CN110257389A

CN110257389A - A kind of J subgroup avian leucosis virus duplication reinforcing agent

Info

Publication number: CN110257389A
Application number: CN201910386917.2A
Authority: CN
Inventors: 成子强; 庞宇; 周德方; 张利
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2019-09-20

Abstract

The present invention provides a kind of J subgroup avian leucosis virus to replicate reinforcing agent, it is three spiral repetitive proteins 1 of collagen that the J subgroup avian leucosis virus, which replicates reinforcing agent, the albumen can be used as virus replication reinforcing agent, directly, significantly and continuously facilitate ALV-J duplication, virus load increases 2 times or more compared with the control group when virus of receiving, and to cytotoxic side effect, inventor further constructs CTHRC1 recombinant plasmid for promoting ALV-J to replicate, make CTHRC1 can be with continuous expression in cell, persistently promote ALV-J duplication, improves its expression quantity.

Description

A kind of J subgroup avian leucosis virus duplication reinforcing agent

Technical field

The present invention relates to molecular immunologies and field of virology, and in particular to a kind of J subgroup avian leucosis virus duplication Reinforcing agent.

Background technique

ALV-J is typical oncogenicity retrovirus, and main clinical manifestation is the white blood of myelocytome sample in chicken group Disease, and mostly tumour etc. occur with growth retardation, high mortality, immune tolerance and more histoorgans for main feature, ALV-J since 1988 since Britain is separated to for the first time, to China and or even the aviculture in the world cause huge loss, in recent years, I Though the generation of state's J subgroup avian leucosis in being gradually reduced trend, 2018 the beginning of the year China six province broken out again typically The Asia myelocytome sample J fowl group's leukaemia, find through inventor's analysis: this time the outburst of J subgroup avian leucosis is made a variation by ALV-J Strain causes.Continuous prevalence with ALV-J in field, pathogenic also continuous enhancing, and host range are only infected from initial Commodity egg, local breeder flock and other poultry have been gradually expanded in commercial broiler group.Meanwhile ALV-J is infecting new host When occur falling ill it is more anxious；Infection rate, morbidity and mortality are higher；Also the features such as more diversified, occurs for tumour.In addition, a large amount of Research report display, the fowl tumor disease based on J subgroup avian leucosis can also be with Marek's disease and reticular endothelium hyperplasia The mixing of the tumor diseases such as disease occurs, and this considerably increases the prevention and control difficulty of J subgroup avian leucosis, bring to aviculture huge Economic loss and potential hidden danger.

With the continuous expansion of ALV-J research and the application successively of deep and all types of ALV-J biological agents, to ALV- The demand of J is increasing, however the virus has typical slow virus characteristic, and ALV-J replicative cycle is up to 1 week；And In the state of ALV-J natural infection, the virus load generated is lower, is far from satisfying in fast-developing every research Relevant enterprise and the unit researcher demand growing to ALV-J.

Virus genomic composition is fairly simple for host, viral due to lacking required enzyme system, so Its reproduction process for needing to complete itself in a replication process using the albumen of host.For virus, it be must adapt to The locating environment complicated and changeable containing thousands of host proteins；However, ten hundreds of host proteins is also virus replication Provide a large amount of available resources.After host infection virus, virus can kidnap the host's egg for being conducive to life cycle It is white or utilize its function with host protein interaction, so that itself reproduction process of virus is completed, to maintain virus in place Persistent infection state in main body.

The related product for improving virus load listed at present, there are apparent technological deficiencies for action principle；Its effect It can not achieve the purpose that significantly improve virus load.Most products are deposited in the form of viruses contact reinforcing agent on the market at present In technical principle are as follows: virus is largely enriched in cell surface by physical action by virus, greatly increases virus and cell Contact, promotes virus infected cell to greatest extent.And the method for this physics can only promote virus to contact with each other with cell Probability, not can guarantee whether really promote virus duplication；Since cell has density dependency and most cells The characteristic of adherent generation is needed, so the surface area of cell is limited, causes limitation to the effect of the product；Meanwhile this The physical means that class product uses cannot continuously promote the duplication of virus.Illustrated by principles above, such product mentions The ability of high ALV-J carrying capacity is than relatively limited and be not routinely to promote virus replication.

Therefore how to obtain a kind of ALV-J duplication reinforcing agent promotes ALV-J to replicate significantly, and solution is producing at present The problem low with the viral yield encountered in practice becomes one of prior art urgent problem to be solved.

Summary of the invention

For the existing above problem, the present invention provides a kind of J subgroup avian leucosis virus to replicate reinforcing agent, should J subgroup avian leucosis virus duplication reinforcing agent is three spiral repetitive proteins 1 of collagen, and the albumen is for the first time in the egg of infection ALV-J It is found in white matter group, is found for the first time through follow-up study, which can be used as virus replication reinforcing agent, directly, significantly Ground is replicated with ALV-J is continuously facilitated, and virus load increases 2 times or more compared with the control group when collecting virus, and to thin Born of the same parents have no toxic side effect, and inventor further established CTHRC1 recombinant plasmid for promoting ALV-J to replicate, and make CTHRC1 in cell In can persistently promote ALV-J duplication, improve its expression quantity, very good solution is at present in production and practice with continuous expression The low problem of the viral yield encountered.

The present invention is that the further investigation carried out based on following theoretical basis is obtained:

Three spiral repetitive proteins 1 of collagen (collagen triple helix repeat containing 1, CTHRC1) thin in the Adventitial fibroblasts and smooth muscle in Damage of Rats vascular repair process in 2005 by Pyagay first It is found in born of the same parents.CTHRC1 relative molecular weight about 25KDa is a kind of outer secreting type glycoprotein, with the albumen homology being currently known It is extremely low, but the albumen between species than more conservative.CTHRC1 is transcribed in nucleus, is secreted into cytoplasm after being activated In, finally reach in extracellular matrix.CTHRC1 albumen mainly consists of three parts, including the N- containing cell exocrine function Terminal signal peptide and three spiral sequence of collagen and C- distal ball structure being made of 36 amino acid.In the prior art simultaneously There is no three spiral repetitive proteins 1 of collagen report relevant to ALV-J to occur, the research of the two is in space state；

Many effort based on inventor finally utilize proteomics method, and having screened, there is ALV-J duplication to rely on The key difference albumen " CTHRC1 " of property；Followed by experimental results demonstrate CTHRC1 to have ALV-J replication-dependent, and And it establishes the albumen and can be used as the preparation and application of ALV-J duplication reinforcing agent.For grinding for subsequent ALV-J biological products System provides most important and most essential Research foundation --- virus.

