CN101935675B - Construction and application of cell membrane expression ALV-env fluorescin eukaryotic transgenic expression plasmid - Google Patents

Abstract

The invention relates to the construction and application of a fluorescent eukaryotic transgene expression plasmid expressing ALV-env protein on the cell membrane. Fluorescent eukaryotic transgene expression plasmid of env protein, the plasmid contains ALV-env gene. After the plasmid is transfected into the host cell, the ALV-env gene and the green fluorescent protein gene (EmGFP) gene can be integrated into the host cell chromosome, and the envelope protein of avian leukemia is expressed on the surface of the host cell membrane. The reporter gene-green fluorescent protein was expressed in the plasma. The membrane-expressed ALV-env protein fluorescent eukaryotic transgene expression plasmid can be used to prepare avian leukosis pseudovirus, and the prepared avian leukemia pseudovirus can be used to detect J-subtype avian leukemia neutralizing antibody.

Description

Construction and application of fluorescent eukaryotic transgene expression plasmid expressing ALV-env protein on cell membrane

(1) Technical field

The invention relates to the construction and application of a cell membrane-expressed ALV-env protein fluorescent eukaryotic transgene expression plasmid, which belongs to the field of biotechnology.

(2) Background technology

The envelope glycoprotein (env) encoded by the avian leukosis virus envelope glycoprotein gene (ALV-env) includes the envelope surface protein gp85 and the penetrating protein gp37, both of which first form the gp85-gp37 polymeric precursor protein in the host cell, Then processed and sheared to form mature envelope glycoproteins gp85 and gp37. The envelope protein encoded by the ALV-env gene is the main surface protein of this type of virus. The envelope protein recognizes and binds to the specific receptor on the surface of the target cell, and is necessary for the virus to enter the target cell for replication and proliferation.

The pLenti7.3 plasmid is a lentiviral eukaryotic expression plasmid vector (Lentiviral vector) constructed by Invitrogen using HIV gene fragments. The pLenti7.3 plasmid can introduce the target gene recombined into its multiple cloning site into the transfected host cell nucleus and integrate into the host cell chromosome. The pLenti7.3 plasmid can transcribe the DNA sequence between its HIV long terminal hybrid promoter (RSV/5'LTR) and SV40pA into a long mRNA, and then reverse transcribe it into cDNA. After this part of the DNA sequence is integrated into the host cell chromosome, under the action of the CMV promoter, the inserted target gene can be transcribed in the host cell nucleus, and the target gene can be expressed in the cytoplasm; at the same time, the green fluorescent protein carried by pLenti7.3 can be expressed ( EmGFP).

(3) Contents of the invention:

The first object of the present invention is to construct a fluorescent eukaryotic transgene expression plasmid (pLenti7.3/ALV- env).

Another object of the present invention is the application of pLenti7.3/ALV-env in preparing avian leukosis pseudovirus.

The present invention is realized through the following technical solutions:

A cell membrane expressing ALV-env protein fluorescent eukaryotic transgene expression plasmid, its nucleotide sequence table is shown in SEQ ID NO: 1; its ALV-env protein amino acid sequence is shown in SEQ ID NO: 2.

A cell membrane expressing ALV-env protein fluorescent eukaryotic transgene expression plasmid is to use specific primers containing promoter sequences: upstream primer: 5'ATGGAAGCCGTCATAAAGGCATTTCTGACTGGGCAC 3'; downstream primer: 5'GACAGCTGCTCCCTAATTCTATG 3', from the genome of avian leukosis virus by PCR (extracted from chicken embryo fibroblast culture) to amplify the env gene. The avian leukosis env gene was recombined with the commercialized pLenti7.3 expression plasmid to form a fluorescent eukaryotic transgene expression plasmid (pLenti7.3/ALV-env) expressing the ALV-env protein on the cell membrane. The Escherichia coli containing the pLenti7.3/ALV-env plasmid was deposited in the General Microbiology Center (CCTCC) of the China Committee for Culture Collection of Microbial Cultures (CCTCC) on November 06, 2009. The preservation number is CGMCC NO: 3415, and the classification name is Escherichia coli Bacteria Escherichia coli.

