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Preparation method of porcine parvovirus genetic engineering vaccine

A technology of genetically engineered vaccine and parvovirus, which is applied in the field of preparation of porcine parvovirus genetically engineered vaccine, can solve the problems of loose virus, difficult to popularize and apply, and short immunity period, so as to prevent degradation, ensure biological activity, and be easy to separate The effect of purification

Active Publication Date: 2012-01-18
嘉兴卓十生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In clinical application, inactivated vaccines have been found to have the following disadvantages: (1) large doses of vaccination or application of concentrated antigens are required, the immunization period is short, and booster vaccination is often required; (2) local immunity cannot be induced, so that the effect of cellular immunity is weak; (3) ) It takes 2-3 weeks to produce complete immunity, which is not conducive to emergency vaccination and lower vaccine costs; (4) There is a possibility of incomplete inactivation and loose virus
However, the attenuated vaccine has the risk of strong virulence, recombination and potential infection, so it is difficult to promote and apply in practice.
Therefore, traditional vaccines have exposed more and more problems in the prevention and control of PPV, and a new type of safe and reliable genetic engineering vaccine is imminent

Method used

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  • Preparation method of porcine parvovirus genetic engineering vaccine
  • Preparation method of porcine parvovirus genetic engineering vaccine
  • Preparation method of porcine parvovirus genetic engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Construction of recombinant yeast expression vector: the expression vector is pGAPZαA, and the host strain is yeast strain SMD1168. The whole gene sequence of PPV VP2 was amplified by PCR, the upstream primer P1 was shown in SEQ ID NO:1, the downstream primer P2 was shown in SEQ ID NO:2, and the enzyme cutting sites were XhoI and NotI, AAAAGA It is the Kex2 protease cleavage site.

[0025]2. Construction of recombinant yeast engineering bacteria: Transform yeast strain SMD1168 by linearization electroporation with the recombinant yeast expression vector constructed above, and the linearization enzyme is AvrⅡ.

[0026] 3. Non-induced expression of recombinant yeast engineered bacteria: Pick the single Zeocin-resistant colony screened with a sterilized pipette tip, and carry out primary culture in 5mL YPD liquid medium, shake overnight at 30°C and 200 r / min , to OD 600 =2-6, that is, the cells are in the logarithmic growth phase. Take 1mL of primary culture solution...

Embodiment 2

[0030] Animal experiment: The PPV genetic engineering vaccine prepared above was used to immunize piglets with basically no difference in body weight and good mental status and PPV negative. At the same time, a positive control, an empty vector control, a normal saline control, an adjuvant control and a blank control without any treatment were set up. Immunization was boosted 15 days later, and after antibodies were detected by ELISA kits, a virulent strain of porcine parvovirus was used for challenge experiments (except group F) to detect whether the antibodies produced had protective power. The experiment is as follows:

[0031] test group Inoculum dose Group A PPV Genetic Engineering Vaccine 3 servings Group B Positive control (PPV attenuated vaccine) 3 servings Group C Empty vector control 3 servings Group D saline control 3 servings Group E Adjuvant control 3 servings Group F Blank control without any treatm...

Embodiment 3

[0034] PPV VP2 expression measurement: VP2 expression supernatant protein concentration was determined using Bradford protein concentration determination kit. Since there is a small amount of bacterial protein in the expression supernatant, the measured concentration is the total protein concentration. The specific steps are as follows:

[0035] 1. Completely dissolve protein standard BSA (5 mg / mL), take 10 μL and dilute to 100 μL, so that the final concentration is 0.5 mg / mL. Standards were diluted with PBS.

[0036] 2. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the diluted standard to the standard wells of the 96-well plate, and add PBS diluent to make up to 20 μL.

[0037] 3. Add 10 μL of the supernatant of VP2 expression time of 48h, 72h, and 96h to the sample wells of the 96-well plate, and add PBS diluent to 20 μL.

[0038] 4. Add 200 μL of G250 staining solution to each well and let stand at room temperature for 3-5 minutes.

[0039] 5. Measure the absorbance at a wavelength...

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Abstract

The invention discloses a preparation method of porcine parvovirus genetic engineering vaccine, which comprises the following steps: constructing and recombining a yeast expression carrier p GAPZ alpha-VP2; then carrying out electric shock to convert pichia pastoris SMD1168, so that VP2 protein is efficiently secreted and expressed in a YPD culture medium to form viruslike particles without virusnucleic acid; purifying protein, carrying out SDS-PAGE and Western Blot and electric microscope detection; and adding a corresponding immunologic adjuvant to prepare the porcine parvovirus genetic engineering vaccine. The porcine parvovirus genetic engineering vaccine prepared by the invention solves the defects in the current porcine parvovirus genetic engineering vaccine, can be used for preventing and treating porcine parvovirus genetic infection and relative diseases caused by the porcine parvovirus genetic infection, and is also suitable for preparing and detecting the envelope antigen of a porcine parvovirus antibody ELISA kit.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a preparation method of porcine parvovirus genetic engineering vaccine. Background technique [0002] Porcine parvovirus (PPV) is a self-replicating virus, and it is one of the main factors that cause deformed fetuses in sows. Usually, primiparous sows infected with PPV can cause reproductive disorders, mainly manifested as abortion and infertility. , produce stillbirths and mummified fetuses, etc., bring significant economic losses to the pig industry. PPV, porcine circovirus (PCV), and porcine reproductive and respiratory syndrome virus (PRRSV) have always been hot spots in swine disease research. Recent studies have found that PPV plays an important role in postweaning multisystemic wasting syndrome (PMWS). [0003] The PPV genome is a single-stranded linear DNA molecule with a size of about 5,000 nucleotides, and mature virions contain only negative-strand DNA genomes. ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/23A61P31/20
Inventor 黄毓茂郭春和刘德辉
Owner 嘉兴卓十生物科技有限公司
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