CN101220348A - Pig replication and respiration syndrome virus attenuated vaccine strain and uses thereof - Google Patents
Pig replication and respiration syndrome virus attenuated vaccine strain and uses thereof Download PDFInfo
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- CN101220348A CN101220348A CN 200710001253 CN200710001253A CN101220348A CN 101220348 A CN101220348 A CN 101220348A CN 200710001253 CN200710001253 CN 200710001253 CN 200710001253 A CN200710001253 A CN 200710001253A CN 101220348 A CN101220348 A CN 101220348A
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Abstract
The invention discloses a porcine reproductive and respiratory syndrome virus attenuated vaccine strain, and the microorganism collection number is CGMCC NO.1883. The attenuated vaccine strain of the invention has stable genetic variation and good safety, which is safe and non-pathogenic for pregnant sows, piglets and the pigs at various ages. The immunized piglets after using the attenuated vaccine strain of the invention for immunization can resist the attack of the strong virus of the porcine reproductive and respiratory syndrome virus.
Description
Technical field
The present invention relates to a kind of attenuated vaccine strain, relate in particular to a boar breeding and respiration syndrome virus attenuated vaccine strain and application thereof, belong to biological technical field.
Background technology
(Porcine Reproductive and Respiratory Syndrome PRRS) causes by PRRSV porcine reproductive and respiratory syndrome, is characteristics with breeding difficulty, respiratory symptom, has the virus disease of hyperinfection.The U.S. at first found this disease in 1987, Wensvoort equals separation in 1991 and has identified this disease pathogen, PRRSV belongs to the Arterivirus of Arteriviridae (Arteriviridae), it is a kind of normal chain single-stranded RNA virus that cyst membrane is arranged, because PRRSV has the characteristic of antigenic drift and variation, and its main target cell is the pulmonary alveolar macrophage (PAM) with important immunization, so the infection of PRRSV is often with secondary infection, the especially respiratory pathogens of various cause of diseases.Cause heavy losses to pig industry at present, caused international extensive concern.For the generation of controlling this disease with spread, the research of carrying out the vaccine of PRRS seems particularly important.External deactivation vaccine and the weak malicious seedling of successively developing prevention PRRS, and preventing and preventing the generation of system PRRS and spread having brought into play enormous function.
PRRS has spread in China and has come in recent years, has seriously hindered the development of China's pig industry, has caused the attention of China relevant department.Harbin Veterinary Medicine Inst., China Academy of Agriculture in 1996 separates at home first and has identified this disease pathogen, set up the IFA detection method simultaneously, therefore PRRS has spread all over the most provinces, cities and autonomous regions of China through generaI investigation, and the research at the vaccine of domestic epidemic isolates seems very urgent.Though developed commercial deactivation vaccine and weak malicious seedling abroad, but because this virus has the characteristic of antigenic variation and drift, the specific aim of therefore external vaccine is relatively poor relatively, and often immune effect is unsatisfactory, and external existing report is because of malicious seedling a little less than the use PRRS causes the outburst of this disease.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a strain new pig breeding and respiration syndrome virus attenuated vaccine strain, this attenuated vaccine strain inheritance stability, security are good, have good immunogenicity for the pig breeding with breathing syndrome virus.
Technical problem to be solved by this invention realizes by following technological approaches:
The present invention selects domestic isolated strain PRRSV CH-1a strain for use, after low temperature goes down to posterity on the Marc-145 cell, when passing to for the 110th～170 generation for 37 ℃, identifies comprehensively again, detects its pure property TCID
50Virulence and immunogenicity determining, and carry out rflp analysis by RT-PCR test with to the ORF5 of the virus of each generation, the proof 110～170 generation PRRSV CH-1a kind poison except that its virus titer on the Marc-145 cell improve a lot and pathogenic obvious reduction, other characteristic is basic consistent with low generation PRRSV CH-1a strain characteristic, 110～170 generation PRRSV CH-1a are caused low virulent strain kind poison (Porcine Reproductive and Respiratory Syndromevirus) called after PRRSV CH-1R strain, and its microbial preservation number is: CGMCC NO.1883; The preservation time is: on December 11st, 2006; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
110 generation PRRSV CH-1a strains are defined as 1 generation of PRRSV CH-1R strain, PRRS living vaccine (CH-1R strain) primordial seed generation is defined as PRRSV CH-1R strain 25～35 generations (135～145 generations of PRRSV CH-1a strain); Basis seed generation is 36～45 generations (146～155 generations of PRRSV CH-1a strain); The seeding generation is 46～55 generations (156～165 generations of PRRSV CH-1a strain).
