CN104531687A - Method for dissociation of Cap protein in porcine circovirus II alumina gel adjuvant vaccine - Google Patents
Method for dissociation of Cap protein in porcine circovirus II alumina gel adjuvant vaccine Download PDFInfo
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- CN104531687A CN104531687A CN201410742117.7A CN201410742117A CN104531687A CN 104531687 A CN104531687 A CN 104531687A CN 201410742117 A CN201410742117 A CN 201410742117A CN 104531687 A CN104531687 A CN 104531687A
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Abstract
The invention provides a porcine circovirus II alumina gel adjuvant vaccine. A used dissociation agent has a nucleotide sequence shown in the formula of SEQ ID NO: 1. The provided dissociation agent can effectively dissociate a Cap protein antigen in the porcine circovirus II alumina gel adjuvant vaccine so that vaccine effects can be accurately and effectively evaluated. The method is a good method for alumina gel adjuvant vaccine effect detection. The method has the advantages of simple operation, low equipment requirement and large sample treatment amount.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of method that in porcine circovirus 2 type aluminium glue Adjuvanted vaccines, Cap protein is dissociated.
Background technology
Porcine circovirus desease 2 type can cause immunosuppressive disease.This virus can bring out multiple virus, the polyinfection of bacterium and secondary infection, causes huge financial loss, particularly just day by day receive publicity to the research of its vaccine to the research of this disease to pig industry.Vaccine is as the Main Means of prevention and control PCV2, and domestic and international investigator is is researching and developing various vaccine, and up to the present, part vaccine obtains successfully in clinical trial, and the application of input had, other candidate vaccines are also just in active research.
But the Cap protein antigenic content in porcine circovirus type 2 subunit vaccine determines the quality of vaccine, therefore in direct-detection vaccine, Cap antigenic content is one of the most frequently used, the most direct method of vaccine potency inspection, but owing to adding aluminium glue adjuvant in porcine circovirus type 2 subunit vaccine, in agar diffusion test, directly affect the result of Detection of antigen.Therefore, be necessary to provide a kind of method that in aluminium glue Adjuvanted vaccines, Cap protein is dissociated, thus better evaluate vaccine effect.
Summary of the invention
The object of this invention is to provide a kind of easy, can be used for the method that in aluminium glue vaccine, Cap protein is dissociated efficiently, thus check the fine jade of the albumen (expand bioactivity) to establish solid basis for the finished product vaccine potency of porcine circovirus 2 type aluminium glue Adjuvanted vaccines.
First the present invention provides one to dissociate agent, and this agent of dissociating is oligonucleotide, and its nucleotide sequence is as follows:
5 '-TCGTCGTTTTGTCGTTTTGTCGTT-3 ' (SEQ ID NO:1), each nucleotide base carries out thioated modification;
Above-mentioned agent of dissociating is for dissociating out from aluminium glue Adjuvanted vaccines by Cap protein;
The present invention also provides a kind of method of dissociating out by Cap protein from aluminium glue Adjuvanted vaccines, is add agent of dissociating in vaccine, and lower 37 DEG C of earthquake condition processes 1 hour or more.
The present invention adopts agar diffusion test to evaluate the dissociation effect of aforesaid method.
Cap protein antigen in porcine circovirus 2 type aluminium glue Adjuvanted vaccines can effectively dissociate out by agent of dissociating provided by the invention, thus more can evaluate the effect of vaccine by accurate and effective valency, and the effect inspection for aluminium glue Adjuvanted vaccines provides a kind of well method.Adopt technological operation of the present invention simple, low for equipment requirements, and quantity of sample handling is large.