The present invention also includes the application of the above cell membrane expressed ALV-env protein fluorescent expression transgene plasmid on avian leukosis pseudovirus.

Cell membrane expression ALV-env protein fluorescent expression transgenic plasmid is transfected into host cells (such as HEK293 cells), the env gene can express the envelope protein of avian leukemia, and colonize the surface of the host cell membrane, while expressing the reporter gene in the host cell plasma -Green fluorescent protein. The ALV-env gene and the green fluorescent protein gene (EmGFP) are integrated into the host cell chromosome, and the envelope protein of avian leukosis is expressed on the surface of the host cell membrane, and the reporter gene-green fluorescent protein is expressed in the host cell plasma. Utilize this HEK293 cell that shows green fluorescence and expresses ALV-env protein on the cell membrane, can prepare avian leukosis pseudovirus by means of helper plasmids (pLP1, pLP2, Invitrogen Company).

The prepared pseudovirus can be used to detect the neutralizing antibody of avian leukosis virus (ALV-J subtype): when there is a sufficient amount of anti-ALV-J subtype neutralizing antibody in the sample to be tested, the pseudovirus can be prevented from interacting with the host Cell binding, when the sample has no anti-avian leukemia virus (ALV-J subtype) neutralizing antibody or less neutralizing antibody, the pseudovirus can be combined with the host cell, and after 24 hours, check whether the host cell has green color under a fluorescent microscope Fluorescence, if there are host cells that can emit green fluorescence, it means that there is no neutralizing antibody in the sample, and the sample test result is negative; if there are no host cells that can emit green fluorescence (negative control sample cells can emit fluorescence) cells, it means that there are cells in the sample Neutralizing antibodies, the sample tested positive.

The beneficial effects of the present invention are: use the pLenti7.3/ALV-env plasmid constructed in the present invention to co-transfect HEK293 cells with the helper plasmid to prepare ALV-J pseudovirus, which can replace the use of live ALV-J virus for neutralization test, The pollution of the environment by the live ALV virus is avoided. Since the pseudovirus can fluoresce automatically, the fluorescent antibody staining part can be omitted in the neutralization experiment to reduce the non-specific fluorescent reaction.

(4) Description of drawings

Figure 1 Electrophoresis results of ALV-env gene amplified by PCR: 1 is env amplified fragment; M is DNA molecular weight marker 2K DNA Marker.

Figure 2 The identification results of T-env digestion of ALV-env gene and Peasy-T1 cloning vector: 1 is single digestion of T-env clone; 2 is double digestion of T-env; 3 is DNA molecular weight marker 2K plus DNA Marker.

Figure 3 The results of pLenti7.3/ALV-env enzyme digestion and identification of ALV-env gene pLenti7.3 vector construction: 1 is the double digestion product of ALV-env/ALV-env expression vector; M is the DNA molecular weight marker 15K DNA Marker.

Fig. 4 Structural diagram of fluorescent eukaryotic transgene expression plasmid expressing ALV-env protein on cell membrane: ALV-env is an inserted exogenous gene, and the rest is pLenti7.3 vector.

The structural features of the pLenti7.3/ALV-env expression plasmid are as follows:

HIV long terminal hybrid promoter (RSV/5'LTR hybrid promoter): 1-410

RSV promoter: bases 1-229

HIV-1 5'LTR: bases 230-410

HIV-1 psi(ψ)packaging signal: 521-565

HIV Response Element (HIV-1 Rev response element (RRE)): 1075-1308

Cppt: bases 1801-1923

CMV promoter: 1935-2519

Avian Leukemia Membrane Protein Gene (ALV-env): 2570-4327

V5 label (V5epitope): 4345-4386

WPRE: bases 4405-5002

SV40 promoter (SV40promoter): 5013-5321

Green fluorescent protein gene (EmEMGFP): 5380-6099

HIV long terminal repeat (ΔU3/3'LTR): 6170-6404

ΔU3: bases 6170-6223

3'LTR: bases 6224-6404

SV40 polyadenylation signal: 6476-6607

Ampicillin (bla) resistance gene: 7565-8425

Plasmid backbone (pUC origin): 8570-9243

Among them, the avian leukosis membrane protein gene (ALV-env) is the gene prepared by the present invention, and the rest is from the commercialized plasmid pLenti7.3.