Specific step is as follows:

Favorable albumen is replicated to ALV-J in order to screen, to the DF-1 cell for infecting ALV-J virus and normally DF-1 cell carries out proteomics detection (Tandem Mass Tag, TMT method), to the difference egg in two histone matter groups White progress bioinformatic analysis and pertinent literature are consulted, and the crucial up-regulation closely related with ALV-J virus replication has been filtered out Differential protein " CTHRC1 "；

First demonstration that direct interaction between ALV-J and CTHRC1；Prove that ALV-J infection causes simultaneously Subcellular localization variation of the CTHRC1 from nucleus to cytoplasm, the two interaction in cytoplasm promote ALV-J duplication；

It is tried by quantitative fluorescent PCR test, Western Blot Assay, enzyme-linked immunosorbent assay and immunohistochemistry It tests, the expression quantity of CTHRC1 is verified in vivo and in vitro, the results showed that ALV-J has activated the expression of CTHRC1；

Whether there is ALV-J replication-dependent for verifying CTHRC1, inventor constructs CTHRC1 over-express vector, the load Body carries out codon optimization to the base sequence of CTHRC1, rare bases is substituted, to improve its expression quantity；DF-1 cell crosses table Up to after CTHRC1 recombinant plasmid, be inoculated with ALV-J, experimental result show: be overexpressed CTHRC1 can extremely significant raisings cell with carefully Virus load in born of the same parents' supernatant；On the contrary, the virus load of ALV-J is remarkably decreased after striking low CTHRC1；We further pass through and exempt from Epidemic disease co-precipitation and this phenomenon of laser co-focusing verification experimental verification, as the result is shown: with ALV-J SU albumen interaction can occur for CTHRC1, This experiment further illustrates feasibility of the CTHRC1 as ALV-J duplication reinforcing agent.

The above process more specifically steps are as follows:

It infects CTHRC1 expressing quantity in the proteomics of ALV-J and significantly raises (change > 1.5 fold):

J subgroup avian leucosis virus (NX0101 plants of ALV-J, virus titer TCID₅₀=10^-4.5/ 100 μ L) infection After DF-1 cell, 72h is maintained, carries out proteomics detection.Bioinformatics is carried out to the differential protein in proteomics Analysis, analyzes the function of its differential protein.Through analyzing, have found that one is in 1.5 times of differential proteins of infection ALV-J The albumen " CTHRC1 " that fowl field was not reported, after testing, the nucleotide sequence of the albumen such as SEQ ID NO.1 institute Show, the amino acid sequence of coding is as shown in SEQ ID NO.2；By sequence alignment discovery chicken source CTHRC1 and source of people CTHRC1 Sequence homology is 75.6%, it was demonstrated that it belongs to CTHRC1 albumen；

Also, find that there is close for differential protein CTHRC1 and virus replication rate by bioinformatic analysis Connection；Meanwhile inventor has found that the high expression of CTHRC1 can inhibit the deposition of collagen, the decline of collagen expression It can change the permeability of host cell membrane, the change of permeability is conducive to the duplication of virus.Therefore, inventor speculates CTHRC1 It is ALV-J replication-dependent albumen.

The expression of ALV-J activation CTHRC1: to the potential target gene that screens carry out fluorescence quantification PCR primer design, The purchase of corresponding antibodies and ELISA kit；DF-1 infect ALV-J after, detect respectively for 24 hours, 48h, 72h and 96h CTHRC1 The dynamic change of mRNA level in-site and protein expression amount.As the result is shown: after infection ALV-J, CTHRC1 expression quantity, which is presented, to be risen Trend；ALV-J can activate the expression of CTHRC1.It illustrates the dynamic rule of CTHRC1 in vivo, is mentioned for later period research High strong data supporting.

It is overexpressed CTHRC1 and dramatically increases ALV-J virus load:

Firstly, carrying out the building of carrier for expression of eukaryon, pcDNA3.1 eukaryon expression plasmid is selected, will be sequenced completely without mutation Gene order be inserted into plasmid, then the plasmid built is transformed into competent cell DH5 α, conversion after the completion of shake Bacterium extracts plasmid, and is sequenced, and errorless rear progress cell transfecting is sequenced；After DF-1 transfection CTHRC1 is overexpressed plasmid 8h, inoculation ALV-J virus verifies the carrying capacity of virus after maintaining 72h.As the result is shown: compared with transfecting pcDNA3.1 empty carrier group, Transfection is overexpressed in the cell of CTHRC1 plasmid, virus load significant (2 times or more) up-regulation of ALV-J.This illustrates that CTHRC1 has There is viral replication-dependent.

Interference CTHRC1 significantly inhibits the duplication of ALV-J: promoting the result of ALV-J virus replication to carry out reversely CTHRC1 Verifying --- shRNA is used, two CTHRC1 interference plasmids (shCTHRC1-1, nucleotide sequence such as SEQ ID are constructed Shown in NO.3；And shCTHRC1-2, nucleotide sequence is as shown in SEQ ID NO.4).Interference plasmid is transfected into cell, then It is inoculated with ALV-J, as a result, it has been found that: after interference CTHRC1, the virus load of ALV-J is significant compared with the unloaded group of transfection to be lowered.This is again Secondary explanation, CTHRC1 are host proteins indispensable in ALV-J reproduction process.

CTHRC1 can interact with ALV-J SU albumen, and it is multiple to further illustrate that CTHRC1 promotes ALV-J System:

After DF-1 cell infection ALV-J, 72h is maintained.Co-Immunoprecipitation (Co-IP) experiment is carried out, is made It is bait with ALV-J SU protein monoclonal antibody (1D4), then precipitate C THRC1 carries out the protein sample of precipitating Western blot detection.It was found that there is interaction in the two.Inventor uses laser confocal microscope again, observes CTHRC1 How to interact in cell with ALV-J.As a result, it has been found that: normal group DF-1 cell, the distribution that CTHRC1 is mainly dispersed in In nucleus；And ALV-J group is connect, CTHRC1 is distributed mainly in cytoplasm, and around nucleus.Meanwhile connecing poison In group, ALV-J and CTHRC1 can overlap, and carry out supplement verifying to CO-IP experiment.