Embodiment
Different fine jade is expanded aluminium glue Adjuvanted vaccines prepared by the antigen of tiring, by adding the even rear Fang Ru Oscillating bed 37 DEG C of the agent Oscillating that dissociates, 200 revs/min, act on 1 hour, to get before dissociating and supernatant after dissociating carries out agar diffusion test, found by research, before dissociating, fine jade expands and tires and be feminine gender, and after dissociating, fine jade expands and tires and join the fine jade before seedling and expands good dependency of having tired, and after dissociating, the expansion of antigen fine jade is tired before tiring and matching well seedling low 0 ~ 1 titre.The present invention can better evaluate the effective protein content in vaccine, can evaluate the effect of vaccine more accurately.
Agent of dissociating used in the present invention, it is oligonucleotide, its sequence: 5 '-T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T-3 ' (* represent carry out thioated to each base), molecular weight is 7696.8, and annealing temperature is 57.3 DEG C.This agent of dissociating can synthetic, and the agent of dissociating in the embodiment of the present invention is synthesized by the precious biotechnology company limited in Dalian.
Below in conjunction with embodiment, the present invention is specifically described.
Embodiment 1
The preparation of 1 different antigen valence vaccine gets the carrying Cap gene of porcine circovirus type 2 liquid (AGP tires as 1:64) of deactivation as seedling antigen, being diluted to AGP respectively with sterile saline tires as after 1:16,1:8,1:4,1:2 and 1:1, and the ratio adding 1 part of aluminium glue adjuvant in 4 parts of antigen liquids makes PCV2 inactivated vaccine.
2 vaccines dissociate 5 of above-mentioned preparation kinds of vaccines, get the vaccine 5ml shaken up respectively, add 0.25g and dissociate after agent, put into setting 200r/min Oscillating bed, 37 DEG C are dissociated 1 hour, within centrifugal 10 minutes, get supernatant prepare to carry out agar diffusion test and immunohistochemical assay with 5000r/min.
3 Cap protein fine jades expand titration
The preparation precision of 3.1 pH value 7.2,0.01mol/L PBS solution takes NaH2PO42H2O 1.56g, and Na2HPO412H2O 3.58g, NaCl 9.0g, after adding distil water 400ml fully dissolves, then adding distil water is to 1000ml, adjust pH to 7.2.
Preparation pH value 7.2, the 0.01mol/L PBS 100ml of 3.2 agar plates, adds 1.0g agarose, dissolves completely with microwave-oven-heating to agarose.Get aseptic diameter 90mm plate, add the agar 20ml after dissolving, make the agar plate that about 3mm is thick, after agar solidifies completely, plate is added a cover and is inverted, and 2 ~ 8 DEG C of refrigerations are for subsequent use.
3.3 test samples diameter 4mm, medium pore and outer perimeter holes are punched apart from the quincunx punch tool being 4mm, back cover.Medium pore adds standard positive serum, and periphery holes adds the Cap protein antigen to be checked of doubling dilution successively, and liquid volume added is advisable not overflow well, after application of sample, leave standstill 5 ~ 10 minutes, plate is inverted gently, put into the built-in 37 DEG C of incubator effects of wet box 24 ~ 48 hours, observations.Tire to occur that fine jade that the most highly diluted multiple of Cap protein antigen is measuring samples expands.
4 vaccine immunity groupizations are tested these 5 groups of vaccines healthy susceptible piglets 5 of immunity 14 ~ 28 ages in days respectively, each musculi colli vaccinate, 2.0ml/ head; Contrast pig 5, each musculi colli injection sterile saline, 2.0ml/ head, isolated rearing.Exempt from latter 35 days, with porcine circovirus 2 type FJ strain, (viral level is 10
6.25tCID
50/ ml) inspection poison attack, every collunarium 1.0ml, intramuscular injection 2.0ml, isolated rearing.Attack poison latter 28 days, cut open to kill and get inguinal lymph nodes, carry out immunohistochemical methods detection.Immune swine should at least 4 be negative, contrast pig should at least 4 be the positive.
5 dissociate standard determination dissociate after fine jade expand and tire and be not less than 1:2, than the fine jade before dissociating expand tire low 1 titre or titre do not reduce be judged to dissociate qualified.
