CN101863977B - J substock lymphoid leuoosis-resistant polyclonal antibody and preparation method thereof - Google Patents
J substock lymphoid leuoosis-resistant polyclonal antibody and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a J substock lymphoid leuoosis-resistant polyclonal antibody and a preparation method thereof. The J substock lymphoid leuoosis-resistant polyclonal antibody can effectively inhibit the infection of J substock avian leukosis virus, and is obtained on the basis of highly conserved sequence synthesized small peptides formed by 27 amino acid in transmembrane protein coded with J substock avian leukosis virus gp 37 gene through a coupled keyhole hemocyanin immune rabbit; and the polyclonal antibody blocks the invasive J substock avian leukosis virus reproduction by being combined with the nucleus non-specifity, and is combined with and eliminates the specificity of the J substock avian leukosis virus, thereby effectively preventing the generation and spreading of the J substock lymphoid leuoosis, greatly reducing the economic loss of aviculture caused by infection of J substock avian leukosis virus, and providing a novel method and thinking for the lymphoid leuoosis preventing and controlling.
Description
(1) technical field
The present invention relates to polyclonal antibody of a kind of J substock lymphoid leuoosis-resistant and preparation method thereof
(2) background technology
The J subgroup avian leucosis is a kind of neoplastic disease that is caused by J subgroup avian leucosis virus (ALV-J).ALV-J is the retrovirus with cyst membrane, and fowl clinical infection main manifestations is myelocytome, immunological tolerance, high mortality and growth retardation.Between 1997-1998, ALV-J is global outburst, and world's meat kind chicken industry has been caused crushing blow.ALV-J infects except causing myelocytome, and erythroblastoma, vascular tumor, nephroncus and sarcoma are everlasting and are infected the later stage and follow generation, and mortality ratio is 1%-5% usually, and the peak period can reach 50%.Studies confirm that, ALV-J has lasting detrimental effect to immunne response, makes productivity low, and secondary infection and polyinfection take place frequently, and the outburst of J subgroup avian leucosis will produce great harm to aquaculture.
1, the J subgroup avian leucosis takes place frequently in China
China white plumage meat kind chicken is all from external introduction, and emerging ALV-J do not taked any precautionary measures, and the cultivating condition of China is relatively backward in addition, makes loss that ALV-J causes in China higher than other any country.2000, the J subgroup avian leucosis is outburst comprehensively in China white plumage meat kind chicken, has caused huge financial loss; Between 2002-2005, ALV-J virus breaks through species barrier, causes outburst J subgroup avian leucosis in Guangdong, the yellow chicken group in Guangxi three, Chinese numb chicken group, and many plants are closed down; 2007, and the closely-related angiomatosis of J subgroup avian leucosis breaks out in egg breeder flock and commodity egg group, and sweep over the country very soon.Avian leukosis J subgroup is given birth in China's fowl mass-sending has following features:
(1) infection rate and sickness rate are high.Over nearly 10 years, infection rate is all in 10% left and right in meat kind chicken, egg kind chicken.
(2) general, the morbidity age in days of early infection obviously shifts to an earlier date.At present fowl J subgroup leukemia has become common disease young chicken group or in opening antenatal chicken group.
(3) polyinfection impels viral recombination frequency to increase.The investigation demonstration, the polyinfection of avian leukosis and Marek, reticuloendotheliosis disease is on the rise, for clinical diagnosis has brought difficulty.
(4) host range expansion.Break out in angiomatous laying hen, China peculiar chicken kind fiber crops chicken, thin and small hen and reed catkins chicken and also detected ALV-J.This shows J subgroup leukemia to laying hen, the propagation of indigenous chicken kind, and its host range obviously enlarges.
(5) tumour is tending towards variation.At present, China infects and has occurred many rare tumours in the past in the fowl group, as vascular tumor, erythroblastoma, hepatocellular carcinoma, gland cancer, fibroma, fibrosarcoma, skin carcinoma and carcinoma of fallopian tube etc.
(6) pathogenic enhancing.Many tumours of finding in laying hen and indigenous chicken are even more serious than the pathology in meat kind chicken before, have never seen myelocytome in some organ body of Broiler Chicken, and more common in laying hen, recommend myelocytome, intestines myelocytome etc. as neck myelocytome, waist.