Based on the above-mentioned technical proposal, compared with prior art, the invention has the advantages that

1, inventor has found that ALV-J replication-dependent PROTEIN C THRC1, the albumen can be used as virus replication reinforcing agent, Directly, significantly and continuously facilitate ALV-J duplication, when collecting virus compared with the control group virus load increase 2 times with On, and to cytotoxic side effect.

2, the new method that CTHRC1 recombinant plasmid promotes ALV-J duplication is established, this method makes CTHRC1 can in cell With continuous expression, persistently promote ALV-J duplication.

Detailed description of the invention

Fig. 1 proteomics method screens the schematic diagram of ALV-J duplication dependent protein CTHRC1,

Wherein a: thermal map (Heat-map)；B: volcano figure (Volcano Plot).C:GO is enriched with analysis chart (Gene Ontology),

The expression accompanying drawings of Fig. 2A LV-J activation CTHRC1；

A: after infection ALV-J, the mrna expression amount figure of different time sections CTHRC1；B and c1: different after infection ALV-J The protein expression spirogram of period CTHRC1；C2: can to the CTHRC1 expressing quantity progress of c1 using Image J software Depending on changing；D: after infection ALV-J, different time sections ALV-J absolute fluorescence is quantitatively schemed；E: after infection ALV-J, different time sections ALV- J protein expression spirogram,

Fig. 3 is overexpressed CTHRC1 and dramatically increases ALV-J carrying capacity accompanying drawings,

A, b and c: transfection CTHRC1 be overexpressed plasmid after, infection ALV-J maintain 72h after CTHRC1 mrna expression amount and Protein expression spirogram；C, d and e: after transfection CTHRC1 is overexpressed plasmid, infection ALV-J maintains the absolute fluorescence of ALV-J after 72h Quantitative and protein expression spirogram,

Fig. 4 interference CTHRC1 obviously inhibits the duplication accompanying drawings of ALV-J；

A, b and c: after transfection CTHRC1 interference plasmid, infection ALV-J maintains the mrna expression amount and egg of CTHRC1 after 72h White expression spirogram；C, d and e: after transfection CTHRC1 plasmid, infection ALV-J maintains the absolute fluorescence of ALV-J after 72h quantitative and egg White expression spirogram.

The related experiment schematic diagram that Fig. 5 CTHRC1 can interact with ALV-J SU albumen；

A:CTHEC1 and ALV-J SU protein-interacting figure；B:CTHRC1 positions variation diagram.

Specific embodiment

In the present embodiment unless otherwise specified, other are all made of prior art completion.

The DF-1 cell of 1 couple of embodiment infection ALV-J carries out proteome analysis (TMT method)

1) preparation of cell: DF-1 cell culture is in 25cm²Tissue Culture Flask, adjusting cell density by cell count is 1.0×10⁸/ hole is divided into 2 groups, every group of 3 repetitions:

Group 1 when DF-1 cell reaches 70% degrees of fusion, is added 1ml ALV-J NX0101 virus liquid, maintains 2h, discard Dulbecco ' s Modified Eagle Medium (DMEM) culture medium containing 1% fetal calf serum is added, 37 in venom DEG C 5%CO2 incubator in culture maintain 72h, be added cell pyrolysis liquid RIPA:PMSF (100:1) harvest cell protein；

Group 2, when DF-1 cell reaches 70% degrees of fusion, be added the DMEM containing 1% fetal calf serum, 37 DEG C 5% CO₂Culture maintains 72h in incubator；Cell pyrolysis liquid RIPA:PMSF (100:1) is added and harvests cell protein；

2) after cell is digested with pancreatin, 4 times of volume lysis buffers (8M urea, 1% albumen protein extraction: are separately added into Enzyme inhibitor, 3 μM of TSA, 50mM NAM and 2mM EDTA), ultrasound cracking.4 DEG C, 15294 × g is centrifuged 10min, removes cell Fragment, supernatant are transferred to new centrifuge tube, carry out determination of protein concentration using BCA kit.

3) pancreatin digests: dithiothreitol (DTT) is added in protein solution makes its final concentration of 5mM, 56 DEG C of reduction 30min.It Iodo-acetamide is added afterwards makes its final concentration of 11mM, and room temperature, which is protected from light, is incubated for 15min.Finally the urea concentration of sample is diluted to Lower than 2M.Pancreatin is added with the mass ratio (pancreatin: albumen) of 1:50,37 DEG C of enzymatic hydrolysis are overnight.Again with the mass ratio of 1:100 Pancreatin is added in (pancreatin: albumen), continues to digest 4h.

4) TMT is marked: vacuum freeze drying after peptide fragment Strata X C18 (Phenomenex) desalination of pancreatin enzymatic hydrolysis. Peptide fragment is dissolved with 0.5M TEAB, peptide fragment is marked according to TMT kit operating instruction.It is easy to operate to be described as follows: labelled reagent solution It is dissolved after jelly with acetonitrile, 2h is incubated at room temperature after mixing with peptide fragment, desalination after the peptide fragment mixing after label, vacuum freeze drying.

5) HPLC is classified: peptide fragment is classified with high pH reverse hplc, and chromatographic column is Agilent 300Extend C18 (5 μm of grains Diameter, 4.6mm internal diameter, 250mm long).Operate as follows: peptide fragment stepwise gradient is 8%-32% acetonitrile, pH 9,60min temporal separation 60 components, subsequent peptide fragment merge into 18 components, carry out subsequent operation after the group lease making vacuum freeze drying after merging.

6) EASY-nLC liquid chromatography-mass spectrometry: is used after peptide fragment liquid chromatogram mobile phase A phased soln 1000 ultra high efficiency liquid phase systems are separated.Mobile phase A is the aqueous solution containing 0.1% formic acid and 2% acetonitrile；Mobile phase B be containing The aqueous solution of 0.1% formic acid and 90% acetonitrile.Liquid phase gradient setting: 0-26 min, 7%-25%B；26-34min, 25%- 38%B；34-37min, 38%-80%B；37-40min, 80%B, flow velocity maintain 350nL/min.

