation was carried out at 42 ℃ for 30 minutes and then transferred to ice. After standing for 2-3 minutes, 10. mu.L of the reaction liquid was transferred to competent cells.

Description of the invention: positive control and self-ligation control, the vector added is identical to the ligation group, but the gene fragment added in the positive control is the target gene (with the same homologous recombination exchange arm)

5. The ligation product was used directly to transform DH 5. alpha. competent bacteria and transformed according to the manufacturer's instructions. After the transformation, the transformants grown on the plate were picked and resuspended in 10. mu.L of LB medium, and 1. mu.L of the resulting culture was used as a template for colony PCR identification. And PCR identification is carried out on the bacterial colony; specifically, the method comprises the following steps: the PCR system is shown in the following table:

reagent	Volume (μ L)
		ddH 2 O	13.7
10×Buffer(with Mg 2+ )	2
		dNTPs (2.5 mM each)	1.6
Primer3(+)(10μM)	0.8
		Primer4(－)(10μM)	0.8
Template	1
		Taq enzyme	0.1

The PCR conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.

As shown in FIG. 3, the 1393bp fragment was obtained from the positive clone, while the 0bp fragment was obtained from the negative clone.

6. Monoclonal bacteria were picked, amplified and cultured, and plasmids were extracted with the TIAN prep Mini Plasmid Kit, named PSE5519-NS1, and subjected to PCR sequencing. The sequencing result is shown in SEQ NO: 5, the sequencing result of the positive clone shows that the sequence is basically consistent with the expected target gene sequence.

Example 2 Lentiviral packaging and purification

1. Cell preparation and transfection

The 293T cells of 2T 10cm culture dishes were prepared in advance, and the growth morphology of the cells was good.

The medium was DMEM + 10% FBS, 1% Glutamax, 1% penicillin-streptomycin.

The cells were divided into 10cm dishes at a cell density of about 1X 10 per flask ⁷ And (4) respectively. After 24h, cells were examined under the mirror. The degree of cell fusion should be about80-90%, and is uniformly distributed.

1 hour prior to transfection, the cell plates were removed, the original cell culture medium was removed, 9. mu.L of Opti-MEM medium was added, and the cells were returned to the incubator.

Two sterile 1.5. mu.L centrifuge tubes were taken, and 100. mu.g of PSE5519-NS1 plasmid, 65. mu.g of dR8.9 packaged plasmid and 35. mu.g of VSVG plasmid were added to tube A and made up to 5. mu.L with Opti-MEM medium. Standing for 5 min.

To tube B, 500. mu.L of Trans-EZ solution and 4.5. mu.L of Opti-MEM medium were added and gently mixed by an electropipettor. The Trans-EZ diluted solution is dripped into the tube A, and the tube A is gently shaken while adding.

The incubation was carried out at room temperature for 20 minutes to allow sufficient binding of the DNA and the Trans-EZ to form a transfection complex.

The resulting DNA-Trans-EZ complex was added dropwise to the cell culture plate 1. mu.L each, using 1. mu.L pipette.

Shake the plate back and forth, mix well and return to the 5% carbon dioxide incubator.

After 6 hours, the cell supernatant was removed and replaced with 10. mu.L of DMEM complete medium. Cells were observed 24h after transfection, and all cells should be healthy and at a density of approximately 60-80%.

The cells were returned to the incubator for further 2 days (36-48 hours). During this process, the cells gradually fuse to form a polykaryon, and most of the cells remain attached.

All supernatants were collected and aliquoted into 50 μ L centrifuge tubes. The cells were centrifuged at 500g for 10min at 4 ℃ to remove exfoliated cells and large cell debris. The total supernatant was about 160. mu.L and filtered through a 60. mu.L 0.22 μm PVDF filter unit. If the filter membrane is found to be clogged, it appears that the filtration rate becomes slow and the filter is replaced with a new one.

2, concentration and purification of lentivirus

Taking 6 μm Ltra-clear SW28 centrifuge tubes, sterilizing with 70% ethanol, placing in an ultra-clean bench, turning on an ultraviolet lamp, and sterilizing for 30 min.

Approximately 32 μ L of pre-treated virus supernatant was added to each m Ltra-clear SW28 centrifuge tube.