Figure 5 Fluorescence detection results 48 hours after expression vector transfection: the cells that emit fluorescence are HEK293 cells that have been transfected with pLenti7.3/ALV-env and are about to release pseudoviruses.

Observation of the neutralization test results in Fig. 6: after culturing for 24 hours after the combination of the pseudovirus and the host cells, the cells that can emit green fluorescence observed under a fluorescent microscope, the sample is now negative.

(5) Specific implementation methods

1. Acquisition of ALV-env gene

The avian leukemia ALV-env gene is amplified from the cell culture of the avian leukemia virus by using the ALV-env specific primer containing the promoter sequence through the PCR method.

1. Extraction of DNA template

The ALV-J virus strain was inoculated in a 100 mL cell culture flask to grow into a 70%-80% chicken embryo fibroblast (CEF) monolayer. The cells were maintained for 7-10 days after inoculation, and the cells inoculated with the virus were collected, and the tissue DNA was extracted according to the following procedures:

1) Collect the cells in the cell bottle inoculated with the virus, blow and wash the cells several times with Hanks solution, then add 0.5-1.0ml of 0.25% trypsin to each bottle, place in a 37°C incubator for 5-10min, digest the cells after blowing Collected in 5ml centrifuge tube.

2) Wash with 1×PBS solution, transfer the pellet by centrifugation at 2000rpm to a 1.5ml centrifuge tube, then add 300μl extract solution (100mmol/L NaCl, 10mmol/L Tris.Cl pH 8.0, 25mmol/L EDTA pH 8.0, 0.5% SDS ) after suspension, proteinase K (100 μg/ml) was added and digested overnight in a water bath at 55°C.

3) Add an equal volume of phenol/chloroform solution (phenol: chloroform: isoamyl alcohol = 25:24:1) to extract about 400 μl once, transfer the upper layer liquid to another 1.5ml centrifuge tube, add 1/10 volume of 3mol /L of sodium acetate and 2 times the volume of absolute ethanol, and then cooled at -20°C for 2 hours or longer.

4) After taking out, centrifuge at 12000rpm for 10min to precipitate the DNA, suspend the DNA precipitate with 70% ethanol, centrifuge at 12000rpm for 5-10min, and discard the supernatant.

5) The DNA after ethanol precipitation, after natural drying at room temperature in the air, was dissolved in TE Buffer (10mmol Tris-Cl, 1mmol EDTA, pH 8.0) in a volume of 50μl, and the nucleic acid quantification was about 50-100ng/μl, which was the template DNA to amplify the proviral DNA template of ALV-J integrated into the cellular genome.

2. ALV-env gene amplification

ALV-env gene-specific primers:

Upstream primer: 5'ATGGAAGCCGTCATAAAGGCATTTCTGACTGGGCAC 3'

Downstream primer: 5'GACAGCTGCTCCCTAATTCTATG 3'

The PCR reaction system is as follows:

ddH ₂ O 15.8 μL

10×Buffer 2.5μL

_MgCl2 2.0 μL

4dNTP 2.0μL

Upstream primer (20pM) 1.0μL

Downstream primer (20pM) 1.0μL

Taq enzyme 0.2μL

Model DNA 1μL

The PCR reaction procedure is as follows

94℃ 10min

94℃ 1min

57℃ 1min

72℃ 2min

72℃ 10min

Store at 4°C

After the reaction, agarose electrophoresis was performed, and the length of the target gene was 1710bp, as shown in Figure 1.

3. PCR product and Peasy-T1 clone

The amplified PCR product was connected to the Peasy-T1 cloning vector to obtain the T-env cloning vector. Its construction process is:

To establish a 5 µl connection system:

PCR product 0.5μL

Peasy-T1 4.5μL

The connection system reacts at room temperature for 10-15 minutes

Transformation of Competent Cells:

Take 5 μL of the ligation product to transform into TOP10 competent cells.

