CN102188702A - Compound immunological adjuvant of subunit vaccine for swine fever - Google Patents
Compound immunological adjuvant of subunit vaccine for swine fever Download PDFInfo
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- CN102188702A CN102188702A CN2011100234956A CN201110023495A CN102188702A CN 102188702 A CN102188702 A CN 102188702A CN 2011100234956 A CN2011100234956 A CN 2011100234956A CN 201110023495 A CN201110023495 A CN 201110023495A CN 102188702 A CN102188702 A CN 102188702A
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Abstract
The invention relates to a compound immunological adjuvant of subunit vaccine for swine fever. The compound immunological adjuvant is prepared from recombinant swine alpha interferon and an aluminum hydroxide adjuvant, and the content of the recombinant swine alpha interferon in subunit vaccine for swine fever for each swine is 1*106U; the recombinant swine alpha interferon is swine alpha interferon protein with an antivirus activity generated by high expression of swine alpha interferon gene in various expression systems by a recombinant DNA (Deoxyribose Nucleic Acid) technology; and the compound immunological adjuvant integrates the granulation sustained-release effect of the aluminum hydroxide adjuvant and the immunoregulation effect of the swine alpha interferon.
Description
Technical field
The present invention relates to a kind of composite adjuvant that is used for swine fever subunit antigen intramuscular injection immunity, belong to biological technical field, be exclusively used in the immune effect that improves the swine fever subunit vaccine.
Background technology
Swine fever (Classical Swine Fever) is a kind of acute heat sexually transmitted disease that is caused pig by swine fever virus, and this disease transmission is strong, and the case fatality rate height causes heavy economic losses for China's pig industry.One of in 16 kinds of zoonosis of category-A are listed it by OIE.
Swine fever virus has only a serotype.Swinery is consistent with neutralizing antibody to the immunity of swine fever, based on humoral immunization.The immunity sow can be passed to piglet with antibody by colostrum, and makes it obtain certain passive immunity, can continue 1-2 month.Maternal antibody has certain interference effect to the immunity of swine fever attenuated vaccine.China carries out the prophylactic immunization of swinery from 1956 with regard to extensive use rabbitization attenuated vaccine, has obtained significant effect.But immune swinery has still the time swine fever to take place, swinery congenital infection or immunosuppressant takes place to cause immuning failure be one of major reason.Still do not have at present method to distinguish attenuated vaccine inoculation and the wild virus infection of commodity swinery, so some developed countries ban use of attenuated vaccine to carry out the prevention of swine fever.In recent years, the swine fever virus E2 protein subunit vaccine of some country's developments of Europe demonstrates good prospects for application.
Although genetic engineering subunit vaccine since safety good, be subjected to maternal antibody disturb little, can the matched reagent box distinguish advantages such as vaccine immunity and wild virus infection and have broad application prospects, but gene engineered subunit antigen immune originality is relatively weak, antibody response is slow, in order better to excite the immune system of pig body, make it enough resistances be arranged, be necessary to seek the immunocompetence of the genetic engineering subunit vaccine of method raising safely and efficiently swine fever virus.
Improve the improvement that the immunocompetent method of subunit vaccine is not limited only to vaccine antigen at present, select suitable immunological adjuvant to come stimulating immune system, the enhancing animal immune system also is an important approach to the immunne response such as picked-up, processing and submission of antigenic substance.An ideal immunological adjuvant should be assisted the vaccine antigen activating immune system by specificity, can not have side effects to the motivation body simultaneously.
Interferon-alpha is a kind of cytokine with antiviral and immunoloregulation function that animal body produces when suffering pathogen infection.Discovering in recent years, IFN-α plays crucial effect in the innate immunity of animal in the development of acquired immunity.Suppress the propagation and the propagation of cause of disease on the one hand, the differentiation of stimulator antigen presenting cell and ripe, activation lymphocytic immunity cell on the other hand, as improve the killing activity of NK cell, the activate the phagocytic capacity of macrophage etc., thereby set up efficiently cause of disease or the antigenic specificity acquired immunity is replied.External recent research is found to compare with independent flu immunization vaccine; the interferon-alpha of doses cooperates the influenza vaccines immune mouse can significantly improve the level of specific IgG 2 α and IgA in the serum; and the attack of 10 times of fatal dose influenza virus produced immunoprotection completely, and the protective rate of influenza vaccines immune mouse closely has 60% separately.But the half-life of interferon-alpha in animal body is shorter, is difficult to bring into play the immunostimulation that comparatively continues, and the immunological adjuvant effect is stable inadequately sometimes.
