Adjuvant for improving effect of porcine reproductive and respiratory syndrome inactivated vaccine, preparation method thereof and application thereof

A technology of inactivated vaccines and immune efficacy, applied in biochemical equipment and methods, medical preparations containing active ingredients, antiviral agents, etc., to achieve high safety, wide application range, and small side effects

Inactive Publication Date: 2011-01-12
朱善元 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research of immune adjuvant is the focus of immunology research, yet the research using HVJ-E as immune adjuvant has not been reported yet, based on the current situation of this fact, the present invention adopts HVJ-E as novel immune adjuvant, observes its It can not only promote the improvement of animal anti-PRRSV antibody level, but also efficiently induce the production of cytokines, reduce the clinical symptoms of animals after being challenged by virulence, and reduce the generation of animal viremia and the time and quantity of virus excretion

Method used

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  • Adjuvant for improving effect of porcine reproductive and respiratory syndrome inactivated vaccine, preparation method thereof and application thereof
  • Adjuvant for improving effect of porcine reproductive and respiratory syndrome inactivated vaccine, preparation method thereof and application thereof
  • Adjuvant for improving effect of porcine reproductive and respiratory syndrome inactivated vaccine, preparation method thereof and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0045] The preparation method of the present invention comprises the following steps:

[0046] 1) Prepare chicken embryo allantoic fluid containing Sendai virus;

[0047] 2) Concentrate the allantoic fluid prepared in step 1) with PEG6000, put 20% sucrose on the bottom, and centrifuge at 20000r / min for 2h;

[0048] 3) Collect the precipitate, wash the precipitate with PBS, and centrifuge again at 20000r / min for 2h to remove sucrose;

[0049] 4) Collect the precipitate and suspend it in pyrogen-free saline;

[0050] 5) at 99mJ / cm 2 The intensity of ultraviolet rays inactivated the collected Sendai virus for 2h;

[0051] 6) The inactivated Sendai virus was named Sendai virus capsule, and the Sendai virus capsule was inoculated into 9-day-old SPF chicken embryos. After 72 hours, the allantoic fluid of the chicken embryos was collected, and the hemagglutination value of the allantoic fluid was determined.

[0052] The described inactivation method is 99mJ / cm 2 UV inactivation...

Embodiment 1

[0053] The preparation of embodiment 1.HVJ-E

[0054] 1. Propagation of HVJ: Take a frozen HVJ virus seed (Z strain) from a -70°C refrigerator, dissolve it slowly on ice, and after dissolving, centrifuge at 2000r / min for 10min to collect the supernatant. The collected supernatant was diluted 1000 times with a PBS solution containing 100 U / ml penicillin and 0.1 mg / ml streptomycin, etc., and 100 μL of the diluted virus was drawn with a disposable sterile syringe and inoculated into 10-day-old SPF chicken embryo chorioallantois membrane cavity. After 24 hours, the death of chicken embryos was observed, and the SPF chicken embryos that died within 24 hours were discarded. After 72 hours, the allantoic fluid of chicken embryos was aseptically collected.

[0055] 2. Concentration of HVJ: Centrifuge the collected allantoic fluid at 10,000 rpm for 1 hour to remove impurities in the allantoic fluid, collect the chicken embryo allantoic fluid after removing impurities, put it into a d...

Embodiment 2

[0063] Example 2. HVJ-E enhances porcine reproductive and disorder syndrome virus inactivated vaccine immune effect research

[0064] 1. Reagents

[0065] Culture medium (RPMI-1640), concanavalin A (ConA), polyethyleneimine (PEI, 25kDa), MTT (purchased from Sigma); fetal bovine serum (purchased from Hyclone); PRRSV antibody detection kit ( Purchased from IDEXX Company); Porcine IFN-γ Immunization Kit (purchased from Invitrogen International Inc.) Porcine IL-10 and IL-12 Immunization Kit (purchased from R&D International Inc.), vacuum pump containing lithium heparin anticoagulant Blood vessels are products of Hunan Pharmaceutical Company, 96-well plates (purchased from Corning Co.LTD.) Ficoll-Paque (purchased from DingGuo Biotech), DNA / RNA extraction kits, and one-step RT-PCR kits are all purchased from TAKARA Company.

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Abstract

The invention discloses an adjuvant for improving the effect of a porcine reproductive and respiratory syndrome (PRRSV) inactivated vaccine, a preparation method thereof and application thereof and relates to the preparation method and steps of Hemagglutinating virus of Japan envelope (HVJ-E) and application of a medicament for improving the effect of the PRRSV inactivated vaccine. The preparation method of adjuvant comprises the following steps of: breeding Hemagglutinating virus of Japan (HVJ) by using an SPF chick embryo, and then concentrating, purifying, counting and inactivating the HVJ to prepare the Hemagglutinating virus of Japan envelope (HVJ-E); immunizing PRRSV negative pigs with the HVJ-E and the PRRSV inactivated vaccine together, and setting a PBS contrast group and a PRRSV inactivated vaccine group at the same time, wherein compared with the contrast group, the HVJ-E and PRRSV inactivated vaccine group can obviously improve the response level of hunoral immunity and cellular immunity of the pigs; and after immunizing, expelling toxin by using a PRRSV virulent strain (JXA1), wherein the result shows that the clinical manifestations and the weight increasing of the HVJ-E and PRRSV inactivated vaccine group are better than those of the contrast group, and the generated viremiavirusemia rate, toxin expelling rate and virus distribution rate are obviously reduced when compared with the contrast group. The adjuvant of the invention has the advantages of convenient production and low cost and is suitable for large-scale production.

Description

Technical field [0001] The invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to the preparation and application of Sendai virus envelope (HVJ-E) as an immune adjuvant, in particular to the use of Sendai virus envelope as a porcine reproductive and respiratory syndrome virus inactivation Vaccine immunization adjuvant. Background technique [0002] Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS) has occurred in the United States since 1987, with the main symptoms of sow abortion, weak birth, stillbirth, mummified fetuses and respiratory disorders of pigs of all ages. The world pig industry has brought huge economic losses. Taking China as an example, the economic loss caused by porcine respiratory and reproductive disorder syndrome reaches more than 20 billion yuan every year. The pathogen of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV). In the report o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/12C12N7/00A61P31/14
Inventor 朱善元张泉徐向明左伟勇洪伟鸣王健成大荣蒋春茂郁杰王永娟王安平杨海峰黄文强秦枫
Owner 朱善元
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