A dual inactivated vaccine against porcine parvovirus and porcine epidemic diarrhea
A dual inactivated vaccine and porcine epidemic diarrhea technology, which is applied in the direction of viruses, vaccines, antiviral agents, etc., can solve the problems of ineffective prevention of porcine parvovirus and poor clinical application effect, and achieve resistance to attack and maintenance Long time, quick effect of antibody production
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Embodiment 1
[0015] Embodiment 1: Screening of porcine parvovirus (PPV) VP2 gene
[0016] In 2010, symptoms of porcine parvovirus disease appeared in many farms in Shandong Province, and the individual pigs with the disease had been injected with the existing parvovirus vaccine before, and it was speculated that the infected virus had mutated; Screening; the porcine parvovirus PPV1 was finally screened out.
[0017] In order to verify the antigenicity of the screened virus, five virus strains from different sources, including the screened PPV1 strain, were used as antigens to prepare vaccines. After immunizing SPF pigs, the screened virus liquid was used to challenge the virus. The results showed that Compared with other porcine parvovirus vaccines, the vaccine prepared by itself has a better immune effect (p<0.05), so it is confirmed that it has a genetic variation.
[0018] According to the antigenic characteristics and amino acid sequence of porcine parvovirus VP2 protein, a pair of pr...
Embodiment 2
[0020] Example 2: Preparation of recombinant porcine parvovirus VP2 protein
[0021] The method comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying the recombinant VP2 protein. The details are as follows: The positive cloned plasmid pMD18-T-VP2 and the expression vector pPICZα vector were digested with KpnI and NotI respectively, and after 1.2% agarose gel electrophoresis, they were recovered with a DNA gel recovery kit, and about 1.7 kb were obtained respectively. and 3.3kb fragments were oriented at 16°C to construct pPICZα-VP2 expression vector, after the correct identification by restriction enzyme digestion; after the plasmid was linearized, it was electrotransformed into Pichia pastoris competent cells to construct Pichia pastoris expression strain X33-VP2, The genome of the expressed strain X33-VP2 was extracted and identified correctly by PCR using primers primer 1 and primer 2...
Embodiment 3
[0030] Embodiment 3: the preparation of subunit inactivated vaccine
[0031] The semi-finished VP2 protein antigen after passing the inspection is used for vaccine preparation (the liquid components in the following preparations are calculated by volume ratio).
[0032] (1) Preparation of oil phase Take 95 parts of veterinary white oil and 1 part of aluminum stearate, put them in the oil phase preparation tank and heat to 80°C, then add 5 parts of Siben-80, until the temperature reaches 115°C, Keep it for 30 minutes, and set aside after cooling.
[0033] (2) Preparation of aqueous phase The porcine parvovirus VP2 protein was diluted with physiological saline to 150 μg / 0.1 ml. Take 5 parts of sterilized Tween-80 and add them to the liquid mixing tank, and at the same time add 95 parts of protein solution for seedling production, and stir for 20-30 minutes to completely dissolve the Tween-80.
[0034](3) Emulsification Take 2 parts of the oil phase and put it in a high-speed s...
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