Aluminum hydroxide adjuvant is the fibrous particle that exists with 4.5nm * 2.2nm * 10nm, and the form with loose 1-10 μ m size behind this particle accumulation exists.The adsorption of antigen and aluminum hydroxide adjuvant mainly is to rely on electrostatic attraction, hydrophobic interaction and ligand exchange.Therefore, aluminum hydroxide adjuvant and the degeneration and the inactivation that can not cause activated protein after interferon and antigen protein etc. cooperates, thereby the collaborative enhanced immunoloregulation function of performance.
Summary of the invention
At the above, the invention provides a kind of complex immunity adjuvant of swine fever subunit vaccine, be used for cooperating with the swine fever subunit antigen, improve the immunogenicity of swine fever subunit vaccine by the intramuscular injection immunity, strengthen the immunne response of pig body immune system to swine fever antigen, safer, prevent the generation of swine fever efficiently.
For reaching above purpose, the present invention has adopted following technical scheme: a kind of complex immunity adjuvant of swine fever subunit vaccine, this complex immunity adjuvant is cooperated by recombinant porcine alpha interferon and aluminum hydroxide adjuvant and forms, and the content of recombinant porcine alpha interferon is 1 * 10 in every part swine fever subunit vaccine
6U; Described recombinant porcine alpha interferon is the porcine alpha interferon protein with antiviral activity that porcine alpha-IFN gene is efficiently expressed generation in various expression systems by recombinant DNA technology; The complex immunity adjuvant combines the immunoregulation effect of aluminum hydroxide adjuvant granulating slow releasing function and porcine alpha-IFN.
After adopting above technical scheme, the present invention has following beneficial effect: the complex immunity adjuvant can significantly strengthen the immunne response of piglet to swine fever E2 proteantigen; The complex immunity adjuvant significantly improves the swine fever specificity neutralizing antibody level that produces behind the swine fever E2 proteantigen cervical region intramuscular injection immunity piglet; The complex immunity adjuvant can significantly strengthen the immunological memory that produces behind the swine fever E2 proteantigen immune swine body.
Description of drawings
Fig. 1 induces the influence of antibody growth and decline to swine fever subunit vaccine single immunization for complex immunity adjuvant of the present invention.
Fig. 2 just exempts from and two influences of exempting from back generation neutralizing antibody level the swine fever subunit vaccine for complex immunity adjuvant of the present invention.
Fig. 3 is the influence of complex immunity adjuvant of the present invention to swine fever subunit vaccine inoculation pig immunological memory.
The specific embodiment
A kind of swine fever subunit vaccine of the present invention complex immunity adjuvant, this complex immunity adjuvant are cooperated by recombinant porcine alpha interferon and aluminum hydroxide adjuvant and form, and the content of recombinant porcine alpha interferon is 1 * 10 in every part swine fever subunit vaccine
6U.Described recombinant porcine alpha interferon is by the porcine alpha interferon protein with antiviral activity of recombinant DNA technology with porcine alpha-IFN gene expressed generation in expression system.
1, the preparation of recombinant porcine alpha interferon:
Reference literature [Zhou Guodong. secreting, expressing and the immunologic adjuvant function thereof of porcine alpha-IFN in Pichia sp.. Agricultural University Of Nanjing's Master's thesis, 2006] method fermentation obtains recombinant porcine alpha interferon, remove macromole impurity by the 50kDa filter membrane, diluting with physiological saline solution after 5kDa filter membrane ultrafiltration desalination concentrates is 107U/mL again.
2, the preparation of aluminum hydroxide adjuvant:
Get 5% aluminum sulfate solution 250ml, adding 5% sodium hydroxide solution 100ml precipitates 2 times with the normal saline centrifuge washing under strong agitation, is hung into to make in the normal saline to reach 250ml again.During immunity inoculation, get an amount of aluminum hydroxide adjuvant add equal-volume antigen can the immunity.
3, the preparation of reorganization swine fever subunit antigen:
Reference literature [(1) Xu Xueqing, Cao Ruibing, Cai Meihong, Ren Xuefeng, Zhou Bin, Chen Puyan. the swine fever virus E2 protein A/expression of D antigenic domain in Pichia sp.. Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal (natural science edition) .2005,33 (3): 11-15. and (2) Xu Xueqing, Zheng Qisheng, Cao Ruibing, Su Xiaoyun, Chen Puyan. swine fever virus E2 protein B/expression and the evaluation of C antigenic region gene in Pichia sp.. Chinese biological engineering magazine .2004,24 (9): 53-57.] method fermentation obtain swine fever virus E2 protein A/D antigen and B/C antigen protein, remove macromole impurity by the 50kDa filter membrane respectively, concentrate through 5kDa filter membrane ultrafiltration desalination, with physiological saline solution dilution configuration swine fever subunit antigen, swine fever virus E2 protein A/D antigen and B/C antigen protein content are 0.5mg/mL again.