6 test-results
6.1 dissociate with the PCV vaccine of the dissociating method established to the different antigen valences of preparation, and Cap antigen fine jade before it is dissociated in vaccine supernatant expand tire and protein content and after dissociating the Cap antigen fine jade of vaccine supernatant expand and to tire and protein content detects, result is that the vaccine supernatant fine jade that do not dissociate expands to tire and is feminine gender, Cap antigen protein content is 0ug/ml, after dissociating, the expansion of antigen fine jade is tired as 1:2 ~ 1:16 titre, Cap antigen protein content is 44.1ug/ml ~ 331.2ug/ml, after dissociating fine jade expand tire match well seedling before low 0 ~ 1 titre, good dependency is had with protein content detected result, illustrate that this method may be used for antigen amount in detection by quantitative finished product vaccine.The results are shown in Table 1.
6.2 when the antigen fine jade that dissociates expand tire be not less than 1:2 time; immunohistochemical assay result equal 4/5 and above negative (protection ratio of vaccine is not less than 4/5); illustrate that the antigen fine jade after dissociating expands to tire and has good dependency with immunohistochemical assay, may be used for the efficacy test of vaccine.After finished product vaccine dissociates, in supernatant, Cap antigen protein fine jade expands and tires that to be judged to vaccine when being not less than 1:2 qualified.The results are shown in Table 1.
Table 1: dissociation effect proof test result
Embodiment 2
1 porcine circovirus 2 type aluminium glue Adjuvanted vaccines 2012001,2012002,2012003,2012004,2012005,2012006 batches is by Qingdao Yi Bang company scale up test.
2 vaccines dissociate above-mentioned 6 batches of vaccines, get the vaccine 5ml shaken up respectively, add 0.25g and dissociate after agent, and put into setting 200r/min Oscillating bed, 37 DEG C are dissociated 1 hour, within centrifugal 10 minutes, get supernatant prepare to carry out agar diffusion test with 5000r/min.
3 Cap protein fine jades expand titration
The preparation precision of 3.1 pH value 7.2,0.01mol/L PBS solution takes NaH
2pO
42H
2o 1.56g, Na
2hPO
412H
2o 3.58g, NaCl 9.0g, after adding distil water 400ml fully dissolves, then adding distil water is to 1000ml, adjust pH to 7.2.
Preparation pH value 7.2, the 0.01mol/L PBS 100ml of 3.2 agar plates, adds 1.0g agarose, dissolves completely with microwave-oven-heating to agarose.Get aseptic diameter 90mm plate, add the agar 20ml after dissolving, make the agar plate that about 3mm is thick, after agar solidifies completely, plate is added a cover and is inverted, and 2 ~ 8 DEG C of refrigerations are for subsequent use.
3.3 test samples diameter 4mm, medium pore and outer perimeter holes are punched apart from the quincunx punch tool being 4mm, back cover.Medium pore adds standard positive serum, and periphery holes adds the Cap protein antigen to be checked of doubling dilution successively, and liquid volume added is advisable not overflow well, after application of sample, leave standstill 5 ~ 10 minutes, plate is inverted gently, put into the built-in 37 DEG C of incubator effects of wet box 24 ~ 48 hours, observations.Tire to occur that fine jade that the most highly diluted multiple of Cap protein antigen is measuring samples expands.
These 6 batches of vaccines are distinguished the healthy susceptible piglets 5 of immunity 14 ~ 28 ages in days by 4 immunohistochemical assay, each musculi colli vaccinate, 2.0ml/ head; Contrast pig 5, each musculi colli injection sterile saline, 2.0ml/ head, isolated rearing.Exempt from latter 35 days, with porcine circovirus 2 type FJ strain, (viral level is 10
6.25tCID
50/ ml) inspection poison attack, every collunarium 1.0ml, intramuscular injection 2.0ml, isolated rearing.Attack poison latter 28 days, cut open to kill and get inguinal lymph nodes, carry out immunohistochemical methods detection.Immune swine should at least 4 be negative, contrast pig should at least 4 be the positive.