Immunogenicity test shows that behind low virulent strain immunity piglet of the present invention, the immune piglet of institute all can be resisted pig breeding and the strong malicious attack of breathing syndrome virus.
The security of low virulent strain of the present invention is good, and the toxicity test test-results shows, low virulent strain of the present invention to each such as pregnant sow, piglet pig equal safety only, had no pathogenicity at age.
Analysis revealed by RFLP, PRRSV CH-1a strain is passed through the low temperature continuous passage and is repeatedly cloned, when reaching for 110 generations in the stable point mutation of PRRSV ORF5 generation, this sudden change is stable to remain to for 170 generations, illustrated that low virulent strain of the present invention is the low virulent strain that causes that a strain has the genetic stability variation.
The wet poison of low virulent strain of the present invention is 12 months-20 ℃ preservation period, is 24 months-70 ℃ preservation perives; It is 24 months-20 ℃ of preservation perives with-70 ℃ that low virulent strain of the present invention freezes in poison.
The using method of vaccine of the present invention:
(1) route of inoculation: musculi colli injection.
(2) dosage of inoculation: piglet is 3～4 week immunity in age, 1 part of every incidence intramuscular injection vaccine; Sow is in the preceding 1 week immunity of breeding, 2 parts of every incidence intramuscular injection vaccine.
Low virulent strain range of application broad of the present invention for example, can be applicable to be prepared into the diagnostic reagent of diagnosis breeding and breathing syndrome virus, also can be applicable to be prepared into single seedling or connection seedling (living or inactivated vaccine) vaccine etc.
Description of drawings
Fig. 1 PRRSV CH-1a strain passage number and virus titer relation.
The rflp analysis result of Fig. 2 ORF5 gene fragment.
Fig. 3 cuts the electrophorogram of ORF5 with TspE I enzyme.
M:Marker DL2000 1:MLV; 2,3:CH-1a strain; The 4:VR2332 strain; The 5:GD-3 strain; 70 generations of 6:CH-1a; 90 generations of 7:CH-1a; 8:CH-1a110 generation; 9:CH-1a130 generation; 10CH-1a150 generation; 155 generations of 11:CH-1a; 160 generations of 12:CH-1a; 170 generations of 13:CH-1a.
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
Embodiment
1.1 test uses pig available from the negative pig farm of the PRRS serum antibody of Pest-or disease-free area.
1.2 virocyte is cultivated and cloning
1.2.1 cell cultures Marc-145 clone is cultivated with the DMEM cell culture fluid, wherein contains the penicillin of 4% deactivation calf serum, 100U/ml, the Streptomycin sulphate of 80 μ g/ml, and uses NaHCO
3Adjust about pH value to 7.2 filtration sterilization.With recovery of this cell culture fluid or continuous cell line Marc-145 is in the little flat bottle of sterilization of 100ml to capacity, is used for virus inoculation after waiting to grow up to good cell monolayer.
1.2.2 planting the cloning of poison goes down to posterity at the Marc-145 cell from domestic isolating PRRSV CH-1a strain, when reaching for 30,50,70,90,110,130 generations, carried out virus clone respectively, except that 30 generation kind poison clones select the 2mm plaque, all select the 1mm plaque with rear clone.30,50 generation the kind poison choose colony's plaque (3～7 single plaque mixed culture), since 70 generation the kind poison choose 1mm with the cultivation of going down to posterity of interior single plaque, it is malicious with planting with interior vaccine at 1mm to have obtained more single plaque diameter through 90,110,130 generations, in 5 generations of every biography, carried out 1 the big minor inspection of plaque, as occur more than 5% then carrying out clone purification once more greater than the plaque of 2mm.
Cultivate the PRRSV CH-1a strain of being carried out the clone 30 generations 1.2.3 plant the low temperature of poison, place 36 ℃ of 10 generations of following subculture, place 35 ℃ of 20 generations of following subculture then, with being placed on 34 ℃ of 30 generations of following subculture, reached for 90 generations, recover 37 ℃ and continue down to cultivate to be passaged to for 170 generations, choose different generations and measure TCID
50Choose the virus of 10,30,50,70,90,110,130,150,155,160,170 generations, do virus titer and measure.
