CN104306995A - Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine - Google Patents
Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine Download PDFInfo
- Publication number
- CN104306995A CN104306995A CN201410593612.6A CN201410593612A CN104306995A CN 104306995 A CN104306995 A CN 104306995A CN 201410593612 A CN201410593612 A CN 201410593612A CN 104306995 A CN104306995 A CN 104306995A
- Authority
- CN
- China
- Prior art keywords
- alv
- vaccine
- epitope
- cenv
- epitope vaccine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the fields of animal virology and immunology, and provides an epitope vaccine for resisting subgroup J avian leukosis virus infection. The vaccine is prepared from screened and purified high-activity recombinant protein His-cENV after prokaryotic expression with the combination of a freund's adjuvant, wherein the nucleotide sequence of the encoding recombinant protein His-cENV is as shown in SEQ ID NO.1. 7-day breeding poultry chicks by utilizing epitope vaccine immunity can produce neutralizing antibodies of 1 to 128000; in-vitro virus neutralization experiments and animal experiments show that the epitope vaccine can neutralize different ALV-J separation strains, and effectively protect the chicks so as to resist ALV-J strain infection. Env source based multi-epitope antigen gene sequence of the epitope vaccine overcomes the virus variation of ALV-J, develops the ALV-J vaccine new era, provides a new means for resisting ALV-J infection, and provides technical support for ALV-J prevention and control.
Description
Technical field
The present invention relates to field of immunology, specifically provide epiposition vaccine of a kind of J substock lymphoid leuoosis-resistant viral infection and its preparation method and application.
Background technology
J subgroup avian leucosis is a kind of tumprigenicity infectious disease caused by J subgroup avian leucosis virus (ALV-J), main common with myelocytic leukemia clinically, occur that tumor etc. is its principal character mainly with immunosuppressant, growth inhibited and multiple organ tissue.The generation of China J subgroup avian leucosis is quite general, and clinical case develops into commodity egg, local breeder flock and other poultry from initial commercial meat bird group, and strengthens along with oncogenicity and the generation of tumor multiplicity.Research display, the J subgroup avian leucosis poison of China presents high infection rate, high incidence, early infection is general, morbidity age in days obviously shifts to an earlier date, mixed infection is multiple/takes place frequently, host range expansion, tumor spectrum expand, the features such as pathological change is complicated, cause huge loss to the aviculture of China.Based on the hypermutation characteristic of J subgroup avian leucosis poison envelope protein, in addition the immune evasion ability of self and the immunoselection pressure of host, make the antigen mobility of avian leukosis virus very big, become its traditional inactivated vaccine, bottleneck that attenuated vaccine development cannot be gone beyond.At present, there is no the prevention and control for J subgroup avian leucosis of related drugs and vaccine clinically.The external generation being controlled primary disease by purification, because purification cycle is long, cost is high, also fail at present effectively to implement in China, jumpbogroup purification can only be carried out by eliminating infection chicken, the infection of uncontrollable ALV-J, constantly occur the report of ALV-J intensive infection on a large scale, the situation is tense in the J subgroup avian leucosis prevention and control of China.All there is the report of the vaccine research and development method for J subgroup avian leucosis both at home and abroad; be mainly traditional formalin-killed vaccine, attenuated vaccine; based on subunit vaccine, the nucleic acid vaccine of SU; these vaccines can provide protection to body to a certain extent; but virulence all can be caused to recover and loose malicious possibility, immune effect instability cannot clinical practice.Based on above present situation, develop epiposition vaccine safely and efficiently, for the prevention and control of J subgroup avian leucosis virus, there is Great significance.