5 dissociate standard determination dissociate after fine jade expand and tire and be not less than 1:2, than the fine jade before dissociating expand tire low 1 titre or titre do not reduce be judged to dissociate qualified.
6 test-results
The method that 6.1 agar diffusion test results are established is to 2012001 of scale up test, 2012002, 2012003, 2012004, 2012005, 2012006 batches of PCV vaccines dissociate, and fine jade is carried out to the vaccine supernatant before dissociating and after dissociating and expands bioactivity, result is that the fine jade expansion of vaccine before dissociating is tired as feminine gender, after dissociating, the expansion of antigen fine jade is tired between 1:2 ~ 1:4, match well seedling proantigen fine jade to expand and tire and have dropped 0 ~ 1 titre, with join Miao Qianqiong and expand good dependency of having tired, it is reliable that immunohistochemical assay result further illustrates method of the present invention, effectively.The results are shown in Table 2.
6.2 immunohistochemical assay result 6 batches vaccine immunity groupization tests equal 5/5 are negative, and control group result is set up, after dissociating, antigen fine jade expands and tires and be all not less than 1:2, demonstrates Cap antigen fine jade when dissociating after fully and expands and tire that to be not less than the judgement of 1:2 time vaccines qualified be accurately and effectively.
In table 26 batches of PCV vaccines, the expansion of antigen fine jade is tired and immunohistochemical assay result
Claims (6)
1. dissociate an agent, it is characterized in that, described agent of dissociating is oligonucleotide, and its sequence is SEQID NO:1.
2. dissociate as claimed in claim 1 agent, it is characterized in that, the nucleotide base in described oligonucleotide carries out thioated modification.
3. the agent of dissociating of claim 1 is in the application of dissociating out by Cap protein from aluminium glue Adjuvanted vaccines.
4. by the method that Cap protein dissociates out from aluminium glue Adjuvanted vaccines, it is characterized in that, described method is the agent of dissociating adding claim 1 in vaccine, and lower 37 DEG C of earthquake condition processes 1 hour or more.
5. method as claimed in claim 4, it is characterized in that, described vaccine is porcine circovirus 2 type aluminium glue Adjuvanted vaccines.
6. detect a method for porcine circovirus 2 type aluminium glue Adjuvanted vaccines effect, it is characterized in that, described method is that first using right profit requires that Cap protein dissociates out by the method described in 4 from aluminium glue Adjuvanted vaccines, then the fine jade carrying out albumen expands bioactivity.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468957A (en) * | 2002-07-19 | 2004-01-21 | 中国人民解放军第二军医大学 | Plasmid used as human therapeutic vaccine adjuvant |
| CN101643496A (en) * | 2008-08-07 | 2010-02-10 | 长春华普生物技术有限公司 | Oligonucleotide with immune suppression function |
| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
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2014
- 2014-12-07 CN CN201410742117.7A patent/CN104531687A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468957A (en) * | 2002-07-19 | 2004-01-21 | 中国人民解放军第二军医大学 | Plasmid used as human therapeutic vaccine adjuvant |
| CN101643496A (en) * | 2008-08-07 | 2010-02-10 | 长春华普生物技术有限公司 | Oligonucleotide with immune suppression function |
| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
Non-Patent Citations (4)
| Title |
|---|
| WEI WANG ET AL.: "Selection of Adjuvants for Enhanced Vaccine Potency", 《WORLD JOURNAL OF VACCINES》 * |
| 梅兴国: "《生物技术药物制剂 基础与应用》", 31 October 2004 * |
| 程凯慧等: "含CpG基序系列猪圆环病毒核酸疫苗的构建", 《中国兽医科学》 * |
| 谢庆阁等: "《畜禽重大疾病免疫防制研究进展》", 31 May 1996 * |
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