Test-results: when PRRSV CH-1a when Marc-145 clone reached for 30 generations, its virus titer is 10
5.0TCID
50About/ml, when cultivating under being lower than 37 ℃ condition, virus titer improves not obvious, up to reach 90 generation virus titer just reach 10
6.3TCID
50/ ml.PRRSV CH-1a is when the Marc-145 cell was uploaded to for 110～170 generations after measured, and virus titer reaches 10
7.0TCID
50More than/the ml; When reaching for 170 generations, still stable maintenance is 10
7.0TCID
50More than/the ml.The results are shown in Table 1, Fig. 1.
Table 1 passage number drips malicious relation with virus
| |
10 | 30 | 50 | 70 | 90 | 110 | 130 | 150 | 170 |
| TCID
50/ |
10 3.5 | 10 4.8 | 10 4.6 | 10 5.3 | 10 6.3 | 10 7.5 | 10 7.3 | 10 7.6 | 10 7.5 |
1.3 physicochemical property test get isolated strain 90,110,130,150,155,160,170 generation cell toxicant suspension (10
7.1～10
7.5TCID
50/ ml) 5ml, average mark is loaded in 5 test tubes, and test tube 1 is in 56 ℃ of water-bath effect 45min; After test tube 2 added 20% ether concussion 10min, 4 ℃ were spent the night, the aseptic ether of flinging to; Test tube 3 is transferred pH to 3.0 with 0.1MHCl earlier, transfers pH to 7.2 with 0.1NaOH behind 4 ℃ of effect 2h; Test tube 4 adds 5-iodine IUDR (5-IUDR) 50 μ l, 4 ℃ of effect 12h; Measure TCID through processing and undressed cell toxicant suspension
50, test-results sees Table 2
Table 2 physicochemical property test-results
1.4 the micro plate method is adopted in hemagglutination test (HA test), respectively with red cell suspension mensuration 110,130,150,155,160, the 170 generation cell toxicant suspension (10 of 0.5% chicken, cavy, rat, rabbit and sheep
7.1～10
7.5TCID
50/ ml) HA titre, with the high dilution of 50% red cell agglutination as judging terminal point.Test-results show 110,130,150,155,160,170 generation the cell toxicant suspension red corpuscle of 0.5% chicken, cavy, rat, rabbit and sheep is not all had agglutination.
1.5 toxicity test is selected 85～93 days sow of 26 pregnancies for use, with 10,30,50,70,90,110,130,150,170 generation PRRSV CH-1a (4 * 10
7.2TCID
50) collunarium, observe incidence.Test-results sees Table 3.
Table 3 toxicity test result
| Generation | Dosage of inoculation (TCID 50/ml) | Number | The morbidity | Sickness rate | |
| 10 | 4×10 4.2 | 4 | 4 | 100％ | |
| 30 | 4×10 5.6 | 4 | 3 | 75％ | |
| 50 | 4×10 6.2 | 4 | 2 | 50％ | |
| 70 | 4×10 7.2 | 5 | 3 | 60％ | |
| 90 | 4×10 7.1 | 5 | 1 | 20％ | |
| 110 | 4×10 7.4 | 4 | 0 | 0％ | |
| 130 | 4×10 7.3 | 6 | 0 | 0％ | |
| 150 | 4×10 7.5 | 3 | 0 | 0％ | |
| 170 | 4×10 7.2 | 3 | 0 | 0％ |
Find that from table 3 virulence that is uploaded to 110～170 generation PRRSV at the Marc-145 cell obviously reduces, and virus titer maintains higher level.
1.6PRRSV the amplification of ORF5 gene is increased to the ORF5 gene of 90,110,130,150,155,160,170 generation viruses respectively, uses primer to see Table 4, reaction conditions sees Table 5.
The table 4ORF5 gene the primer that increases
| The primer title | |
| P5S | |
| 5′AAGAATTCACCATGGTGGGGAAATGCTTGAC 3′ | |
| |
5′AAACTAGTATAGAGACGACCC CATTG 3′ |
The table 5ORF5 gene PCR reaction conditions that increases
Amplified production is through 1% agarose gel electrophoresis, ethidium bromide staining, observations under the ultraviolet lamp, the result special purpose band (see figure 2) that all increases.