Epiposition vaccine is that developed recently gets up ripe new generation vaccine, and in the virus of people source, research deeply.Epiposition vaccine utilizes genetic engineering means, the epi-position of vivoexpression or synthetic pathogenic microorganism, then develops preventative or therapeutic vaccine.Epiposition vaccine has more advantage relative to traditional vaccine, epitope peptide is short and small, immunogenicity is strong, the heredity restriction of major histocompatibility complex (MHC) molecule can be overcome, itself is safe, nontoxic, stable, can directly stimulate body to produce specific immune response, its maximum advantage is exactly can overcome virulence that traditional vaccine causes to recover or the possibility of loose poison, has boundless development prospect.At present, the epiposition vaccines such as effective HIV (human immunodeficiency virus), hepatitis B virus, nerpes vinrus hominis have been developed.ALV-J belongs to retrovirus retrovirus.Viral envelope proteins (env) can divide for by the surface protein of gp85 gene code (surface protein, and the transmembrane protein of gp37 gene code (transmembrane protein SU), TM), the two forms dimer together, and viral envelope proteins determines the specificity of subgroup.Gp85 is the main component on viral envelope proteins surface, infect absorption host cell at ALV and induce body to produce in specific antibody and there is important function, containing numerous epitopes, be responsible for identifying the specific receptor on target cell membrane, the fusion process of TM primary responsibility mediate retroviral and cell.Based on the virus characteristic of ALV-J, the achievement in research of epiposition vaccine is the hypermutation characteristic of antagonism ALV-J, successfully develops ALV-J vaccine and provides scientific basic and technical support.
Summary of the invention
For correlation circumstance of the prior art, inventor considers that epiposition vaccine being applied to anti-fowl J subgroup avian leucosis virus infects vaccine, and then after testing after deliberation, provide a kind of epiposition vaccine of J substock lymphoid leuoosis-resistant viral infection, this vaccine screens purification highly active recombiant protein His-cENV after adopting prokaryotic expression combines Freund adjuvant preparation and obtains, the wherein nucleotide sequence of coding recombiant protein His-cENV, as shown in SEQ ID NO.1, utilize these epiposition vaccine immunity 7 age in days kind fowl chickling can produce the neutralizing antibody of 1:128000, external viral Neutralizing test, zoopery shows this epiposition vaccine can neutralize different ALV-J isolated strain, available protecting chicken group resists ALV-J virus strain infection, the multi-epitope gene sequence that this epiposition vaccine is originated based on env, overcome the virus variation of ALV-J, open the ALV-J vaccine New Times, provide the new tool that anti-ALV-J infects, for the prevention and control of ALV-J provide technical support.
Inventor finds that ALV-J exists different epitopes at whole env after deliberation, can develop effective polyepitope vaccines by the epitope on screening env.Early stage the deep research of system is carried out to the pathogenic of ALV-J by inventor; in its immunosuppression mechanism of system analysis, across on the basis of kind of mechanism of transmission and immune evasion mechanism; this theory of wide spectrum epitope information can be transmitted in conjunction with multi-epitope; finally prepare anti-fowl J subgroup avian leucosis virus and infected vaccine; the immuning failure caused due to virus variation can be avoided; be conducive to provide more completely and more targetedly immunoprotection, the development that the achievement in research of epiposition vaccine is opens new direction and important technical support.
Concrete technical scheme of the present invention is as follows:
The epitope on the ALV-J cyst membrane that NCBI delivers is obtained by analysis; filter out the antigen epitope genes sequence representing epidemic isolates; connected by the codon of employing encodes glycine, serine; finally determine synthetic gene fragment 998bp; its nucleotide sequence is as shown in SEQ ID NO.1; prokaryotic expression goes out recombiant protein; its aminoacid sequence is as shown in SEQ ID NO.2; epiposition vaccine prepared by associating Freund adjuvant, and the epiposition vaccine obtained can realize protection body and support J substock lymphoid leuoosis-resistant infection.
Its detailed process is as follows:
Search the ALV-J env gene sequence that NCBI delivers, in conjunction with both at home and abroad to the progress of ALV-J epitope and immune evasion mechanism, DNAman software analysis is used to obtain the 22 sections of epitopes deriving from env gene, filter out and represent epidemic isolates envelope antigen epitope gene sequence, the codon of employing encodes glycine, serine is connected, obtain restructuring env gene sequence 998bp, its nucleotide sequence is as shown in SEQ ID NO.1.Described gene order 5 ' is held containing Nco I restriction enzyme site, and 3 ' end is containing Xho I restriction enzyme site.
Said gene sequence is synthesized by Shanghai Sheng Gong biological engineering company limited, and synthetic product cENV is connected to pUC57 carrier.
Synthetic product checks order, and its nucleotide sequence is as shown in SEQ ID NO.1;
This genetic fragment is connected to pET30a carrier, obtains recombinant vector pET30a-cENV, use 1-1.5mmol/L IPTG abduction delivering obtains the recombiant protein His-cENV with HIS label, and then utilizes this albumen associating Freund adjuvant to prepare epiposition vaccine.