Pick a single positive colony, inoculate it into a test tube containing 5 mL of LB (100 μg/mL) medium, and incubate overnight at 37°C with shaking. The plasmid was extracted, digested with restriction endonucleases BamH1 and Xho1, and electrophoresed on 1% agarose gel. Two DNA fragments of 1710bp and 3928bp were seen, as shown in Figure 2.

Two, ALV-env and pLenti7.3 connection:

The T-env cloning vector was digested with restriction endonucleases BamH1 and Xho1. 1% agarose gel was used for electrophoresis, and ALV-env fragments with sticky ends were recovered from the gel.

Using restriction endonucleases BamH1 and Xho1 to digest the plasmid pLenti7.3, connect the linearized vector and the recovered DNA fragment with T4DNA ligase, and obtain the fluorescent eukaryotic transgene expression plasmid pLenti7.3/ Alv-env plasmid.

Its construction process is:

Double digestion of T-env vector and pLenti7.3 expression vector

Establish a 50 microliter enzyme digestion reaction system:

BamH1 2 μL

Xho1 2 μL

10×K Buffer 5μL

Plasmid 30μL

ddH2O 11μL

37 ℃ water bath for 4h. Electrophoresis gel recovery.

The ALV-env fragment was connected to the pLenti7.3 expression vector:

Recover the expression vector and ALV-env fragment, and establish a ligation reaction system:

PCR product 2μl

pLenti7.3 8μl

Solution I 10μl

Ligation overnight at 16°C.

5. Transformation and enrichment

Transform Stbl3 competent, pick a single colony, inoculate into a test tube containing 10mL LB (containing 100μg/mL) medium, culture overnight at 37°C with shaking, extract the plasmid and digest it with restriction endonucleases BamH1 and Xho1, and use 1 % agarose gel electrophoresis, two DNA fragments of 1710bp and 7935bp were seen, as shown in Figure 3. DNA sequence determination confirmed that ALV-env had been recombined with the pLenti7.3 expression vector, and the reading frame was correct, and the pLenti7.3/ALV-env expression vector plasmid was obtained.

6. Bacteria enrichment and plasmid extraction

Mass culture, use QIAGEN DNA plasmid purification kit, extract plasmid, electrophoresis identification. The specific process is as follows:

(1) Re-plate the screened clones, pick a single colony, inoculate it into a test tube containing 10mL LB medium, culture it with shaking at 37°C overnight, take 1 mL of the culture and inoculate it into a medium containing 100mL LB (containing 100μg/mL) Incubate overnight at 37°C with shaking in a test tube.

(2) Use the QIAGEN DNA plasmid purification kit to extract the plasmid:

1) Take 30mL of bacterial culture in a 50mL centrifuge tube and centrifuge to precipitate the bacteria.

2) Add 1mL Lysis Solution I, shake vigorously, and suspend the bacteria.

3) Add 1mL Lysis Solution II, mix gently, and ice-bath for no more than 5 minutes.

4) Add 1mL Lysis Buffer III, shake up and down, and ice-bath for 10 minutes.

5) Centrifuge at 12000rpm for 10min, and take the supernatant.

6) Put the supernatant into the chromatographic column, let it stand vertically, and wait until the supernatant completely passes through the chromatographic column.

7) Add 2mL for elution, and stand vertically until the eluent passes through the column completely.

8) Collect the eluate, detect the DNA concentration, and store it in a -20°C refrigerator for later use.