1.7ORF5 the rflp analysis of gene fragment
Adopt TspE I restriction enzyme that the ORF5 of purified each generation is carried out enzyme and cut, undertaken by TspE I restriction enzyme operation instruction.More than reaction all under 37 ℃, was reacted 2 hours, got 10 μ l reaction product then, carried out the agarose gel electrophoresis analysis, and concrete agarose gel electrophoresis the results are shown in Figure 3.
1.8 the immunogenicity test, is divided into 5 groups (5/group) at random with 25 28～35 age in days PRRSV negative antibody piglets, the 1st～4 group is test group, and the 5th group is control group.Get 150,155,160,170 generation cell toxicant suspension (10
7.1～10
7.5TCID
50/ ml) each the immunity 5 piglets, the 2ml/ head, the 5th group is made as the not control group of vaccinate.Inoculated 2 * 10 in the intranasal in back 28 days in immunity
5.0TCID
50PRRSV CH-1a strain is poison by force, measures piglet body temperature and observes the piglet clinical manifestation, observes altogether 14 days.
Test-results with 150,155,160,170 generation cell virus suspension (10
7.1～10
7.5TCID
50/ ml) behind the inoculation piglet, all do not have bad clinical response and occur.Back 28 days of immunity with pig breeding and respiratory syndrome strong virus attack; immune group piglet body temperature is normal; there is not other abnormal response yet; and fervescence and dyspneic symptom have all appearred in the pig of control group; illustrate that this 4 generation cell virus suspension has good immunogenicity; all can protect the attack of the strong poison of piglet opposing fully, concrete outcome sees Table 6.
Table 6PRRS living vaccine (CH-1R strain) is to the active immunity test-results of piglet
1.9 steriling test get isolated strain 110,130,150,155,160,170 generation cell toxicant suspension (1 * 10
6.6TCID
50/ ml) to carry out according to pressing " Chinese veterinary drug allusion quotation ", test-results shows no living contaminants.
1.10 mycoplasma check get isolated strain 110,130,150,155,160,170 generation cell toxicant suspension (1 * 10
6.6TCID
50/ ml) to be undertaken by " Chinese veterinary drug allusion quotation ", test-results shows no mycoplasma contamination.
1.11 detecting by " Chinese veterinary drug allusion quotation ", exogenous virus carries out.
Utilize pig trivalent (HCV, PPV, BVDV) fluorescence antibody, PRV (Pseudorabies virus) fluorescence antibody to detect, all do not detect specificity fluorescent; Adopt cytopathy (CPE) method to detect, do not see that CPE appears in the Vero cell; Adopt the red corpuscle absorption method to detect, do not see that the Vero cell hemocyte adsorption phenomena occurs and occurs.Evidence, CH-1R strain PRRS kind poison do not detect exogenous virus and pollute.
1.12 the test of seed culture of viruses preservation period was made regular check on virus titer at-20 ℃ in 6,12,24,30 months with-70 ℃ of preservations respectively with freeze-drying seed culture of viruses and wet seed culture of viruses, and the record result, test-results sees Table 7.
Preservation period test-results under the table 7 seed culture of viruses different condition
As known from Table 7, the freeze-drying seed culture of viruses the longlyest reaches 24 months under-20 ℃, under-70 ℃, preserve 24 months virus titers and do not have considerable change.Wet seed culture of viruses is below-20 ℃, the longlyest reaches December, below-70 ℃, preserves 24 months viral levels and do not have considerable change.
Claims (3)
1. pig breeding and breathing syndrome virus (Porcine Reproductive and Respiratory Syndromevirus) attenuated vaccine strain, its microbial preservation number are: CGMCC NO.1883.
2. a vaccine composition is characterized in that: be made up of the attenuated vaccine strain of claim 1, pharmaceutically acceptable adjuvant or carrier.