2, the prevention and control present Research of J subgroup avian leucosis
All the time, due to the singularity of ALV-J virus self and the complicacy that causes disease, the study on prevention of ALV-J is had no remarkable one-tenth, so far without medicine, the vaccine of the effective ways of controlling ALV-J and control ALV-J infection.
To the prevention and control of ALV-J, developed country mainly takes to purify and eliminates, 1997-1998, after whole world outburst ALV-J, the world 4 greatly foster fowl company successively drops into huge fund research ALV-J strategy anti-processed, finally makes a whole set of strict control techniques, has progressively controlled the lasting outburst of ALV-J; After 2002, ALV-J has purified in breeder flock clean for generations substantially, but also has fragmentary outburst in commercial meat bird, its weekly average attack rate still in 1.5% left and right.Controlling ALV-J is a long-term and unremitting job.Purification has also brought some problems comprehensively on a large scale, and except aquaculture cost rolled up, the most potential impact was the evolution that has promoted ALV-J.
Under As-Is, China there is no effective prevention and control measure to the J subgroup avian leucosis, and government is subsidy not, does not also have which company can afford to bear high purification expense, due to the characteristics of ALV-J itself and the cultivation present situation of China, the Biosafety measure produces little effect to it.Simultaneously, science, irrational control techniques, also can not accelerate sudden change and the evolution of virus, makes the disease more sophisticated.
Because ALV-J is togavirus, and virus envelope has determined viral antigenicity, and antigenicity has the characteristics of continuous change, makes it to become the bottleneck that the avian leukosis vaccine development can't be gone beyond, therefore, the development of the leukemic medicine of anti-J subgroup becomes present focus and focus.
(3) summary of the invention
The purpose of this invention is to provide polyclonal antibody of a kind of J substock lymphoid leuoosis-resistant and preparation method thereof, for the control of J subgroup avian leucosis provides new effective means, reduce aviculture and infect the financial loss that causes because of ALV-J.
The little peptide of main contents of the present invention: ISU (immunosuppression district) is synthetic; Coupling; Antigen emulsification, immune rabbit; Antibody titer detects; Collect serum and obtain the polyclonal antibody Preservation in sterile condition.
Determine gp37 gene conserved sequence according to early-stage Study basis, by the synthetic little peptide of ISU of this sequence and with little peptide respectively with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) coupling.
Get the little peptide of ISU of phosphate buffer soln (PBS) dilution-KLH conjugate, fully emulsified with the complete Freund's adjuvant of equivalent, in rabbit neck part or back subcutaneous injection, the dosage that every rabbit press antigen 1 00-500 μ g is injected.Head exempts from 3-4 booster immunization for the first time after week, and the antigen dose of every rabbit immunity is with immunity for the first time, and the full freund's adjuvant that toos many or too much for use is fully emulsified; Later on every 1-3 week booster immunization 1 time; Since the 1st booster immunization, each immune rear 7-10 days, measure tiring of antibody through ear vein or arteria auricularis blood sampling, after detection met the requirements through booster immunization, jugular vein was taken a blood sample, and separation of serum obtains polyclonal antibody ,-20 ℃ of aseptic subpackaged preservations.
The little peptide conjugate storage temperature of described ISU is 4 ℃, and the little peptide conjugate storage temperature of ISU after the PBS dilution is-20 ℃.
Compared with prior art, advantage of the present invention is:
1, the prepared polyclonal antibody of the present invention can effectively suppress the infection of J subgroup avian leucosis virus, and can be because the variation of J subgroup avian leucosis virus does not lose its validity, and this at home and abroad still belongs to the first time.
2, the polyclonal antibody of the present invention's preparation has the advantages such as high specificity, efficient disease-resistance toxic action, stable in properties, long quality-guarantee period.
3, the invention solves aviculture and effectively control the difficult problem that the J subgroup avian leucosis virus infects, for aviculture control J subgroup avian leucosis has been started new situation, can decrease J subgroup avian leucosis cause directly and indirect economic loss, this is the inaccessiable effect of means of prevention of existing avian leukosis.
(4) description of drawings
Fig. 1 is product innovation production technological process of the present invention.