3. Preparation of HEK293 cells expressing ALV-env protein in cell membrane and green fluorescent protein in cytoplasm

HEK293 cells were inoculated in a 24-well plate, 1 ml MEM+10% FBS (fetal bovine serum) per well, and cultured until 70%-80% cell confluence. Add liposome-DNA transfection solution (pLenti7.3/ALV-env 0.8μg; helper plasmids PLp1, PLp20.8μg; liposome 2μg) expression plasmid mixed solution to each well, oscillate slightly to mix, and store at 37°C, Incubate in an incubator with 5% CO ₂ for 6 hours, discard the transfection solution, add 1ml of DMEM containing 10% FBS to each well, and observe for 60-72 hours that green fluorescence can appear in the host cell plasma (excitation light wavelength 488nm, emission light wavelength 567nm), see Figure 5. The supernatant was collected, and after titration of the virus titer (ID ₅₀ ), it could be used to detect neutralizing antibodies against avian leukosis virus. The specific method is: serially dilute the serum sample, mix 50-100*ID ₅₀ avian leukosis pseudovirus with the same amount of diluted sample to be tested, if the sample contains a sufficient amount of anti-ALV-J subtype neutralizing antibody , can prevent the pseudovirus from combining with the host cell; when the sample has no anti-avian leukosis virus (ALV-J subtype) neutralizing antibody or less neutralizing antibody, the pseudovirus can combine with the host cell. After 24 hours, check whether the host cells have green fluorescence under a fluorescent microscope. If there are host cells that can emit green fluorescence, it means that there is not enough neutralizing antibody in the sample (as shown in Figure 6). If there are no host cells that can emit green fluorescence ( Negative control sample cells can emit fluorescence) cells indicate that there are neutralizing antibodies in the sample.

<110> Shandong Agricultural University

<120> cell membrane expression ALV-env fluorescent eukaryotic transgene expression vector

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attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat 180

aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact 240

ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc 300

aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag 360

agacagatcc attcgattag tgaacggatc tcgacggtat cggttaactt ttaaaagaaa 420

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acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttatcg ataagcttgg 540

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tagtaacggc cgccagtgtg ctggaattgc cctt atg gaa gcc gtc ata aag gca 1195

                                            5’UTP Met Glu Ala Val Ile Lys Ala

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ttt ctg act ggg cac cct gga aag gtg agc aag aag gac tct aag aag 1243

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Phe Cys Leu Ser Leu Gln Ser Ala Thr Ser Pro Phe Arg Thr Cys Leu

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Asn Val Thr Ala Cys Asp Asn Asp Ala Asp Leu Ala Ser Gln Thr Ala

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Cys Leu Ile Gln Ala Leu Asn Thr Thr Leu Pro Trp Asp Pro Gln Glu

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Leu Asp Ile Leu Gly Ser Gln Met Ile Lys Asn Gly Thr Thr Arg Thr

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Cys Val Thr Phe Gly Ser Val Cys Tyr Lys Glu Asn Asn Arg Ser Arg

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gtc tgt cac aat ttt gat ggg aat ttt aat ggg act ggt ggg gcg gaa 1771

Val Cys His Asn Phe Asp Gly Asn Phe Asn Gly Thr Gly Gly Ala Glu

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gca gaa ttg cgt gac ttc ata gca aaa tgg aaa agt gat gac ctt ctt 1819

Ala Glu Leu Arg Asp Phe Ile Ala Lys Trp Lys Ser Asp Asp Leu Leu

200 205 210 215

ata agg ccc tat gtc aac caa tca tgg acg atg gta agt cca ata aac 1867

Ile Arg Pro Tyr Val Asn Gln Ser Trp Thr Met Val Ser Pro Ile Asn

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Thr Glu Ser Phe Ser Ile Ser Ser Ser Arg Tyr Cys Gly Phe Thr Ser Asn

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Glu Thr Arg Tyr Tyr Arg Arg Asn Arg Ser Ser Trp Cys Ser Ser Lys

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Asn Thr Arg Gly Cys Gly Gly Asn Cys Thr Ala Glu Trp Asn Tyr Tyr

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Gly Thr Ala Lys Ala Leu Pro Pro Gly Ile Phe Leu Ile Cys Gly Asp

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Leu Gly Gln Leu Thr Met Leu Ser Pro Asn Phe Thr Thr Trp Ile Thr

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Pro Glu Lys Thr Pro Leu Leu Pro Ser Arg Gly Tyr Phe Phe Phe Gln

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Thr Ile Leu Val Cys Val Val Ile Ile Ser Val Val Pro Gly Val Gly

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Gly Val His Leu Leu Arg Gln Pro Gly Asn Val Trp Val Thr Trp Ala

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Asn Met Thr Gly Arg Thr Asp Phe Cys Leu Ser Leu Gln Ser Ala Thr

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Ser Pro Phe Arg Thr Cys Leu Ile Gly Ile Pro Gln Tyr Pro Leu Asn

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Thr Phe Glu Gly Tyr Val Thr Asn Val Thr Ala Cys Asp Asn Asp Ala