3. the attenuated vaccine strain of claim 1 is preparing the purposes of preventing and treating in pig breeding and the breathing syndrome virus medicine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102107003A (en) * | 2011-01-05 | 2011-06-29 | 重庆大学 | Porcine reproductive and respiratory syndrome Virosome vaccine and preparation method thereof |
| CN103695465A (en) * | 2013-12-16 | 2014-04-02 | 中国农业科学院哈尔滨兽医研究所 | CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof |
| CN104388399A (en) * | 2014-08-18 | 2015-03-04 | 张澍 | PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and neutralizing antibody and possesses wide-spectrum immunogenicity |
| WO2016119079A1 (en) * | 2015-01-29 | 2016-08-04 | 山东省农业科学院畜牧兽医研究所 | Sx-105 strain of porcine reproductive and respiratory syndrome virus (prrsv) and use thereof |
| CN111849924A (en) * | 2020-06-23 | 2020-10-30 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Similar NADC30 porcine reproductive and respiratory syndrome virulent strain, attenuated strain and application thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6042830A (en) * | 1992-08-05 | 2000-03-28 | Boehringer Ingelheim Vetmedica, Inc. | Viral agent associated with mystery swine disease |
| DE69426228T2 (en) * | 1993-02-08 | 2001-03-01 | Bayer Ag | Method of propagating the swine virus causing reproductive and respiratory syndrome and using it as a vaccine. |
| ES2102971B1 (en) * | 1996-01-25 | 1998-03-01 | Hipra Lab Sa | NEW ATTENUATED STRAIN OF THE VIRUS CAUSING THE RESPIRATORY AND REPRODUCTIVE SYNDROME (PRRS), THE VACCINES AND DIAGNOSTIC MEDIA OBTAINABLE WITH THE SAME AND THE PROCEDURES FOR ITS OBTAINING. |
| US5866401A (en) * | 1996-03-01 | 1999-02-02 | Schering Corporation | Porcine reproductive and respiratory syndrome vaccine |
| US5976537A (en) * | 1996-07-02 | 1999-11-02 | The United States Of America As Represented By The Secretary Of Agriculture | Porcine reproductive and respiratory syndrome vaccine |
| SG83740A1 (en) * | 1999-08-10 | 2001-10-16 | Inst Of Molecular Agrobiology | An attenuated porcine reproductive and respiratory syndrome virus strain and methods of use |
| ATE479440T1 (en) * | 2004-11-19 | 2010-09-15 | Intervet Int Bv | PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME (PRRS) SYNDROME VIRUS STRAINS AND COMPOSITIONS OF SWINE |
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2007
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102107003A (en) * | 2011-01-05 | 2011-06-29 | 重庆大学 | Porcine reproductive and respiratory syndrome Virosome vaccine and preparation method thereof |
| CN103695465A (en) * | 2013-12-16 | 2014-04-02 | 中国农业科学院哈尔滨兽医研究所 | CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof |
| CN104388399A (en) * | 2014-08-18 | 2015-03-04 | 张澍 | PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and neutralizing antibody and possesses wide-spectrum immunogenicity |
| CN104388399B (en) * | 2014-08-18 | 2017-02-15 | 张澍 | PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and neutralizing antibody and possesses wide-spectrum immunogenicity |
| WO2016119079A1 (en) * | 2015-01-29 | 2016-08-04 | 山东省农业科学院畜牧兽医研究所 | Sx-105 strain of porcine reproductive and respiratory syndrome virus (prrsv) and use thereof |
| US10883085B2 (en) | 2015-01-29 | 2021-01-05 | Institute Of Animal Science And Veterinary Medicine, Shandong Academy Of Agricultural Sciences | PRRSV SX-105 strain and use thereof |
| CN111849924A (en) * | 2020-06-23 | 2020-10-30 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Similar NADC30 porcine reproductive and respiratory syndrome virulent strain, attenuated strain and application thereof |
| CN111849924B (en) * | 2020-06-23 | 2022-05-10 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Similar NADC30 porcine reproductive and respiratory syndrome virulent strain, attenuated strain and application thereof |
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Application publication date: 20080716 Assignee: Harbin Weike Biotechnology Development Co., Ltd. Assignor: Harbin Veterinary Medicine Inst., China Academy of Agriculture Contract record no.: 2014230000506 Denomination of invention: Pig replication and respiration syndrome virus attenuated vaccine strain and uses thereof Granted publication date: 20090805 License type: Exclusive License Record date: 20140911 |
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