4, the preparation of complex immunity adjuvant swine fever subunit vaccine:
With above-mentioned aluminum hydroxide adjuvant, recombinant porcine alpha interferon and reorganization swine fever E2 proteantigen normal saline by 5: 1: 4 mixed, join even.Contain swine fever virus E2 protein A/D antigen and B/C antigen protein each 200 μ g and recombinant porcine alpha interferon 10 in the 1mL Adjuvanted vaccines
6U.
With reference to the accompanying drawings, the present invention is divided into 4 groups at random with 11 (broods) 30 age in days piglets, respectively 3 of swine fever cell vaccine group (A), no adjuvant swine fever subunit vaccine group (B), complex immunity adjuvant swine fever subunit vaccine groups (C), 2 of normal saline matched groups (D).The antigen protein content of no adjuvant swine fever subunit vaccine equates with complex immunity adjuvant swine fever subunit vaccine, is to prepare with normal saline.With at 35 ages in days cervical region intramuscular injection vaccine or normal saline 2mL behind ear respectively; Respectively organize booster immunization once after 3 weeks, immune vaccine is with dosage and just exempt from identical; Just exempting from back each group of 7 weeks all exempts from commercialization swine fever cell vaccine three.Each is organized piglet and exempts from back 0,3,5,7 and 9 all blood-sample withdrawal chemical examinations respectively just.
Complex immunity adjuvant swine fever subunit vaccine immune effect of the present invention is measured the cell micro virus neutralization tests and is detected the swine fever neutralizing antibody, adopts the fixed virus amount, the method for dilute serum.Blood serum sample is used the PBS doubling dilution through 56 ℃ of deactivations after-filtration degerming in 30 minutes.On 96 porocyte plates the swine fever virus of each dilution blood serum sample of 50 μ L with 50 μ L, 200 TCID50 mixed, at 37 ℃ of effect 1h, each serum dilution repeats 2 holes.Add 100 μ L 1 * 10
4Individual PK15 cell continues to cultivate 72h for 37 ℃.Application swine fever monoclonal antibody is carried out the swine fever virus in the indirect immunofluorescence detection cell.The blood serum sample greatest dilution of the virus that can neutralize fully is NAT.
Result of the test (table 1) shows: test pig is the commodity piglet of large-scale pig farm, has low-level swine fever neutralizing antibody in the part piglet body when 35 ages in days.3 pigs of just exempting from no immunological adjuvant swine fever subunit vaccine immune group of 3 week of back all do not produce neutralizing antibody; 3 piglets of complex immunity adjuvant swine fever subunit vaccine immune group all have the rising of swine fever neutralizing antibody, but similar with commercialization swine fever cell vaccine immune group, the level of neutralizing antibody is also lower; The raising of swine fever neutralizing antibody level all appears in the three groups of vaccinated piglets in two immunity backs, the swine fever neutralizing antibody level of complex immunity adjuvant swine fever subunit vaccine immune group piglet is significantly higher than no Adjuvanted vaccines group, but a little less than commercialization swine fever cell vaccine immune group.Exempt from 9 weeks of back one, each vaccination group piglet is under swine Fever Vaccine is lived malicious stimulation, the neutralizing antibody level significantly improves, complex immunity adjuvant swine fever subunit vaccine immune group has produced the immunological memory of similar clause swine fever cell vaccine, and swine fever neutralizing antibody lifting amplitude is significantly higher than no Adjuvanted vaccines group.The result shows that the complex immunity adjuvant can promote the immunne response of pig body to the swine fever subunit antigen, produces antigen immune memory more efficiently.
The immune protective effect of rabbit body heating protection test model evaluation swine fever subunit vaccine
Swine fever virus rabbitization low virulent strain (HCLV) claims the C strain again, is the weakening strain that crossdrift strain swine fever virus is gone down to posterity for a long time at the rabbit body.HCLV does not have pathogenic to the rabbit body, but can cause the characteristic heating of rabbit behind intravenous inoculation 48-96h first, and secondary inoculation can not cause typical heat again; If inoculation HCLV is tested the hog cholera antibody neutralization in the porcine blood serum, then inoculate the rabbit body and can not cause heating, behind the counteracting toxic substances typical heat takes place once more.China Veterinary Drugs Supervisory Inst.'s rabbit body heating commonly used protection test is as the animal model of judging test swine Fever Vaccine effectiveness.