Then peptide fragment ionize in injection NSI ion source into Orbitrap after separating via ultra high efficiency liquid phase systems Fusion^TMMass spectrum is analyzed.Ion source voltage is set as 2.0kV, and peptide fragment parent ion and its secondary fragment all use high-resolution Orbitrap be detected and analyzed.First mass spectrometric scanning range is set as 350-1550m/z, and scanning resolution is set as 60,000；It is 100 m/z that second order ms scanning range, which then fixes starting point, and Orbitrap scanning resolution is set as 30,000.Number (DDA) program is scanned using data dependence type according to acquisition mode, i.e., highest preceding 10 peptide of selection signal intensity after level-one scanning Section parent ion sequentially enters HCD collision cell and carries out fragmentation using 35% fragmentation energies, equally successively carries out second order ms point Analysis.In order to improve mass spectrographic effective rate of utilization, setting automatic growth control (AGC) is 5E4, and signal threshold value is set as 5000 Ions/s, maximum injection length are set as 100ms, and the dynamic exclusion time of tandem mass spectrum scanning is set as 30 s and avoids parent ion Multiple scanning.

7) database search: second order ms data are retrieved using Maxquant (v1.5.2.8).Retrieval parameter setting: Database is UniProt Gallus (29480 sequences), is added to anti-library to calculate false positive rate caused by random fit (FDR), it and in the database joined common pollution library, for eliminating the influence of contaminating protein in qualification result；Digestion Mode is set as Trypsin/P；Leakage enzyme site number is set as 2；Peptide fragment minimum length is set as 7 amino acid residues；Peptide fragment is maximum Modification number is set as 5；The level-one parent ion quality error tolerance of First search and Main search are set to 20ppm And 5ppm, the quality error tolerance of secondary fragment ions are 0.02Da.Fixed modification is set by cysteine alkylation, it can Become the oxidation for being modified to methionine, the acetylation of albumen n end.Quantitative approach is set as TMT-10plex, Identification of Fusion Protein, PSM The FDR of identification is both configured to 1%.

8) proteome analysis: the GO annotation of GO enrichment analysis, albumen is divided into 3 major class: biological processes, groups of cells At, molecular function.The accurate both-end method of inspection of Fischer (Fisher's exact test) is used to verify that differentially expressed protein Using the albumen identified as background.GO enrichment inspection p-value value is considered significant less than 0.05.

Clustering is based on 1.3 times of differential proteins, we collect the function classification letter that albumen grouping used is enriched to first Breath and corresponding enrichment p-value value, then filter out at least be in the grouping of albumen significant enrichment (p-value < 0.05) function classification.Obtained p-value data matrix is screened to first pass around with-log₁₀Logarithmic transformation, then will transformation Data matrix afterwards uses transform to each function classification.It is finally that the data set obtained after transform is (European using hierarchical cluster Distance, average connection cluster) method does unilateral clustering.Clustering relationships use the function in R language pack ggplots The thermal map that heatmap.2 is drawn out is visualized.

By above-mentioned analysis, inventor infection ALV-J 1.5 times of differential proteins in have found one poultry field not The albumen " CTHRC1 " reported is [as shown in Figure 1, have 88 in (a) Heatmap analysis of agglomeration ALV-J infection DF-1 cell The significant differentially expressed protein of kind.Group learns data with log₂Ratio input.Red indicates the protein raised in DF-1 cell, blue Color table shows the protein of downward.(b) volcano figure, group are learned each albumen in data and are handled with-log (p), ALV-J infection cell Middle protein expression abundance and normal cell protein gene expression abundance are with log₂Ratio mapping.Red dot indicates the cell infected in ALV-J The albumen of middle expression up-regulation, Bluepoint indicate the albumen that expression is lowered.(c) GO functional analysis, blue represent cell composition, orange generation Table molecular function, yellow represent bioprocess.Wherein CTHRC1 is labelled in the figure of the volcano (b).]

As shown in Figure 1a, ALV-J infection DF-1 cell shares 88 differential expressions compared with the protein abundance of normal cell Albumen (multiple variation > 1.3), there is 40 (45%) upregulated proteins, 48 (55%) down-regulation proteins；As shown in Figure 1 b, group learns number Each protein value progress-log (p) conversion in, the upper protein quantity lowered that reconciles is similar as the result is shown, but lowers (green to select) egg It is white to be more than up-regulation (red dot) egg；GO functional analysis discovery, differentially expressed protein are carried out using 6.8 software of DAVID as illustrated in figure 1 c Cell outer room seam is mainly enriched in cell component；Molecular function relates generally to protein-glutamine gamma glutamyltransferase Activity；Bioprocess relates generally to peptide crosslinking.By carrying out bioinformatic analysis to proteomics, the height of CTHRC1 is found Expression, which is likely to replicate with ALV-J, has close contact.The CTHRC1 nucleotide sequence is shown in SEQ ID NO.1, amino acid sequence Column are shown in SEQ ID NO.2.It is found through comparing, is 75.6% with source of people CTHRC1 base sequence homology.

The expression of 2 ALV-J of embodiment activation CTHRC1:

In order to further confirm ALV-J to the facilitation of CTHRC1, the present embodiment establishes the external thin of ALV-J infection Born of the same parents' model.The variation of CTHRC1 expression quantity after ALV-J infection is detected by qPCR, western blot and ELISA.

(1) quantitative fluorescent PCR/qPCR

Design qPCR specific primer: fowl host protein CTHRC1 qPRC primer sequence is F:5 '- ACGCTGGCTTGGTGGA-3 ', R:5 '-CAGTTCTTCAATGATGATACGG-3 '；

The primer sequence of fowl reference gene GPADH is F:5 '-GAACATCATCCCAGCGTCCA-3 ', R:5 '- CGGCAGGTCAGGTCAACAAC-3’。

ALV-J fluorescent quantitation primer sequence is F:5 '-TGCGTGCGTGGTTATTATTTC-3 ', R:5 '- AATGGTGAGGTCGCTGACTGT-3′

Nucleotide sequence is sequentially shown in SEQ ID NO.5-10；

Primer is synthesized by Qingdao Hua Da Genetic Biotechnologies Co., Ltd.

By ALV-J infection for 24 hours, the cell of 48h, 72h and 96h harvest, using the total extraction reagent kit of cell RNA (QIAGEN, America cell total rna) is extracted, is then cDNA by RNA reverse transcription, this detects CTHRC1 mRNA's by qPCR for template Expression variation.

Reaction system are as follows: carried out in 20 μ L reaction systems: 12.5 μ L SYBR Premix Ex TaqTM (2 ×), 0.4 μ L upstream primer, 0.4 μ L downstream primer, 1 μ L cDNA, 8.2 μ L ddH₂O。

Reaction condition are as follows: use 2 footwork PCR reaction conditions: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s；64 DEG C of annealing 34s；95 DEG C of denaturation 10s；65 DEG C of annealing 60s；97 DEG C of extension 1s, [as shown in Fig. 2 a and d (a and d) extracts ALV-J to 38 circulations Infect DF-1 cell and normal DF-1 cell RNA.CTHRC1 mRNA and ALV-J copy number is detected by qPCR.]