This epiposition vaccine of the present invention is safe and efficient, cost is low is easy to large-scale mass production, the infection of 45%ALV-J strain can be resisted, two exempt from rear antibody holds time and reaches 11 weeks, can realize opening antenatal protection to kind of chicken, thus cut off the vertical transmission route of virus, the prevention and control purification for China J subgroup avian leucosis is significant, and high specificity can realize the protection to fowl J subgroup avian leucosis; Simultaneously due to its epitope information containing wide spectrum, the immuning failure caused due to virus variation can be avoided, the control for J subgroup avian leucosis provide more completely and more targetedly, the vaccine protection of more highly effective and safe.
Accompanying drawing explanation
Fig. 1 is recombiant plasmid double digestion qualification result electrophoretogram,
In figure, M is DL10000Marker, and 1 is the genetic fragment obtained after using restricted enzyme EcoR I, Xho I double digestion pUC57-cENV;
Fig. 2 is SDS-PAGE analysis result before and after recombinant expressed bacterium expression of results and protein purification,
In figure, M is 170KD Protein Ladder; 1 is abduction delivering bacterium liquid precipitate, and 2 is supernatant, and 3 is great expression albumen precipitation, and 4 is result after great expression protein purification;
Fig. 3 is that Western Blot method detects recombiant protein,
In figure, A uses HR1 multi-resistance, and B uses HR2 multi-resistance, and C uses His tag antibody, and D uses the anti-SU multi-resistance of chicken, and E uses mouse-anti gp85 multi-resistance, the result.
Detailed description of the invention
The concrete reagent adopted in the present embodiment and instrument are:
(1) main agents
HRP ELIAS secondary antibody is Bo Aosen biological company limited product;
TMB one-component substrate chromophoric solution, DAB colour reagent box, it is sky root Products that PCR primer glue reclaims test kit;
Albumen pre-dyed Marker, Taq polymerase, restricted enzyme EcoR I, Xho I are Thermo Products;
T4 ligase, pMD18-T carrier, DNA Marker are the precious biological company limited in Dalian product;
Plasmid extraction test kit is OMEGA Products;
His tag antibody is Beijing Quanshijin Biotechnology Co., Ltd's product;
Tryptone (Tryptone) and yeast extract (Yeast Extract) are Oxiod Products;
All the other reagent are domestic analytical reagent.
(2) key instrument
Pipettor, spectrophotometric count Eppendorf Products;
Low-temperature and high-speed centrifuge is Hunan, Hubei instrument Products;
Gel imaging system is that Beijing monarch anticipates Products;
Microplate reader is Thermo Products;
Constant-temperature table is Shanghai Shen Neng betting office product;
Constant incubator, superclean bench are that fast Products is won in Shanghai;
Electrophresis apparatus, SDS-PAGE electrophoresis tank, Western-blot film transfer printing device are Beijing 61 Products;
Cell supersonic wave pulverizer is Ningbo new sesame biological company limited product.
List except content except above-mentioned, the NM part of the present invention all adopts prior art.
The screening synthesis of embodiment 1ALV-J multi-epitope gene
Search the ALV-J env gene sequence that NCBI delivers, bioinformatics method analysis is used to obtain envelope antigen epi-position 22 sections, filter out representative Chinese epidemic isolates envelope antigen epitope gene sequence in recent years, employing encodes glycine, serine codon are connected, obtain restructuring env gene sequence 998bp, its gene order is as shown in SEQ ID NO.1.Described gene order 5 ' is held containing Nco I restriction enzyme site, and 3 ' end is containing Xho I restriction enzyme site.
Said gene sequence is synthesized by Shanghai Sheng Gong biological engineering company limited, and utilizes common process to be connected to pUC57 carrier synthetic product cENV further by it.
Synthetic product checks order, and its gene order is as shown in SEQ ID NO.1;
PET30a and the above-mentioned pUC-57 cloning vehicle being connected to genes of interest are carried out double digestion 60min with restricted enzyme Nco I and Xho I respectively under 37 DEG C of water bath condition, reclaims large fragment and genes of interest fragment with after 1% agarose gel electrophoresis respectively according to the requirement of glue recovery test kit description;
Double digestion result as Fig. 1,
Spent the night by the object 22 DEG C of above-mentioned acquisition connection, wherein linked system is: pET30a carrier 0.5 μ l, cENV genetic fragment 1 μ l, ddH2O20 μ l, T4DNA ligase 1 μ l, 10 × Green Buffer2.5 μ l, cumulative volume is totally 25 μ l, obtains and connects product pET30a-cENV recombiant plasmid.