Get two porcine blood serums of exempting from 2 weeks of back and carry out the protection test of rabbit body counteracting toxic substances; the blood serum sample 500 μ L and the isopyknic HCLV that contains 100 times of rabbit precursor reactant heat that take normal saline dilution in 1: 16 act on 1h in room temperature (15-25 ℃); 2 monthly ages of intravenous inoculation cleaning level White Rabbit; every 6h surveys a rectal temperature, is higher than 40 ℃ and is judged to be heating.Recover the HCLV second time counteracting toxic substances of normal back with 100 times of rabbit precursor reactant heat, record temperature check data at rabbit body temperature degree.
Swine fever NAT testing result in table 1 pig anteserum sample
A: the test group piglet is immunoprophylaxis for the second time, vaccine kind and dosage with just exempt from identical.
B: the test group piglet is immunity inoculation for the third time, all inoculates the swine fever cell vaccine.
Result of the test (table 2) shows: 3 piglets of complex immunity adjuvant swine fever subunit vaccine immune group have all obtained the swine fever protective immunity two after exempting from; suitable with attenuated vaccine immunity group effect, do not have only a piglet to exempt from the protection of back acquisition hog cholera immune and there is immunological adjuvant swine fever subunit vaccine immune group two.The result shows that the complex immunity adjuvant can significantly improve the immune effect of swine fever subunit vaccine.
Table 2 rabbit body typical heat inhibition test result
Claims (2)
1. swine fever subunit vaccine complex immunity adjuvant is characterized in that this complex immunity adjuvant is cooperated by recombinant porcine alpha interferon and aluminum hydroxide adjuvant to form, and the content of recombinant porcine alpha interferon is 1 * 10 in every part swine fever subunit vaccine
6U.
2. a kind of swine fever subunit vaccine complex immunity adjuvant according to claim 1 is characterized in that described recombinant porcine alpha interferon is by the porcine alpha interferon protein with antiviral activity of recombinant DNA technology with porcine alpha-IFN gene expressed generation in expression system.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907776A (en) * | 2016-05-20 | 2016-08-31 | 金福赛（北京）生物科技有限公司 | Subunit vaccine capable of inducing immune response to porcine reproductie and respiratou syndrome virus |
| CN107007828A (en) * | 2017-03-21 | 2017-08-04 | 天津瑞普生物技术股份有限公司 | A kind of canine parvovirus VP2 protein protein subunit vaccine |
| CN117417416A (en) * | 2022-07-19 | 2024-01-19 | 江苏农牧科技职业学院 | Recombinant antigen protein for detecting African swine fever virus, preparation method thereof, ELISA kit and application |
-
2011
- 2011-01-14 CN CN2011100234956A patent/CN102188702A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库农业科技辑》 20080515 周国栋 猪alpha干扰素在毕赤酵母中的分泌表达及其免疫佐剂功能研究 第三章摘要、第5章摘要和表5-1 1-2 , 第05期 * |
| 《中国博士学位论文全文数据库农业科技辑》 20080215 姚清侠 猪alpha干扰素、猪beta干扰素与猪gamma干扰素的抗病毒活性及其免疫佐剂的研究 第5.5节 1-2 , 第2期 * |
| 周国栋: "猪α干扰素在毕赤酵母中的分泌表达及其免疫佐剂功能研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 姚清侠: "猪α干扰素、猪β干扰素与猪γ干扰素的抗病毒活性及其免疫佐剂的研究", 《中国博士学位论文全文数据库农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907776A (en) * | 2016-05-20 | 2016-08-31 | 金福赛（北京）生物科技有限公司 | Subunit vaccine capable of inducing immune response to porcine reproductie and respiratou syndrome virus |
| CN105907776B (en) * | 2016-05-20 | 2019-10-29 | 金福赛（北京）生物科技有限公司 | The subunit vaccine of the immune response for pig blue-ear disease poison can be induced |
| CN107007828A (en) * | 2017-03-21 | 2017-08-04 | 天津瑞普生物技术股份有限公司 | A kind of canine parvovirus VP2 protein protein subunit vaccine |
| CN117417416A (en) * | 2022-07-19 | 2024-01-19 | 江苏农牧科技职业学院 | Recombinant antigen protein for detecting African swine fever virus, preparation method thereof, ELISA kit and application |
| CN117417416B (en) * | 2022-07-19 | 2024-05-28 | 江苏农牧科技职业学院 | Recombinant antigen protein for detecting African swine fever virus, preparation method thereof, ELISA kit and application |
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Application publication date: 20110921 |