Fig. 2 a can activate the expression of CTHRC1 after capable of illustrating ALV-J infection；And with the passage of infection time, The expression quantity of CTHRC1 gradually rises.Fig. 2 d illustrates that the present embodiment connects the reliability of poison as auxiliary data.

(2)Western blot

By ALV-J infection for 24 hours, the cell of 48h, 72h and 96h harvest, use PMSF and RIPA mixing thing liquid (100:1) Lytic cell；Adjusting each protein sample is uniform concentration, and albumen sample-loading buffer is added and heats 5min in 100 DEG C of water.Sample Electrophoresis apparatus is added, glue pressure stabilizing under 80V state is concentrated and carries out, separation gel pressure stabilizing under 110V state carries out.It, will after the completion of electrophoresis On protein delivery to 0.22 μm of pvdf membrane on protein adhesive, using being added after 37 DEG C of 5% skimmed milk power closing 2h, TBST washing After 37 DEG C of incubation 1h, TBST washings of primary antibody, 37 DEG C of incubation 1h of secondary antibody are added, are finally seen using Western blot visualizer It examines.[CTHRC1 expressing quantity in (c) Western blot detection ALV-J infection cell and normal cell as shown in Figure 2 c.]

Can be illustrated by Fig. 2 c, ALV-J can in active cell CTHRC1 albumen expression.

(3)ELISA

Collect ALV-J infection for 24 hours, the cell conditioned medium of 48h, 72h and 96h, pass through CTHRC1 albumen check ELISA kit (Senbeijing, China) and ALV-J p27 antigen detection kit (IDEXX, America) are checked to be infected not in ALV-J With period extracellular CTHRC1 albumen expression variation [as shown in Fig. 2 b and e (b and e) by ELISA detect ALV-J infection The expression quantity of CTHRC1 and ALV-J p27 albumen in cell and normal cell supernatant.]

Being illustrated by Fig. 2 b, the expression quantity of extracellular CTHRC1 albumen increases with the passage of ALV-J infection time, this Also illustrate that ALV-J can activate the expression of CTHRC1.Fig. 2 e is as supplementary number it was demonstrated that ALV-J connects poison success.

ALV-J be inoculated with DF-1 cell after, by qPCR, western blot and ELISA detect respectively for 24 hours, 48h, 72h With the dynamic change of 96h CTHRC1 mRNA level in-site and protein expression amount.Test result explanation, ALV-J infection can promote Into the expression of extracellular and intracellular CTHRC1 protein level and intracellular CTHRC1 mRNA.

Embodiment 3 is overexpressed CTHRC1 and dramatically increases ALV-J virus load:

In order to which the activation of clear CTHRC1 can promote ALV-J to replicate, the present embodiment constructs CTHRC1 and is overexpressed plasmid, By being overexpressed CTHRC1, facilitation of the verifying CTHRC1 to ALV-J carrying capacity.

1. constructing CTHRC1 is overexpressed plasmid

(1) building of carrier for expression of eukaryon is carried out.The building of CTHRC1 carrier for expression of eukaryon transfers to Huada gene company to adopt It is completed with the prior art, the present invention selects pcDNA3.1 eukaryon expression plasmid, and the completely CTHRC1 gene sequence without mutation will be sequenced Column are inserted into plasmid, and then the plasmid built is transformed into competent cell DH5 α, and bacterium is shaken after the completion of conversion and extracts matter Grain, and be sequenced, sequencing result and aim sequence exactly match.

(2) then by the CTHRC1 eukaryotic expression plasmids DF-1 cell of building；When DF-1 transiently transfects CTHRC1 mistake Expression plasmid 8h harvests cell after maintaining 72h, extracts cell RNA, lytic cell albumen, harvest cell conditioned medium, passes through respectively QPCR, western blot and ELISA detection CTHRC1 expression quantity [result (a) DF-1 cell transfecting as shown in fig. 3 a-c PcDNA3.1-CTHRC1 or control plasmid (pcDNA3.1) are inoculated with ALV-J afterwards, extract cell RNA after maintaining 72h.Pass through qPCR CTHRC1 mRNA level in-site is detected, the results show that the expression quantity for being overexpressed the intracellular CTHRC1 mRNA of CTHRC1 group is significantly higher than Control group.(b) using transfection pcDNA3.1-CTHRC1 or pcDNA3.1 and be inoculated with ALV-J cell supernatant detect CTHRC1 Expression quantity, the results show that the expression quantity for being overexpressed the extracellular CTHRC1 albumen of CTHRC1 group is significantly higher than control group.(c)DF-1 Cell transfecting pcDNA3.1-CTHRC1 or control plasmid (pcDNA3.1) are inoculated with ALV-J afterwards, maintain 72h.Pass through western Blot detects the expression quantity of intracellular CTHRC1 albumen, the results show that being overexpressed the table of the intracellular CTHRC1 albumen of CTHRC1 group It is significantly higher than control group up to amount.]

Fig. 3 a-c explanation, the good CTHRC1 overexpressing cell model of the present embodiment framework are established for subsequent experimental Basis.

2. being overexpressed CTHRC1 detects the influence replicated to ALV-J

(1) cell culture is in 12 orifice plates before transfecting, it is ensured that cell keeps optium concentration and state；

(2) reagent rewarming: X-tremeGENE HP DNA Transfection Reagent, CTHRC1 are overexpressed plasmid 20 DEG C or so are warming up to dilution, of short duration whirlpool mixes X-tremeGENE HP DNA Transfection Reagent；

(3) solution is prepared: being used Opti-MEM culture medium as dilution, CTHRC1 overexpression plasmid is diluted to dense eventually Degree is 1 μ g/100 μ L culture medium, soft to mix.The 100 μ L dilutions for being overexpressed plasmid containing 1 μ g CTHRC1 are separately added into In sterile centrifugation tube.X-tremeGENE HP DNA Transfection Reagent is directly appended to the training containing dilution DNA It supports in base, the ratio of Plasmid DNA and transfection reagent is 1:3, and in the process, pipette tips are sure not contact centrifugation tube wall；It uses Diluent volume is more than 100 μ L；

(4) be incubated for: transfection composite is incubated for 15min in 20 DEG C or so environment；

(5) transfect: the cell taken out in incubator, without discarding original culture medium, transfection composite is directly added drop-wise to carefully In born of the same parents；

(6) ALV-J infection cell: discarding original culture medium after cell transient transfection 8h, is washed 3 times using PBS, every hole adds Enter NX0101 plants of venom of 600mL ALV-J, venom is discarded after 37 DEG C of maintenance 2h, the DMEM culture containing 1% fetal calf serum is added Base continues to 72h.