Conventionally proceed in competence antibacterial BL21 prepared by 0.1%CaCl2 by connecting product pET30a-cENV recombiant plasmid, coat in the LB flat board containing kanamycin, 37 DEG C of incubated overnight, next day picking list bacterium colony, shake bacterium, extract after plasmid carries out double digestion qualification and send to order-checking, confirm the sequence that wherein has as shown in SEQ ID NO.1, the fragment of the visible restructuring env gene 998bp obtained successful clone enters in pET30a-cENV recombiant plasmid.
The expression and purification of embodiment 2 recombinant envelope protein
Positive BL21 bacterium containing recombiant plasmid after order-checking is inoculated in (commercial conventional medium in the LB fluid medium containing kanamycin (final concentration is 10 μ g/ml), wherein containing 1% (w/v) Tryptone, 0.5% (w/v) Yeast Extract, 1% (w/v) NaCl, 0.1mg/ml Kanamycin), 37 DEG C of vibrations (200rpm), be cultured to OD600=1.0, to add concentration be the IPTG of 500mMol is 1mmol/L to the final concentration of IPTG in whole culture medium, 37 DEG C are continued to cultivate 4h, establish the recombinant plasmid transformed BL21 bacterium do not induced in contrast simultaneously.
Analyze display through SDS-PAGE, detecting step is as follows: collect the engineering bacteria bacterium liquid cultivated, 5000rpm, centrifugal 5min, abandons supernatant, and precipitation utilizes the resuspended acquisition re-suspension liquid of the PBS of standard; Add the ratio of standard P BS3ml according to 100ml initial medium, the PBS adding standard in precipitation obtains re-suspension liquid.Every pipe 3ml in resuspended rear subpackage and 5ml centrifuge tube.Be placed in by centrifuge tube in the beaker of mixture of ice and water, use Ultrasonic Cell Disruptor carrying out ultrasonic bacteria breaking, 120W, work 1s, interval 2s, ultrasonic 90 circulations, repeat once; Proceeded to by carrying out ultrasonic bacteria breaking liquid in 50ml centrifuge tube, trim, puts into high speed refrigerated centrifuges, 8000rpm, 4 DEG C, and then centrifugal 10min uses SDS-PAGE electrophoresis detection; Without albumen (not shown) in result supernatant, albumen is present in precipitation, illustrates that recombiant protein His-cENV expresses with inclusion bodies, and as shown in Figure 2, destination protein size is about 33KD.
The purification step of recombiant protein is as follows:
By the cleaning mixture I resuspended precipitation of the precipitation of above-mentioned centrifugal acquisition with 9 times of volumes, after leaving standstill 5min, 8000rpm, 4 DEG C, 15min, abandons supernatant.By the resuspended precipitation of the cleaning solution II of 9 times of volumes, after leaving standstill 5min, 8000rpm, 4 DEG C, 15min abandons supernatant, retains precipitation.By the resuspended precipitation of the cleaning solution II I of 9 times of volumes, after leaving standstill 5min, 8000rpm, 4 DEG C, 15min, abandons supernatant, retains precipitation.Add 4-10ml lysate by 100ml bacterium liquid, 4 DEG C are spent the night.8000rpm，4℃，15min。Get supernatant, be purifying protein.
Preparation of reagents method described in inclusion body purification:
(1) antibacterial re-suspension liquid (250ml): PBS (NaCl2g, KCl0.05g, Na
2hPO
40.36gKH
2pO
40.06g), 1mmol/L EDTA.
(3) solution I (500ml): 500mmol/L Tris-Cl (pH=6.0), 50mmol/L NaCl, 50mmol/L EDTA
(3) solution II (250ml): solution I, 1M carbamide
(4) solution III (250ml): solution I, 2M carbamide
(5) solubilization of inclusion bodies liquid (250ml): solution I, 8M carbamide
SDS-PAGE method and Western Blot method detect the albumen after purification (shown in Fig. 2 and 3).The albumen obtained measures protein content through Bradford method, and 2ml centrifuge tube subpackage-80 DEG C saves backup.