In figure, 1 is that the little peptide of ISU is synthetic, the little peptide coupling of the 2nd, ISU, and the 3rd, little peptide conjugate emulsification, the 4th, immune animal, the 5th, bioactivity, the 6th, the jugular vein blood sampling, the 7th, serum separates, and the 8th, polyclonal antibody.
Fig. 2 is that indirect immunofluorescence (IFA) detected result: A connects virus to add anti-fluoroscopic examination; B does not add anti-fluoroscopic examination for connecing virus; C does not add anti-negative control for connecing virus.
In figure the presentation of results of B of the present invention many anti-can with the nonspecific combination of nucleus, blocked copying of virus.In figure, A has illustrated the present invention resists the effect that has produced specific binding with ALV more.
Fig. 3 is the fluorescence quantitative PCR detection result: figure post 1 is for first adding anti-rear inoculation ALV-J; Figure post 2 for first inoculate add after ALV-J anti-; Figure post 3 is for only inoculating ALV-J; Figure post 4 is normal control.Experimental result is by 2
-Δ Δ CtMethod of calculation are analyzed.
In figure presentation of results the present invention ALV-J has been produced good antiviral effect.
(5) embodiment
Be described in further detail below in conjunction with the production of accompanying drawing to polyclonal antibody.
1, be LQNRAAIDFLLLAQGHGCQDVEGMCCF according to the definite gp37 gene conserved sequence in early-stage Study basis, by this sequence by the synthetic little peptide of ISU in BeiJing ZhongKe Yaguang Biology Science Co., Ltd and with little peptide respectively with KLH and BSA coupling, adopting high performance liquid chromatography (HPLC) method to carry out synthesizing and purifying identifies, purity reaches more than 97%, and SDS-PAGE (SDS-PAGE) method identifies that coupling effect is good.
2, select about 1.5 months, male stalwartness, the new zealand white rabbit about body weight 2Kg, before immunity, first ear vein blood sampling separation of serum stores for future use as the bioactivity negative control, raises 7 days after animal conforms, and begins to carry out immunity.
3, take the little peptide of ISU of PBS dilution-KLH conjugate 0.5-1.0ml, fully emulsified with the complete Freund's adjuvant of equivalent, in the subcutaneous 6-12 point injection of rabbit neck part or back, every some 0.1-0.2ml, the dosage that every rabbit is pressed antigen 1 00-500 μ g is injected.
4, head exempts from 3-4 and carries out booster immunization for the first time after week, and the dosage of every rabbit immunizing antigen is with immunity for the first time, and the full freund's adjuvant that toos many or too much for use is fully emulsified, injects in the subcutaneous 6-12 point of rabbit neck part or back; Later on every 1-3 week booster immunization 1 time.
5, since the 1st booster immunization, rear 7-10 days of each immunity, through ear vein or arteria auricularis blood sampling 2-4ml, adopt indirect enzyme-linked immunosorbent assay (ELISA) to measure tiring of antibody, the little peptide of the ISU of coupling BSA is used as antigen in the indirect ELISA detection method.
6, detect to tire after the 4th booster immunization and reach 6.4 * 10
4-1.28 * 10
5Between (as table 1), meet the requirements, jugular vein blood sampling, separation of serum, aseptic subpackaged-20 ℃ preservation.
Table 1 indirect ELISA is measured the data that polyclonal antibody is tired
Annotate: P/N 〉=2 are positive, P/N=(positive mean value-blank value)/(negative mean value-blank value)
the immunosuppression that breaks through virus is the key point that the antagonism avian leukosis virus infects, " transmembrane protein (TM) major function district infects at ALV carrying out the National Nature fund project for we, effect in immunity " find in process, TM albumen by the gp37 genes encoding plays a decisive role in virus infection and immunosuppression, wherein in the TM protein subunit, the highly conserved sequence of 27 amino acid compositions is being brought into play keying action, and there is not the otherness between strain in this highly conserved sequence, the present invention based on this, developed the polyclonal antibody that effective inhibition J subgroup avian leucosis virus infects.
This polyclonal antibody is applied to the control of avian leukosis, can the decrease aviculture because ALV-J infects the financial loss that causes, for the prevention and control of avian leukosis provide strong technical guarantee, started simultaneously the new approaches of fowl tumprigenicity prevention and cure of viruses research.