(7) detect ALV-J carrying capacity: harvest cell detects ALV-J by qPCR, western blot and ELISA respectively [(c) DF-1 cell transfecting pcDNA3.1-CTHRC1 as shown in Fig. 3 c-e or control plasmid (pcDNA3.1) are inoculated with ALV- to carrying capacity afterwards J maintains 72h.The expression quantity of intracellular ALV-J SU protein level is detected by western blot, the results show that being overexpressed CTHRC1 group ALV-J SU expressing quantity is significantly higher than control group.(d) DF-1 cell transfecting pcDNA3.1-CTHRC1 or control Plasmid (pcDNA3.1) is inoculated with ALV-J afterwards, extracts cell RNA after maintaining 72h.ALV-J carrying capacity is detected by qPCR, is as a result shown Show, is overexpressed the intracellular ALV-J rna expression amount of CTHRC1 group and is significantly higher than control group.(e) using transfection pcDNA3.1- CTHRC1 or pcDNA3.1 and the supernatant for being inoculated with ALV-J cell detect the expression quantity of p27 albumen, the results show that overexpression The extracellular ALV-J expressing quantity of CTHRC1 group is significantly higher than control group.]

It is proved by Fig. 3 c-e, compared with Mock group, being overexpressed CTHRC1 can promote twice of ALV-J carrying capacity or more.It is logical Cross these results suggest that, be overexpressed CTHRC1 can remarkably promote ALV-J duplication.

It can be seen that compared with prior art, amplification virus can be improved the virus load of 2 times or more in the above manner.

The interference of embodiment 4 CTHRC1 significantly inhibits the duplication of ALV-J

In order to clear CTHRC1 expression quantity to the importance of ALV-J carrying capacity, the present embodiment constructs CTHRC1 interference matter Grain, by striking low CTHRC1, the importance that verifying CTHRC1 replicates ALV-J.

The building of 1.CTHRC1 interference plasmid

(1) result of ALV-J virus replication is promoted reversely to be verified by shRNA CTHRC1, You Jima company structure Build two CTHRC1 interference plasmids --- its nucleotide sequence of shCTHRC1-1:5 '-GCACTGAACTACGGCATAGAC-3 ' As shown in SEQ ID NO.3；Its nucleotide sequence of shCTHRC1-2:5 '-GTCTGTGTGAAGGGATCAACG-3 ' such as SEQ ID Shown in NO.4；

(2) interference plasmid is transiently transfected into cell 8h, maintains 72h, harvest cell；

(3) by qPCR, western blot and ELISA CTHRC1 expression quantity is detected respectively [result is as shown in figures 4 a-c. (a) after shRNA transfects DF-1 cell (shCTHRC1-1, shCTHRC1-2 or shNC), ALV-J infection cell simultaneously extracts cell RNA.CTHRC1 mRNA is detected by qPCR, the results show that the CTHRC1 mrna expression amount of transfection shCTHRC1 group is significantly low In control group.(b) shRNA (shCTHRC1-1, shCTHRC1-2 or shNC) transfects cell, then ALV-J infection cell, leads to ELISA detection CTHRC1 expressing quantity is crossed, the results show that the extracellular CTHRC1 expressing quantity of transfection shCTHRC1 group Substantially less than control group.(c) this is infected with shRNA (shCTHRC1-1, shCTHRC1-2 or shNC) transfection subsequent ALV-J of cell Cell detects CTHRC1 by western blot, the results show that the intracellular CTHRC1 albumen table of transfection shCTHRC1 group Control group is substantially less than up to amount.]

Fig. 4 a-c is able to demonstrate that: after transfection CTHRC1 interference plasmid, the expression of low CTHRC1 can be struck in cell.Cause This explanation, the present embodiment construct good CTHRC1 interference cell model, are ALV-J duplications for the clear CTHRC1 of subsequent experimental Dependence protein is laid a good foundation.

2. interference CTHRC1 detects the influence replicated to ALV-J

(3) solution is prepared: being used Opti-MEM culture medium as dilution, will be contained 1 μ g CTHRC1 interference plasmid 100 μ L dilutions are separately added into sterile centrifugation tube.X-tremeGENE HP DNA Transfection Reagent directly adds It is added in the culture medium containing dilution DNA, the ratio of Plasmid DNA and transfection reagent is 1:3, and in the process, pipette tips are sure not to connect Touching centrifugation tube wall；The diluent volume used is more than 100 μ L；

(7) detect ALV-J carrying capacity: harvest cell detects ALV-J by qPCR, western blot and ELISA respectively [as shown in Fig. 4 c-e (c) transfects cell with shRNA (shCTHRC1-1, shCTHRC1-2 or shNC) and is then inoculated with ALV- carrying capacity J detects ALV-J expressing quantity by western blot, the results show that the intracellular ALV-J egg of transfection shCTHRC1 group White expression quantity is substantially less than control group.(d) after shRNA transfects DF-1 cell (shCTHRC1-1, shCTHRC1-2 or shNC), ALV-J infection cell simultaneously extracts cell RNA.ALV-J virus load is detected by qPCR, the results show that transfection shCTHRC1 group Intracellular ALV-J rna expression amount be substantially less than control group.(e) shRNA (shCTHRC1-1, shCTHRC1-2 or shNC) Cell is transfected, then ALV-J infection cell, ALV-J p27 expressing quantity is detected by ELISA, the results show that transfection The extracellular ALV-J expressing quantity of shCTHRC1 group is substantially less than control group.]

Fig. 4 c-e is proved, is struck low CTHRC1 and is able to suppress ALV-J duplication.By these results suggest that, ALV-J duplication need Want the participation of CTHRC1；CTHRC1 is ALV-J replication-dependent albumen.

With ALV-J SU albumen interaction can occur for 5 CTHRC1 of embodiment

In order to which clear CTHRC1 directly participates in ALV-J duplication, the present embodiment is tried by co-immunoprecipitation and laser co-focusing It tests to illustrate relationship between the two.