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Asp Leu Ala Ser Gln Thr Ala Cys Leu Ile Gln Ala Leu Asn Thr Thr

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Leu Pro Trp Asp Pro Gln Glu Leu Asp Ile Leu Gly Ser Gln Met Ile

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Lys Asn Gly Thr Thr Arg Thr Cys Val Thr Phe Gly Ser Val Cys Tyr

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Lys Glu Asn Asn Arg Ser Arg Val Cys His Asn Phe Asp Gly Asn Phe

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Asn Gly Thr Gly Gly Ala Glu Ala Glu Leu Arg Asp Phe Ile Ala Lys

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Trp Lys Ser Asp Asp Leu Leu Ile Arg Pro Tyr Val Asn Gln Ser Trp

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Thr Met Val Ser Pro Ile Asn Thr Glu Ser Phe Ser Ile Ser Ser Ser Arg

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Tyr Cys Gly Phe Thr Ser Asn Glu Thr Arg Tyr Tyr Arg Arg Asn Arg

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Ser Ser Trp Cys Ser Ser Lys Gly Gly Glu Trp Ser Ala Gly Tyr Ser

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Asn Gly Thr Glu Cys Ser Ser Asn Thr Arg Gly Cys Gly Gly Asn Cys

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Thr Ala Glu Trp Asn Tyr Tyr Ala Tyr Gly Phe Thr Phe Gly Lys Gln

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Pro Glu Val Leu Trp Asn Asn Gly Thr Ala Lys Ala Leu Pro Pro Gly

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Ile Phe Leu Ile Cys Gly Asp Arg Ala Trp Gln Gly Ile Pro Arg Asn

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Ala Leu Gly Gly Pro Cys Tyr Leu Gly Gln Leu Thr Met Leu Ser Pro

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Asn Phe Thr Thr Trp Ile Thr Tyr Gly Pro Asn Ile Thr Gly His Arg

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Arg Ser Arg Arg Ser Pro Asn His Leu Ser Pro Asp Cys Asn Asp Glu

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Val Gln Leu Trp Ser Val Thr Ala Arg Ile Phe Ala Ser Phe Phe Ala

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Pro Gly Val Ala Ala Ala Gln Ala Leu Lys Glu Ile Glu Arg Leu Ala

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Cys Trp Ser Val Lys Gln Ala Asn Leu Thr Ser Leu Ile Leu Asn Ala

420 425 430

Ile Leu Glu Asp Thr Asn Ser Ile Arg His Ala Val Leu Gln Asn Arg

435 440 445

Ala Ala Ile Asp Phe Leu Leu Leu Ala Gln Gly His Gly Cys Gln Asp

450 455 460

Val Glu Gly Met Cys Cys Phe Asn Leu Ser Asp His Ser Glu Ser Ile

465 470 475 480

His Lys Ala Leu Gln Ala Met Lys Glu His Thr Glu Lys Ile Arg Val

485 490 495

Glu Asp Asp Pro Ile Gly Asp Trp Phe Thr Arg Thr Phe Gly Gly Leu

500 505 510

Gly Gly Trp Leu Ala Lys Gly Val Lys Thr Leu Leu Phe Ala Leu Leu

515 520 525

Val Ile Val Cys Leu Leu Ala Ile Ile Pro Cys Ile Ile Lys Cys Phe

530 535 540

Gln Asp Cys Leu Ser Arg Thr Met Tyr Gln Phe Met Asp Glu Arg Ile

545 550 555 560

Arg Tyr His Arg Ile Arg Glu Gln Leu Ser Arg Ala Ile Leu Gln Ile

565 570 575

Ser Ile Thr Leu Ala Ala Ala Arg Val

580 585

Claims (2)

1. an Escherichia coli (Escherichia coli) containing pLenti7.3/ALV-env plasmid, it is characterized in that this bacterial strain has been preserved on November 06, 2009 in the General Microorganism Center of China Committee for Microorganism Culture Preservation, and the preservation number is CGMCC NO: 3415. 2. the application of the pLenti7.3/ALV-env plasmid contained in the escherichia coli described in claim 1 in the preparation of avian leukosis pseudovirus.

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