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Preparation method and application of fusion protein and vaccine composition containing same

A vaccine composition and fusion protein technology, applied in the field of veterinary biological products, can solve the problems of immune failure, long research and development cycle, and slow antibody production

Active Publication Date: 2015-01-14
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the existing HP-PRRS commercial vaccines include inactivated vaccines and attenuated live vaccines, but the inactivated vaccines are often inoculated, the antibody production is slow, and the protective effect is unstable, which often leads to immune failure; it cannot induce heterologous HP-PRRS The strong immune effect of the virus strain; the attenuated live vaccine is made by weakening the strong virus strain through continuous passage, which requires a lot of manpower and material resources, the research and development cycle is long, and there is even a risk of virulence returning to strength. The immune protection between different types and strains with large mutations is also weak

Method used

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  • Preparation method and application of fusion protein and vaccine composition containing same
  • Preparation method and application of fusion protein and vaccine composition containing same
  • Preparation method and application of fusion protein and vaccine composition containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The synthesis of embodiment 1NLNK gene

[0059] In order to make the fusion protein have a good spatial structure and free extension, and improve the pharmacological and biological activities, a hydrophobic flexible peptide (GGGGSGGGGSGGGGS) composed of glutamic acid and serine was designed as a Linker to connect between Nsp9 and Nsp10.

[0060] According to the Nsp9 protein and Nsp10 protein in the highly pathogenic porcine reproductive and respiratory syndrome virus JXA1 strain (accession number: FJ548854.1) reported in NCBI (http: / / www.ncbi.nlm.nih.gov) The gene sequence, as well as the gene sequence of Linker (GGGGSGGGGSGGGGS) and the carboxy-terminal part (KKDELRVELKDEL), were synthesized by Shanghai Bioengineering Co., Ltd. by artificial synthesis, and the synthesized gene was named Nsp9-Linker-Nsp10-KDEL (abbreviated as NLNK ), the synthesized gene fragment has a full length of 243bp, and the sequencing result is shown in SEQ ID No.8 of the sequence table.

Embodiment 2

[0061] Example 2 Preparation, Identification and Purification of Fusion Protein PEA-NLNK

[0062] 2.1 Synthesis and amplification of PEA gene

[0063] According to the gene sequence of Pseudomonas aeruginosa exotoxin type A (Pseudomonas aeruginosa exotoxin type A, accession number: K01397.1) domain I and II reported in NCBI (http: / / www.ncbi.nlm.nih.gov) ( See SEQ ID NO.9), and use PCR to amplify the corresponding target gene. Wherein, the PEA gene primer design is as follows:

[0064] Upstream primer F1: 5'-CGGGATCCGCCGAGGAAGCCTTCGAC-3',

[0065] Downstream primer R1: 5'-CAACTTCCTCTTTGCCGTCGCCGAGGAACT-3',

[0066] PCR amplification program: 95°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 52°C renaturation for 30 s, 72°C extension for 60 s, 35 cycles, and finally 72°C extension for 10 min.

[0067] The obtained PCR amplification product was electrophoresed with 1% agarose gel, and the detection result showed that the corresponding target band appeared at about ...

Embodiment 3

[0085] Embodiment 3 Preparation of highly pathogenic porcine reproductive and respiratory syndrome vaccine composition

[0086] Dilute the fusion protein PEA-NLNK prepared in Example 2 with a pH7.2 PBS solution, prepare the diluted fusion protein solution and Gel adjuvant as shown in Table 1, stir at a speed of 500-800r / min for 10-15min, Add 1% thimerosal solution by volume before terminating the stirring so that the final concentration does not exceed 1 / 10,000, shake and mix well, and the highly pathogenic porcine reproductive and respiratory syndrome vaccine composition is obtained. Store it at 2-8°C after aliquoting.

[0087] Table 1 Components and ratios contained in the highly pathogenic porcine reproductive and respiratory syndrome vaccine composition

[0088]

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Abstract

The invention provides a fusion protein, a highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) vaccine composition containing the fusion protein and an application of the vaccine composition. The fusion protein comprises pseudomonas exotoxin A structural domains I and II, an HP-PRRS antigenic protein and a carboxyl terminal part, wherein the HP-PRRS antigenic protein comprises an Nsp9 protein and an Nsp10 protein. The invention also discloses the vaccine composition containing immune dosage of fusion protein and the application of the vaccine composition to preparation of drugs for preventing and / or treating HP-PRRSs. The vaccine composition has the advantages that recombinant expression can be carried out on the components of the vaccine composition in quantity by means of genetic engineering, thus not only consuming short time but also facilitating large-scale production.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a fusion protein of highly pathogenic porcine reproductive and respiratory syndrome virus, a vaccine composition containing the fusion protein and applications thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS), also known as "pig blue ear disease", is a pig disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Highly contagious disease, mainly characterized by reproductive disorders such as fever, anorexia, early abortion, stillbirth, mummified fetus, and weak piglets in sows, respiratory symptoms and high mortality in piglets (Yin Zhen, Liu Jinghua. Animal Virology. 2nd Edition . Beijing: Science Press. 1997). At the same time, PRRSV can cause immunosuppression in animals, and th...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/63A61K39/12A61P31/14
Inventor 张许科孙进忠王莹田克恭
Owner PU LIKE BIO ENG
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