The SDS-PAGE method of above-mentioned use is conventional method, and NM reagent is the reagent that conventional method specifies to use, and its concrete operation step is as follows:
1) by glass plate wash clean, fix with clip, vertically place, configure 12% separation gel, be injected between two glass plates fast, glue top adding distil water seals, to keep glue face smooth.After gelling to be separated is solid, removes the distilled water on separation gel surface, suck residual water with filter paper.Inject concentrated glue, quick insertion comb, and note the generation preventing bubble.
2) recombiant protein got after purification adds 15-20 μ l4 × SDS loading buffer, boils 10min.
3) add 1 × Tris-glycine buffer toward electrophoresis tank, gelling to be concentrated is solid, carefully pulls out comb, uses wash buffer well, use microsyringe loading, 20 μ l/ holes, exact connect ion electrophoresis tank both positive and negative polarity, opening power.120V electrophoresis, stops electrophoresis when electrophoresis to bromophenol blue arrives the bottom of glue.
4) dye: take off gel, with distilled water flushing, put into coomassie brilliant blue staining liquid, dyeing more than 4h.
5) decolour: gel is put into destaining solution, is placed on shaking table and decolours, period changes more than 3 times destaining solution, treats that gel blue background all sloughs stopping, gel is put into distilled water and stop decolouring.To take pictures preservation, as shown in Figure 2, occurred that size is about the object recombiant protein band of 33KD.
6) cut a NC film identical with Western Blot gel size, soak into deionized water, identical with gel size two filter paper and fiber mat transfer printing buffer are soaked into simultaneously.
7) install transfer device by sandwich, i.e. negative pole folder-fiber mat-filter paper-gel-NC film-filter paper-fiber mat-positive pole folder, make NC film be positioned at the positive pole-face of transfer printing, gel is positioned at negative pole face, therebetween without any bubble gap.Transfer device is put into electrophoretic blotting instrument, add transfering buffering liquid, 140mA electrophoresis 2h.
8) after transfer printing terminates, by the rinsed with deionized water one time of the NC film after transfer, NC film is soaked in confining liquid, closes for 4 DEG C and spend the night.
9) NC film is washed three times, each 10min with PBST.
10) use His tag antibody to be primary antibodie, NC film and primary antibodie 37 DEG C react 1h, wash film three times, 10min/ time with PBST.
11) NC film is placed in the sheep anti-mouse igg of HRP labelling, and 37 DEG C of reaction 1h, wash film three times with PBST, 10min/ time.
12) colour developing of DAB colour reagent box lucifuge is used; Now answer close observation process color, and flowing water color development stopping at more shallow background and when reaching suitable colored intensity.To take pictures preservation, as shown in Figure 3, occurred that size is about the object recombiant protein band of 33KD.
The Activity determination of embodiment 3 recombiant protein
The His-cENV albumen bag using above-mentioned purification to obtain, by elisa plate, uses elisa to detect the biologic activity of this albumen.With the chicken serum of gp85 protein immunization Mus positive serum, HR1 polypeptide, HR2 polypeptide immune chicken positive serum and IDEXX detection kit test positive for positive control, healthy SPF chicken serum is negative control, and HRP labelling sheep anti-mouse igg, goat-anti chicken IgG ELIAS secondary antibody by specification use.The OD value ratio of result display positive control and negative control is greater than 2.1, illustrates that this albumen has good biologic activity and antigenicity.
ELISA concrete steps are as follows:
1) bag quilt: the recombiant protein getting purification, with normal saline dilution to optium concentration, wraps by Sptting plate, and 100 μ l/ holes, 4 DEG C are spent the night.
2) wash: get rid of the coating buffer in ELISA Plate, fill it up with each hole with PBST (PBS, 0.05%Tween-20), shaking table concussion 3min, discards PBST, washs 3 times, 3min/ time, pat dry.
3) close: each hole adds confining liquid (3% skimmed milk), 350 μ l/ holes, puts 37 DEG C of incubators and hatches 0.5h, washs 3 times, 3min/ time, pats dry.
4) application of sample: will add bag by plate after serum to be checked is by gradient dilution, 100 μ l/ holes, set up yin and yang attribute serum control simultaneously.Put 37 DEG C of incubators and hatch 1h, wash 3 times, 3min/ time, pat dry.
5) with confining liquid, HRP labelling sheep anti-mouse igg, goat-anti chicken IgG ELIAS secondary antibody are pressed 1:5000 dilution, 100 μ l/ holes, put 37 DEG C of incubators and hatch 1h, wash 3 times, 3min/ time, pat dry.