1. co-immunoprecipitation

(1) prepare sample: DF-1 cell confluency degree is inoculated with ALV-J when reaching 70%, maintains 2h, uses instead containing 1% DMEM culture medium continues to 72h；Discard culture medium, using PBS wash 3 times, every 1 × 10⁶It is added what 200 μ L kits carried Cracking/equilibration buffer cracks 20min on ice, is then centrifuged 10min under the conditions of 4 DEG C with 17000g, and drawing supernatant is egg Sample is divided to two parts by white sample, and first part for whether, containing the target protein to be detected, second part for connect in test sample The immunoprecipitation assay to get off；

(2) be incubated for bait antibody: ALV-J monoclonal antibody 1D4 is added in the protein sample of cracking, in 4 DEG C of incubation 1h；

(3) immunoprecipitation: firstly, 100 μ L cracking/equilibration buffer are added in adsorption column, at room temperature with 1000g from Heart 1min.It removes and flows through object and abandon, then chromatographic column is put into new collecting pipe；The sample for being incubated for ALV-J antibody is added, 1000g is centrifuged 1min at room temperature, and adsorption column is transferred in a new centrifuge tube；100 μ L washing is added into adsorption column Buffer, 1000g is centrifuged 1min, and adsorption column is transferred in a new centrifuge tube；3-5 μ L neutralization buffer is added to suction In attached column, 35 μ L elution buffers are then added, with 1000g centrifugation 1 time；

(4) Western blot analyzes sample: first group of sample and treated second group of sample are carried out SDS- PAGE and Western blot distinguishes in test sample whether contain CTHRC1 and ALV-J SU albumen.We are public using Abcom As Co-IP test secondary antibody, [as shown in Figure 5 a, (a) cracks the DF-1 cell of ALV-J infection 72h to the VeriBlot of department.Cracking produces Object makes anti-ALV-J SU antibody precipitate C THRC1, by western blot detect in eluted product whether containing CTHRC1 and ALV-J, the results show that the two is implicitly present in interaction.]

Illustrate that there are interactions between ALV-J and CTHRC1 by Fig. 5 a.Because ALV-J genome structure is simpler It is single, required enzyme system needed for lacking self-replication, so ALV-J is needed in life cycle using host protein, in host Self-replication is completed under the assistance of albumen.It is also illustrated by result above, CTHRC1 is that institute is required in ALV-J reproduction process Host protein, CTHRC1 is replicated with facilitation to ALV-J, and result above is that CTHRC1 is mentioned as ALV-J duplication reinforcing agent Solid theoretical basis is supplied.

2. laser co-focusing

(1) cell pretreatment: cell culture is divided into two groups in laser co-focusing plate, by the cell of culture, and first group, ALV-J is infected when cell reaches 70% convergence degree, maintains 72h with the DMEM culture medium containing 1% fetal calf serum；Second group, when Cell uses the DMEM culture medium containing 1% fetal calf serum instead and maintains 72h when reaching 70% convergence degree；

(2) wash: discarding the culture medium in plate, the PBS that 1mL preheating is added is washed 3 times, the culture medium of wash residual and Dead cell；

(3) fixed cell: the ethyl alcohol and acetone mixture (2:3) of 1mL pre-cooling are added into plate, maintains 7min；

(4) it washs: 2mL PBS washing is added three times, each 5min；

(5) it closes: the skimmed milk power of 1mL 5%, 37 DEG C of closing 1h is added；

(6) it washs: with (4)；

(7) it is incubated for primary antibody: being incubated for CTHRC1 and ALV-J antibody, 37 DEG C of incubation 1h respectively；

(8) it washs: with (4)；

(9) it is incubated for secondary antibody: being incubated for secondary antibody, FITC is added in CTHRC1, and ALV-J is incubated for Alexa Fluor 647；

(10) it washs: with (4)；

(11) nucleus, 37 DEG C of 5 min of incubation nuclear targeting: are marked using nucleus specificity fuel DAPI；

(12) it washs: with (4)；

(13) [as shown in Figure 5 b, (b) CTHRC1 uses anti-CTHRC1 primary antibody and FITC to confocal laser scanning microscope Label；ALV-J SU albumen is marked using ALV-J SU protein monoclonal antibody and Alexa Fluor 647；Nucleus uses DAPI dyeing.The combination of the expression of Merge 1 ALV-J infected group DAPI and antiCTHRC1 fluorescence；Merge 2 indicates ALV-J sense The overlapping of DAPI, antiCTHRC1 and antiALV-J fluorescence in dye group.]

Fig. 5 b aids in illustrating CTHRC1 and ALV-J, and there are interactions.Result above also illustrates simultaneously, in normal cell In, CTHRC1 is mainly dispersed in and is distributed in nucleus；And in the DF-1 cell of ALV-J infection, CTHRC1 is around nucleus It is positioned in cytoplasm.Above results proved that CTHRC1 is a shuttling proteins, ALV-J infection can drive CTHRC1 to send out The raw positioning variation from nucleus to cytoplasm.

It can be derived that conclusion by testing above, CTHRC1 is an ALV-J replication-dependent albumen, is overexpressed CTHRC1 After can remarkably promote ALV-J duplication, therefore CTHRC1 can be applied as ALV-J duplication reinforcing agent.The present embodiment building CTHRC1 is overexpressed plasmid, transfects the plasmid and is inoculated with ALV-J, can dramatically increase ALV-J duplication amount.Of the invention is specific Using being exactly to provide most essential most important Research foundation-virus for the correlative study of ALV-J, by being overexpressed CTHRC1 ALV-J duplication can be remarkably promoted.