Claims (1)
1. the preparation method of the polyclonal antibody of a J substock lymphoid leuoosis-resistant is characterized in that:
(1) determine that by the gp37 gene conserved sequence of determining the aminoacid sequence of the little peptide of ISU is LQNRAAIDFLLLAQGHGCQDVEGMCCF, by this sequence by the synthetic little peptide of ISU in BeiJing ZhongKe Yaguang Biology Science Co., Ltd and with little peptide respectively with KLH and BSA coupling, adopting the high performance liquid chromatography method to carry out synthesizing and purifying identifies, purity reaches more than 97%, and the SDS-PAGE method identifies that coupling effect is good;
(2) select 1.5 months, male stalwartness, the new zealand white rabbit of body weight 2Kg, before immunity, first ear vein blood sampling separation of serum stores for future use as the bioactivity negative control, raises 7 days after animal conforms, and begins to carry out immunity;
(3) take the little peptide of ISU of PBS dilution-KLH conjugate 0.5-1.0ml, fully emulsified with the complete Freund's adjuvant of equivalent, in the subcutaneous 6-12 point injection of rabbit neck part or back, every some 0.1-0.2ml, the dosage that every rabbit is pressed antigen 1 00-500 μ g is injected;
(4) head exempts from 3-4 and carries out booster immunization for the first time after week, and the dosage of every rabbit immunizing antigen is with immunity for the first time, and the full freund's adjuvant that toos many or too much for use is fully emulsified, injects in the subcutaneous 6-12 point of rabbit neck part or back; Later on every 1-3 week booster immunization 1 time;
(5) since the 1st booster immunization, rear 7-10 days of each immunity through ear vein or arteria auricularis blood sampling 2-4ml, adopts indirect enzyme-linked immunosorbent assay to measure tiring of antibody, and the little peptide of the ISU of coupling BSA is used as antigen in the indirect ELISA detection method;
(6) detect to tire after the 4th booster immunization and reach 6.4 * 10
4-1.28 * 10
5Between, meet the requirements, jugular vein blood sampling, separation of serum, aseptic subpackaged-20 ℃ preservation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085640A (en) * | 2015-07-03 | 2015-11-25 | 山东农业大学 | PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102304181A (en) * | 2011-10-24 | 2012-01-04 | 山东农业大学 | Monoclonal antibody of avian leukosis virus subgroup J surface protein and preparation method thereof |
| CN106442982B (en) * | 2016-08-31 | 2018-06-08 | 扬州大学 | A kind of avian leukosis virus J subgroup specific antigen polypeptides and its application |
| CN108929881A (en) * | 2018-08-06 | 2018-12-04 | 扬州大学 | A kind of expression vector and its construction method and protein expression of the avian leukosis virus gp37 albumen lacking transmembrane domains |
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| CN101333246A (en) * | 2008-07-30 | 2008-12-31 | 中国农业科学院哈尔滨兽医研究所 | Neutral B cell antigen epitope polypeptide of encephalitis b virus E protein and uses thereof |
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| AU6698900A (en) * | 1999-07-16 | 2001-02-05 | Agricultural Research Service | Live recombinant vaccine against avian leukosis virus using a marek's disease virus (mdv) virus as vector |
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| CN101333246A (en) * | 2008-07-30 | 2008-12-31 | 中国农业科学院哈尔滨兽医研究所 | Neutral B cell antigen epitope polypeptide of encephalitis b virus E protein and uses thereof |
Non-Patent Citations (3)
| Title |
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| Qin AJ et al.Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus.《Avian Diseases》.2001,第45卷(第4期),938-945. * |
| 刘青.RNA干扰J亚群禽白血病病毒跨膜蛋白(TM)表达对病毒复制的影响.《中国优秀硕士学位论文全文数据库农业科技辑》.2010,(第3期),中文摘要,正文11-14、26-52页. * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085640A (en) * | 2015-07-03 | 2015-11-25 | 山东农业大学 | PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide |
| CN105085640B (en) * | 2015-07-03 | 2018-08-21 | 山东农业大学 | A kind of J subgroup avian leucosis virus immunosuppressive polypeptides based on PEG modifications |
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