6) develop the color: use TMB one-component colour reagent box (TIANGEN Products) to add bag by plate by 100 μ l/ holes, under 37 DEG C of conditions, lucifuge colour developing 20min adds stop buffer, 2M H2SO4 color development stopping.
7) survey with enzyme-linked immunosorbent assay instrument it to detect each hole OD value at wavelength 450nm, negative control OD450 value is N, and positive control OD450 value is P.If P/N>2.1 is judged to the positive.
The preparation of embodiment 4 J substock lymphoid leuoosis-resistant viral infection epiposition vaccine
1) buy the 1 blue brown chickling 50 in age in days sea from Eastern Mountain Zhong Qin company limited, under isolation cover condition, grouping is raised, and gathers serum and cotton swab respectively, and use IDEXX test kit detects ALV-J antibody and ALV antigen is feminine gender;
Wherein said is grouped into blank group-O group 10, groups 20, immune His-cENV group-1, groups 20, immune His-cENV+ Freund adjuvant group-2.
2) purifying protein of above-mentioned acquisition is carried out concentration determination, after mensuration concentration reaches 7.6mg/ml, PBS can be used to be diluted to 1mg/ml, by the protein solution after dilution and Freund adjuvant according to the proportions of volume ratio 1:1, mix the syringe subpackage of rear 1ml on the oscillator, often pipe 0.2ml;
The recombiant protein solution pH of wherein said 1mg/ml is that 7.4,1 × PBS buffer dilutions obtain;
7 ages in days carry out multiple spot immunity by the mode of intramuscular injection to lower limb flesh, immune group every chicken immune albumen 0.1g, and matched group injection same dose sterilizing PBS, after two weeks, booster immunization once.
3) antibody dynamic regularity monitoring, gathers weekly determination of serum antibody titer after within after first immunisation 10 days, two, exempting from 10 days;
Wherein said antibody titer measures, mainly by serum to be checked with 10 times of doubling dilutions, other operating procedures detect recombiant protein active process with ELISA.
The external viral neutralization of embodiment 5 detects
Virus neutralization tests example 1
The high-titer serum that inventor adopts booster immunization to prepare carries out viral Neutralizing test;
ALV-J NX0101 strain is the pure J subgroup avian leucosis strain of the strain that is separated to from China's Ningxia, China broiler calendar year 2001, clear background;
By blood serum sample 56 DEG C before experiment, 30min deactivation, remove non-specific material, the aseptic DMEM culture fluid of ALV-J NX0101 virus liquid is diluted to 1TCID50/0.1ml, 10TCID50/0.1ml, 100TCID50/0.1ml, the inactivated serum of the virus liquid and equivalent of getting 250 μ l dilutions fully mixes latter 37 DEG C and hatches 1 hour, be inoculated in 24 orifice plates covering with monolayer DF-1 cell, every hole 200 μ l, and set up the contrast only adding virus liquid and an increase serum, inoculate the DMEN culture fluid changed containing 1% peptide Ox blood serum after 1 hour and maintain 7 days, get cell conditioned medium ALV antigen detection kit and detect viral level in supernatant, result display 100TCID50/0.1ml group, virus liquid matched group is positive, all the other are negative, and the positive value of 100TCID50/0.1ml group is starkly lower than virus liquid matched group, illustrate recombiant protein His-cENV combine Freund adjuvant prepare epiposition vaccine induction body produce antibody can under in vitro conditions with ALV-JNX0101 strain generation neutralization reaction, and Be very effective, prove that epiposition vaccine provided by the present invention can protect chicken group to support the infection of J substock lymphoid leuoosis-resistant.
Virus neutralization tests example 2
ALV-J WS0705 strain is the pure J subgroup avian leucosis strain of the strain that is separated to from Shandong laying hen for 2007, clear background;
Inventor adopts test example 1 same procedure to carry out viral Neutralizing test, operating process is consistent, result display 100TCID50/0.1ml group, virus liquid matched group is positive, all the other are negative, and the positive value of 100TCID50/0.1ml group is starkly lower than virus liquid matched group, illustrate bright recombiant protein His-cENV combine Freund adjuvant prepare epiposition vaccine induction body produce antibody can under in vitro conditions with ALV-J WS0705 strain generation neutralization reaction, and Be very effective, prove that epiposition vaccine provided by the present invention can protect chicken group to support the infection of J substock lymphoid leuoosis-resistant.
