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Subunit vaccine for porcine reproductive and respiratory syndrome as well as preparation method and application of subunit vaccine

A subunit vaccine and porcine PRRS technology, which is applied in the field of porcine PRRS subunit vaccine and its preparation, can solve problems such as irritation, and achieve the effects of improving immunogenicity, good protection effect, and easier separation and purification

Active Publication Date: 2020-11-24
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccines designed with their ideas still need to be further improved to stimulate the body's cellular immune response

Method used

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  • Subunit vaccine for porcine reproductive and respiratory syndrome as well as preparation method and application of subunit vaccine
  • Subunit vaccine for porcine reproductive and respiratory syndrome as well as preparation method and application of subunit vaccine
  • Subunit vaccine for porcine reproductive and respiratory syndrome as well as preparation method and application of subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Construction of target protein expression vector

[0041] 1. Obtain the target protein sequence:

[0042]Get the gene sequence of PRRSV NADC30-like strain from NCBI (Accession: MH651743), select the nucleotide sequence of GP5 protein and optimize it; get the gene sequence of Pseudomonas aeruginosa from NCBI (GenBank: CP039293), select the exotoxin The translocation domain of PEA (remove its DomainⅢ, hereinafter referred to as PEA△DⅢ) sequence; connect PEA△DⅢ and the optimized GP5 gene sequence in series, add 8×His tag sequence, add NdeI and C-terminal respectively XhoI restriction site, gene sequence of biosynthetic recombinant protein (PEA△DⅢ-GP5) (as shown in SEQ ID No.1).

[0043] 2. Link the synthetic fusion protein pUC-PEA△DⅢ-GP5 sequence into the expression vector pFastBacdual with NdeI and XhoI restriction sites:

[0044] (1) Enzyme digestion of pUC-PEA △DⅢ-GP5 and insect baculovirus expression vector pFastBacdual (purchased from Fenghui Biotechnology, produ...

experiment example 1

[0066] Experimental Example 1 Safety Test of PEA△DⅢ-GP5 Subunit Vaccine on Mice

[0067] Ten Balb / C mice aged 4-5 weeks were purchased and divided into 2 groups, 5 mice in each group, which were the immune group and the control group, respectively. The immunized group was inoculated with 200 μL of the PEA△DⅢ-GP5 subunit vaccine prepared in Example 1 by calf intramuscular injection on day 0 and day 14 respectively; the blank control group was injected with the same volume of vaccine control in the same way. Continuous observation for 14 days after immunization showed that the test mice had normal spirit and appetite, and were in good health without any adverse reaction, which indicated that the subunit vaccine was safe for mice. The results of the safety test on mice are shown in Table 1.

[0068] Table 1

[0069]

experiment example 2

[0070] Experimental example 2 PEA△DⅢ-GP5 subunit vaccine safety and effectiveness test on piglets

[0071] 1. Safety and effectiveness test of PEA△DⅢ-GP5 subunit vaccine on piglets:

[0072] Ten 45-day-old PRRSV-negative piglets were selected and divided into two groups, 5 in the immunization group and 5 in the control group. The immunized group was intramuscularly injected with 1.0 ml of the PEA△DⅢ-GP5 subunit vaccine prepared in Example 1 behind the ears on the 0th day and the 14th day, and the blank control group was not immunized. After 14 days of continuous observation, the piglets did not show any clinical symptoms, and their spirit, appetite, and body temperature were all normal, without any adverse reactions. The results showed that the subunit vaccine was safe for piglets.

[0073] The specific results of the piglet safety test are shown in Table 2.

[0074] Table 2

[0075]

[0076] 2. Immune efficacy test of PEA △DⅢ-GP5 subunit vaccine

[0077] Ten 45-day-old...

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Abstract

The invention relates to the fields of genetic engineering and veterinary biological pharmacy, and in particular discloses a subunit vaccine for porcine reproductive and respiratory syndrome as well as a preparation method and application of the subunit vaccine. The invention firstly provides a fusion protein, wherein an amino acid sequence of the fusion protein is as shown in SEQ ID No. 1; and then the invention provides the subunit vaccine for the porcine reproductive and respiratory syndrome, wherein the subunit vaccine comprises the fusion protein. The fusion protein in the subunit vaccinefor the porcine reproductive and respiratory syndrome is obtained by conducting fusion expression on an ORF5 full-length gene of a PRRSV NADC30-like strain, which is optimized by virtue of a codon, and a gene of pseudomonas aeruginosa, from which a Domain III structural Domain is removed, and by virtue of an insect baculovirus / insect cell expression system. The vaccine provided by the inventionis good in protective effect, capable of inducing effective humoral immunity and cellular immunity and is suitable for clinical prevention of the reproductive and respiratory syndrome.

Description

technical field [0001] The invention relates to the fields of genetic engineering and veterinary biopharmaceuticals, in particular to a porcine PRRS subunit vaccine and its preparation method and application. Background technique [0002] Porcine reproductive and respiratory syndrome is a highly contagious disease caused by PRRSV. It can cause reproductive disorders in sows, respiratory symptoms in pigs of all ages, and high mortality in piglets. It is one of the important diseases that cause serious economic losses in the pig industry. The vaccines currently on the market for the prevention of the disease mainly include inactivated vaccines and attenuated vaccines. Inactivated vaccines are safe but cannot induce cellular immunity well. Attenuated vaccines have good immune protection effects but there are risks of dispersing the virus and returning to strong. The subunit vaccine is expected to play a better preventive role. [0003] The new epidemic strain NADC30-like can ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/866A61K39/12A61K39/385A61K47/64A61P31/14
CPCC07K14/005C12N15/86A61K39/12A61K39/385A61K47/6415A61P31/14C12N2770/10022C07K2319/55C07K2319/21C07K2319/50C12N2710/14043C12N2800/105A61K2039/552A61K2039/57A61K2039/6037C12N2770/10034
Inventor 张华伟孙芳徐高原周明光曾小燕朱娴静郝根喜邵伟
Owner WUHAN KEQIAN BIOLOGY CO LTD
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