Sequence table

<110>Shandong Agricultural University

<120>a kind of J subgroup avian leucosis virus replicates reinforcing agent

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1260

<212> DNA

<213>artificial sequence ()

<400> 1

atgggtcctc atttgaagat gttcaggctg aacagccccg gagctctcag cttttcatat 60

gggagatgct tcaggcccct cttcatcctt gtgaccccgt caccatctct gtttccgggc 120

ggtgacacac cacaggctgg ctcagcacaa ggaactcaga gcagacgtcc cgccgctctt 180

tgtagaaccc tcttttacag gaaaagttgg gatgcgggat ttgaaagctt ccagcccggg 240

cccaaagtct cactgttatc aaacccaggt ggggaaacgc gtacagatgg ccgtcgcagc 300

cgtcgtaacg gggcactgac cgcgccgcac ggccgcctta ggggcatacg ggcacgccgc 360

cggcgacaca gcgcgggccg tgcgagccgg gtaccccctc acagcaccgg gggggcggga 420

gcccccggcc cgtgccaatg gctgccgggg ccggcgccgc ccccgctcgg ctgcgggtgg 480

aacgcgccgg ccccggggga gggagcgggg ctcggggccg tgaaagcctc attcatcgcc 540

ggcggcggag aggcacggcc gggccccgcc atgcgccgcg ccccgctgct gctcctggcg 600

ctgctgctgg gctcgggcct cgccgacagc ccgcggggca aacagcgagc ggcgcggccg 660

cgggaggtgc tggaggcgta caacggcgtc tgcctgcagg gccccagcgg cgtcccggga 720

cgggacggga accctggaac caacgggatc ccggggacac cggggatccc ggggcgggac 780

gggcccaaag gggaaaaggg cgagtgcttg cgggagagca ttgaggagtc ctggacgccc 840

aacttcaagc agtgctcgtg gagcgcactg aactacggca tagacctggg gaaaatagcg 900

gaatgtacgt tcacaaagat gcgctccaac agtgctctca gagtcctttt cagtggatcg 960

ctccggctga agtgccgaag cgcctgctgt cagcgctggt acttcacttt caatggagca 1020

gaatgcgccg ggccacttcc catcgaagcc ataatatatt tagatcaagg cagtccggaa 1080

ctgaactcta ctatcaacat acaccgaacc tcctcagtgg aaggtctgtg tgaagggatc 1140

aacgctggct tggtggacat cgccatctgg gtcgggactt gctctgacta ccccagggga 1200

gatgcttcta ctggatggaa ttcagtctcc cgtatcatca ttgaagaact gccaaaataa 1260

<210> 2

<211> 419

<212> PRT

<213>artificial sequence ()

<400> 2

Met Gly Pro His Leu Lys Met Phe Arg Leu Asn Ser Pro Gly Ala Leu

1 5 10 15

Ser Phe Ser Tyr Gly Arg Cys Phe Arg Pro Leu Phe Ile Leu Val Thr

20 25 30

Pro Ser Pro Ser Leu Phe Pro Gly Gly Asp Thr Pro Gln Ala Gly Ser

35 40 45

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

50 55 60

Phe Tyr Arg Lys Ser Trp Asp Ala Gly Phe Glu Ser Phe Gln Pro Gly

65 70 75 80

Pro Lys Val Ser Leu Leu Ser Asn Pro Gly Gly Glu Thr Arg Thr Asp

85 90 95

Gly Arg Arg Ser Arg Arg Asn Gly Ala Leu Thr Ala Pro His Gly Arg

100 105 110

Leu Arg Gly Ile Arg Ala Arg Arg Arg Arg His Ser Ala Gly Arg Ala

115 120 125

Ser Arg Val Pro Pro His Ser Thr Gly Gly Ala Gly Ala Pro Gly Pro

130 135 140

Cys Gln Trp Leu Pro Gly Pro Ala Pro Pro Pro Leu Gly Cys Gly Trp

145 150 155 160

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

165 170 175

Ser Phe Ile Ala Gly Gly Gly Glu Ala Arg Pro Gly Pro Ala Met Arg

180 185 190

Arg Ala Pro Leu Leu Leu Leu Ala Leu Leu Leu Gly Ser Gly Leu Ala

195 200 205

Asp Ser Pro Arg Gly Lys Gln Arg Ala Ala Arg Pro Arg Glu Val Leu

210 215 220

Glu Ala Tyr Asn Gly Val Cys Leu Gln Gly Pro Ser Gly Val Pro Gly

225 230 235 240

Arg Asp Gly Asn Pro Gly Thr Asn Gly Ile Pro Gly Thr Pro Gly Ile

245 250 255

Pro Gly Arg Asp Gly Pro Lys Gly Glu Lys Gly Glu Cys Leu Arg Glu

260 265 270

Ser Ile Glu Glu Ser Trp Thr Pro Asn Phe Lys Gln Cys Ser Trp Ser

275 280 285

Ala Leu Asn Tyr Gly Ile Asp Leu Gly Lys Ile Ala Glu Cys Thr Phe

290 295 300

Thr Lys Met Arg Ser Asn Ser Ala Leu Arg Val Leu Phe Ser Gly Ser

305 310 315 320

Leu Arg Leu Lys Cys Arg Ser Ala Cys Cys Gln Arg Trp Tyr Phe Thr

325 330 335

Phe Asn Gly Ala Glu Cys Ala Gly Pro Leu Pro Ile Glu Ala Ile Ile

340 345 350

Tyr Leu Asp Gln Gly Ser Pro Glu Leu Asn Ser Thr Ile Asn Ile His

355 360 365

Arg Thr Ser Ser Val Glu Gly Leu Cys Glu Gly Ile Asn Ala Gly Leu

370 375 380

Val Asp Ile Ala Ile Trp Val Gly Thr Cys Ser Asp Tyr Pro Arg Gly

385 390 395 400

Asp Ala Ser Thr Gly Trp Asn Ser Val Ser Arg Ile Ile Ile Glu Glu

405 410 415

Leu Pro Lys

<210> 3

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 3

gcactgaact acggcataga c 21

<210> 4

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 4

gtctgtgtga agggatcaac g 21

<210> 5

<211> 16

<212> DNA

<213>artificial sequence ()

<400> 5

acgctggctt ggtgga 16

<210> 6

<211> 22

<212> DNA

<213>artificial sequence ()

<400> 6

cagttcttca atgatgatac gg 22

<210> 7

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 7

gaacatcatc ccagcgtcca 20

<210> 8

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 8

cggcaggtca ggtcaacaac 20

<210> 9

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 9

tgcgtgcgtg gttattattt c 21

<210> 10

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 10

aatggtgagg tcgctgactg t 21

Claims

1. a kind of J subgroup avian leucosis virus replicates reinforcing agent, it is characterised in that: the reinforcing agent is the repetition of three spiral of collagen Albumen 1 (collagen triple helix repeat containing1, CTHRC1), nucleotide sequence such as SEQ ID Shown in NO.1, the amino acid sequence of coding is as shown in SEQ ID NO.2.

2. the duplication reinforcing agent of J subgroup avian leucosis virus described in claim 1 is preparing answering in J subgroup avian leucosis virus With.