Virus neutralization tests example 3
ALV-J DZ1022 strain is the pure J subgroup avian leucosis strain of the strain that is separated to from Shandong laying hen for 2011, clear background;
Inventor adopts test example 1 same procedure to carry out viral Neutralizing test, operating process is consistent, result display 100TCID50/0.1ml group, virus liquid matched group is positive, all the other are negative, and the positive value of 100TCID50/0.1ml group is starkly lower than virus liquid matched group, illustrate bright recombiant protein His-cENV combine Freund adjuvant prepare epiposition vaccine induction body produce antibody can under in vitro conditions with ALV-J DZ1022 strain generation neutralization reaction, and Be very effective, prove that epiposition vaccine provided by the present invention can protect chicken group to support the infection of J substock lymphoid leuoosis-resistant.
Embodiment 6 in vivo test detects
Recombiant protein associating Freund adjuvant immunity 7 age in days kind fowl chickling, 19 age in days booster immunizations once, carry out counteracting toxic substances and observe immune protective effect after 10 days, establish ALV-J to infect positive controls and negative control group simultaneously.
Raise to 14 week age, all cut open and kill, period carries out weekly hematology, pathological study and pathogeny detection antiviral effect;
Result display immunoprotection group chicken group well-grown, blood indices has no significant change, and ALV Detection of antigen is negative, and without toxin expelling phenomenon, histopathology detects and has no tissue injury and inflammatory reaction.And serious viremia appears in ALV-J positive controls, there is significantly damage and inflammation in the important organ such as liver and kidney.Experiment proves that this epiposition vaccine can protect chicken group to resist the infection of ALV-J.
Sequence table
Claims (2)
1. the epiposition vaccine of a J substock lymphoid leuoosis-resistant viral infection, it is characterized in that: this vaccine screens purification highly active recombiant protein His-cENV after adopting prokaryotic expression combines Freund adjuvant preparation and obtains, the wherein nucleotide sequence of coding recombiant protein His-cENV, its nucleotide sequence is as shown in SEQ ID NO.1, prokaryotic expression goes out recombiant protein, and its aminoacid sequence is as shown in SEQ ID NO.2.
2. epiposition vaccine according to claim 1 is as the application of ALV-J vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410593612.6A CN104306995B (en) | 2014-10-29 | 2014-10-29 | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410593612.6A CN104306995B (en) | 2014-10-29 | 2014-10-29 | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104306995A true CN104306995A (en) | 2015-01-28 |
| CN104306995B CN104306995B (en) | 2017-04-12 |
Family
ID=52362396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410593612.6A Active CN104306995B (en) | 2014-10-29 | 2014-10-29 | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104306995B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442982A (en) * | 2016-08-31 | 2017-02-22 | 扬州大学 | Fowl leucovirus subgroup J specific antigenic polypeptide and application thereof |
| CN114349829A (en) * | 2022-01-14 | 2022-04-15 | 华南农业大学 | Identification of ALV-J MHC-B2 restricted epitope peptide and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070172461A1 (en) * | 1994-07-27 | 2007-07-26 | The Council Of The Queensland Institute Of Medical Research | Polyepitope vaccines |
| WO2007062656A3 (en) * | 2005-11-30 | 2008-05-22 | Copenhagen University | A nucleotide vaccine |
| CN101899465A (en) * | 2009-12-15 | 2010-12-01 | 华南农业大学 | Recombinant J subgroup avian leucosis virus infective cloned plasmids and preparation method and application thereof |
| CN103614329A (en) * | 2013-11-06 | 2014-03-05 | 山东农业大学 | Construction method and application of DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J |
| CN103805635A (en) * | 2014-03-07 | 2014-05-21 | 山东农业大学 | J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier as well as preparation method and application thereof |
| CN103937833A (en) * | 2014-03-07 | 2014-07-23 | 山东农业大学 | siRNA recombinant interference vector based on subgroup J avian leukosis virus LTR gene conserved sequence, preparation method and application thereof |
-
2014
- 2014-10-29 CN CN201410593612.6A patent/CN104306995B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070172461A1 (en) * | 1994-07-27 | 2007-07-26 | The Council Of The Queensland Institute Of Medical Research | Polyepitope vaccines |