A10. mu.L pipette was used to aspirate 12. mu.L of 20% sucrose solution. The pipette is inserted all the way to the bottom of the centrifuge tube and the sucrose solution is gently pipetted out 4. mu.L. Similarly, the remaining 8 μ L of sucrose solution was added to two additional centrifuge tubes, respectively.

Another clean pipette was taken and the remaining 3 tubes were treated the same way. The weight of each tube was adjusted with PBS so that the weight difference between the corresponding centrifuge tubes did not exceed 0.1 g.

All 6 tubes were placed in sequence into a Beckman SW28 ultracentrifuge rotor. Centrifuge at 25000rpm (82700g) for 2 hours at 4 ℃.

The tube is carefully removed from the rotor. The supernatant was decanted off and the tube was placed on a paper towel for 10 minutes to allow the remaining supernatant to drain. The remaining droplets were aspirated off. There should be visible precipitation at the bottom of the tube.

The pellet was washed with 200. mu.L of Opti-MEM without serum added to each tube. The SW28 ultracentrifuge tubes were inserted into 50 μ L conical-bottomed centrifuge tubes and the lids were closed. Dissolve for 2 hours at 4 ℃ with gentle shaking every 20 minutes. The solution was concentrated at the bottom of the tube by centrifugation at 500g for 1 minute at 4 ℃. The pellet was resuspended by gentle pipetting using a 200 μ L pipette. Avoiding the generation of foam. The liquid in all tubes was pooled into one SW28 centrifuge tube.

The pooled virus suspension was aliquoted into 50. mu.L portions and stored in finished tubes. The mixture was quickly frozen with crushed dry ice and stored at-80 ℃.

Example 3 screening of BHK cells stably expressing NS1

BHK cells were cultured in a T75 culture dish until the growth state was good, and the culture solution was discarded and washed twice with 2. mu.L of PBS.

mu.L of 0.25% trypsinized cells were added, and after counting the cells, 6 well plates were plated, 30 ten thousand cells per well.

After 12h the cell growth status was observed under a microscope and the infection was performed with lentivirus at an MOI of 50.

And (3) changing the liquid 8H after infection, adding puromycin at 48H after infection for screening, wherein the concentration is 2 ug/mu L, and changing the liquid every two to three days.

The selection was continued for 2 weeks to obtain stable strains, which were sampled for mRNA and WB detection.

Example 4 identification of cell line function by qPCR and western blot

This example was carried out using the stable strain obtained in example 3 (nomenclature: BHK-PSE 5519). In addition, experiments were performed using a blank set (i.e., the plasmid in the transfection process in example 2 was directly replaced with PMT406 plasmid, named BHK-PMT406) to control the experimental results.

First, qPCR experiment

Freezing and freezing the cell strain for 3 times, sucking 300 microliter of Trizol added with 700 microliter of Trizol, and standing at room temperature for 5min to fully crack the cell strain.

2. Centrifuge at 12000rpm for 5min, discard the precipitate.

3. Chloroform was added to 200. mu.L of chloroform/. mu.L of Trizol, followed by shaking and mixing, and then allowed to stand at room temperature for 15 min.

4. Centrifuge at 12,000g for 15min at 4 ℃.

5. The upper aqueous phase was aspirated into another centrifuge tube. No intermediate interface is sucked;

6. adding 0.5 muL isopropanol/muL Trizol into isopropanol, mixing, and standing at room temperature for 5-10 min.

7. Centrifugation at 12,000g for 10min at 4 ℃ removed the supernatant and RNA deposited at the bottom of the tube.

8. Add 75% ethanol to 1. mu.L of 75% ethanol/. mu.L Trizol, gently shake the centrifuge tube, and suspend the precipitate.

9. Centrifuge at 8,000g for 5min at 4 ℃ and discard the supernatant as much as possible.

10. Air drying at room temperature or vacuum drying for 5-10 min.

11. Dissolve 50 μ L EPC in water, incubate at 55-60 deg.C for 5 min.

12. After determination of the RNA concentration according to takara PrimeScript ^TM RT Master Mix (Perfect Real Time) indicates reverse transcription into cDNA using TaKaRa TB Green ^TM Premix Ex Taq ^TM II qPCR was performed. Wherein, the primer sequences and PCR systems are shown in the following two tables

The results of the experiment are shown in the following table and fig. 4, from which it can be seen that: the target virus PSE5519 infects a BHK cell sample, and the QPCR detection result shows that the NS1 gene has obvious overexpression effect. The NS1 gene over-expression stable strain is successfully constructed.

Quantitative values and analysis (2-. DELTA.Ct analysis)

Second, Western Blot experiment

Collecting cells, discarding cell culture solution, adding appropriate amount of 1 × Lysis Buffer according to cell amount, and lysing cells at 4 deg.C for 10-15 min.

And scraping the cells by using a cell scraper, transferring the cells into a correspondingly marked 1.5 mu L EP tube, and carrying out ice bath for 10-15 min.

Centrifuge at 12000g for 5min at 4 ℃ and aspirate the supernatant slowly into the correspondingly labeled 1.5 μ LEP tube. And (4) quantifying the protein by using the BCA method, and calculating the protein concentration.

Treating the protein sample: adding 5 Loading Buffer, heating at 95-100 deg.C for 5min, and storing at-20 deg.C for use.

Formulation of SDS-PAGE

1) The same volume of the separating gel buffer solution and the separating gel solution are mixed evenly, namely, 2.0/2.7/4.0 mu L of each solution is taken.

2) 40/60/80 mu L of modified ammonium persulfate solution is added into the mixed solution in the step 1) and mixed evenly.

3) Injecting the solution obtained in the step 2) into a glue-making glass plate, and adding a proper amount of water or ethanol to cover the separation glue.

4) And pouring out the upper water or alcohol after the separation gel is solidified.

5) Adding 10/15/20 μ L modified ammonium persulfate solution into 1.0/1.5/2.0 μ L concentrated gel premix, and mixing.

6) Injecting the mixture into a glue-making glass plate, and inserting comb teeth. After the concentrated gel is solidified, the comb teeth are pulled out for electrophoresis.

Electrophoresis

1) Loading: the gel was clamped in the electrophoresis chamber and sufficient 1 x electrophoresis solution was added to begin the preparation.

2) Electrophoresis: the constant current of the two pieces of glue is 25mA-30mA for about 3 h.

3) Registering: and recording information such as loading sequence and antibody dilution ratio on the loading record.

Immunoblotting (Wet transfer)

Film transferring: the splint is placed into a transfer electrophoresis device, a proper 1-transfer buffer solution is added, and the membrane is transferred for 120min under the condition of constant current of 400mA in an ice bath.

Immunochromatographic development

And (3) sealing: preparing sealing solution (TBST solution containing 5% skimmed milk), completely immersing PVDF membrane in the sealing solution, sealing at room temperature for 2 hr

Primary anti-incubation: incubate at 4 degrees overnight.

Washing the membrane: taking out PVDF membrane, placing into culture dish, adding appropriate amount of 1 × TBST solution, placing on decolorizing shaker, gently shaking, washing membrane for 3 times, each time for 10min

And (3) secondary antibody incubation: diluting the corresponding secondary antibody with the blocking solution, and incubating the PVDF membrane for 2h at room temperature

Washing the membrane: 1 TBST washing membrane 3 times, each time 10 min.

ECL color development: color was developed on a gel imaging system.

The results are shown in FIGS. 5 to 8, and it can be seen that the test group BHK-PSE5519NS1 protein is well expressed.

The above description is only one preferred embodiment of the present invention, and should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Sequence listing

<110> southern China university of agriculture

HUANONG (ZHAOQING) BIOLOGICAL INDUSTRY TECHNOLOGY RESEARCH INSTITUTE Co.,Ltd.

<120> construction method of cell line stably expressing duck tembusu virus NS1 protein and application thereof

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agccatgacc tttgatttga tc 22

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cggctctaga gcctctgcta 20

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cgtgagtcaa accgctatcc ac 22

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ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg 60

gatccgccac catggacacg gggtgctcaa tcgacttggc taggaaagaa ttgaaatgtg 120

gacaaggcat gtttgtcttc aacgatgttg aggcttggaa ggataattat aagtactatc 180

catccacacc aaggagactt gccaaagtcg tggcagaagc tcatgaggct ggaatttgtg 240

gcatacgatc agtcagcagg ctcgagcaca acatgtgggt aagcatcaaa catgagttga 300

acgcgatctt ggaagacaac gccattgact tgactgtggt ggttgaagaa aatcctggaa 360

gatacaggaa aaccaatcag aggctgccga acgttgatgg agagctcatg tacggatgga 420

agaaatgggg gaaaagtatt tttagcagcc cgaagatgtc aaataataca tttgtcatcg 480

atggaccaaa aactaaagag tgcccagatg agagaagagc atggaatagt atgaagattg 540

aagactttgg gtttggagtg ttgtccacaa aggtatggat ggaaatgcga acagaaaata 600

caactgattg tgacaccgca gtaatgggca cagcaattaa aggaaataga gctgtgcaca 660

gtgacctgag ctattggata gagagcaaga ataatggaag ctggaaactg gagagagctg 720

tgctgggcga ggtgaagtca tgcacatggc cggaaaccca cacactgtgg agtgacagcg 780

ttgtggagag tgaactcatc atacctaaaa cattgggagg accgaagagt catcacaaca 840

cgaggacagg atacaaggtt cagagttccg gaccgtggga tgagaaagag attgtaatag 900

acttcgacta ctgccctgga acaactgtca cagtaacgag ctcgtgccgc gacagagggc 960

cttcagctag gacaacaaca gcgagtggga aactgataac agattggtgt tgtaggtctt 1020

gcaccacccc accactgaga tttgttacaa aaagtggatg ctggtatggg atggaaattc 1080

ggccaactgc tcacggagac gacatgttga tcaaatcaaa ggtcatggct gactacaagg 1140

atgacgatga caaggattac aaagacgacg atgataagga ctataaggat gatgacgaca 1200

aataactcga gactagtatt atgcccagta catgacctta tgggactttc ctacttggca 1260

gtacatctac gtattagtca tcgctattac catggtgatg cggttttg 1308

Claims (10)

1. A primer combination for fishing NS1 gene in duck tembusu virus is characterized by comprising a primer I and a primer II, wherein the primer I is a primer with a nucleotide sequence shown as SEQ ID NO: 1; the second primer has a nucleotide sequence shown as SEQ ID NO: 2.

2. A method for constructing a cell line stably expressing a duck Tembusu virus NS1 protein, which comprises the following steps:

(1) constructing a recombinant plasmid using the primer combination of claim 1;

(2) transfecting cells by using the recombinant plasmid to obtain recombinant cells;

(3) co-culturing the recombinant cells and the lentivirus packaging plasmids to obtain a culture solution of lentivirus-like particles;

(4) infecting host cells with the culture solution of the lentivirus-like particles, screening to obtain stable strains, and culturing to obtain a cell line.

3. The method of constructing as claimed in claim 2, wherein step (1) comprises:

(1.1) performing PCR amplification of the NS1 gene using the primer combination of claim 1 or claim 2;

(1.2) carrying out homologous recombination on the PCR amplification product and a PSE5519 vector obtained by enzyme digestion, and converting the PCR amplification product into DH5 alpha competent bacteria;

(1.3) carrying out monoclonal bacterial amplification culture on DH5 alpha competent bacteria, and isolating to obtain a PSE5519-NS1 recombinant plasmid.

4. The method of claim 3, wherein in step (1.2), the PMT406 vector is digested with BamHI-HF and XhoI, and 8537bp vector fragment is recovered to obtain the PSE5519 vector.

5. The method of claim 2, wherein in the step (2), the cell is a BHK-21 cell.

6. The method of claim 2, wherein in step (3), the lentiviral packaging plasmids are a pCMV-dR8.9 plasmid and a pCMV-VSV-G plasmid.

7. The method of claim 2, wherein in the step (4), the host cell is a BHK-21 cell.

8. Plasmid, characterized in that it is a PSE5519-NS1 recombinant plasmid as claimed in claim 3.

9. A packaged plasmid comprising the PSE5519-NS1 recombinant plasmid of claim 3 and the pCMV-dr8.9 plasmid, pCMV-VSV-G plasmid of claim 6.

10. The use of a cell subline constructed by the method according to claim 2 in any one of the following (1) to (3):

(1) preparing and/or screening a medicine for treating duck tembusu virus;

(2) preparing a vaccine for preventing duck tembusu virus;

(3) a kit for detecting duck tembusu virus.

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