| WO2007062656A3 (en) * | 2005-11-30 | 2008-05-22 | Copenhagen University | A nucleotide vaccine |
| CN101899465A (en) * | 2009-12-15 | 2010-12-01 | 华南农业大学 | Recombinant J subgroup avian leucosis virus infective cloned plasmids and preparation method and application thereof |
| CN103614329A (en) * | 2013-11-06 | 2014-03-05 | 山东农业大学 | Construction method and application of DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J |
| CN103805635A (en) * | 2014-03-07 | 2014-05-21 | 山东农业大学 | J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier as well as preparation method and application thereof |
| CN103937833A (en) * | 2014-03-07 | 2014-07-23 | 山东农业大学 | siRNA recombinant interference vector based on subgroup J avian leukosis virus LTR gene conserved sequence, preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 侯磊: "ALV-J分离株Hrb-1 gp85基因蛋白的单克隆抗体及抗原表位初步分析", 《中国优秀硕士学位论文全文数据库.农业科技辑》 * |
| 杨玉莹 等: "鸡内源性类J亚群禽白血病病毒gp85基因的克隆及分析", 《病毒学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442982A (en) * | 2016-08-31 | 2017-02-22 | 扬州大学 | Fowl leucovirus subgroup J specific antigenic polypeptide and application thereof |
| CN114349829A (en) * | 2022-01-14 | 2022-04-15 | 华南农业大学 | Identification of ALV-J MHC-B2 restricted epitope peptide and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104306995B (en) | 2017-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102636644B (en) | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen | |
| CN103751774B (en) | The recombinant cell lines of stably express CSFV E 2 protein and in the application of preparing in subunit vaccine for swine fever and diagnostic reagent | |
| Castellino et al. | Generating memory with vaccination | |
| CN105331636A (en) | Recombination cell line for stable expression of classical swine fever virus E2 and application thereof | |
| CN104548087B (en) | Epitope vaccine for resisting A/B subgroup avian leucosis virus infection and preparation method and application of epitope vaccine | |
| CN101235085B (en) | Monoclonal antibody of membrane protein E for resisting West Nile virus and application thereof | |
| CN102304181A (en) | Monoclonal antibody of avian leukosis virus subgroup J surface protein and preparation method thereof | |
| CN103751773A (en) | Recombinant BHK cell line for stably expressing classical swine fever virus E0-E1-E2 protein, and applications of the same in preparation of vaccines and diagnosis reagents of classical swine fever | |
| CN107630024A (en) | The gene of encoding hemagglutinin protein of H 5 avian influenza virus and its application | |
| CN107793473A (en) | A kind of antigen protein of secondary poultry bacillus and its application | |
| CN103360498B (en) | Anti-chicken infectious bursal disease recombinant protein subunit vaccine | |
| He et al. | Vaccine design for severe acute respiratory syndrome coronavirus | |
| CN102816246A (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN104974249A (en) | Riemerella anatipestifer OmpA/MotB truncated recombinant protein, antibody and preparation method and application thereof | |
| CN105348386B (en) | The monoclonal antibody cocktail and detection kit of the O-shaped virus of resistant to foot and mouth disease | |
| CN104306995A (en) | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine | |
| Strizki et al. | Differential reactivity of immune sera from human vaccinees with field strains of eastern equine encephalitis virus | |
| CN101591379A (en) | Based on the amino acid mutation of EIAV attenuated live vaccine and the anti-HIV vaccine that makes up | |
| CN101481675B (en) | Anti-swine fever multi-epitope DNA vaccine, construction method and use | |
| CN101082051A (en) | Recombined foot-and-mouth disease virus polypeptide fragment and and uses thereof | |
| CN105296434A (en) | Hybridoma cell strain and monoclonal antibody secreted by hybridoma cell strain for resisting foot-and-mouth disease O-type viruses and application | |
| CN105296435B (en) | The monoclonal antibody specific and application of hybridoma cell strain and its O-shaped (O/GX/09-7) virus of the resistant to foot and mouth disease of secretion | |
| CN102058881A (en) | Gene recombinant vaccine for preventing enterovirus 71 infection and preparation method thereof | |
| CN103983782A (en) | ELISA kit for detecting hog cholera virus Erns IgM antibody | |
| CN102304180A (en) | Monoclonal antibody of avian reticuloendotheliosis virus envelope protein and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |