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Vaccine composition, preparation method and application thereof

A technology of vaccine composition and fusion protein, which is applied in the field of vaccine composition and its preparation, can solve the problems of differences in the pathogenicity of virulent strains of animals, etc., and achieve the effect of easy large-scale production and convenient storage

Active Publication Date: 2015-04-15
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing NDV has only one serotype, but the virulence and pathogenicity to animals are very different among the strains

Method used

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  • Vaccine composition, preparation method and application thereof
  • Vaccine composition, preparation method and application thereof
  • Vaccine composition, preparation method and application thereof

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Experimental program
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Embodiment 1

[0047] The construction of embodiment 1 Newcastle disease virus fusion protein cloning vector

[0048] According to NCBI ( http: / / www.ncbi.nlm.nih.gov The gene sequence in Newcastle disease China / Guangxi9 / 2003 (accession number: JF343539) reported in ), and the nucleotide sequences of F1, F2, and HN protein genes were respectively selected (see the sequence table for details, SEQ ID No.1, SEQ ID No. .2 and SEQ ID No.3), they were connected in series by genetic engineering as a fusion protein of Newcastle disease virus.

[0049] 1.1 Primer design

[0050] According to the selected nucleotide sequences of F1, HN, and F2 protein genes, ie, SEQ ID No.1, SEQ ID No.3, and SEQ ID No.2, primers for the respective fragments were designed respectively, and the details are as follows. Among them, SacI and XbaI restriction endonuclease sites were added to both ends of the upstream and downstream primers of F1 respectively; XbaI and XhoI restriction endonuclease sites were added to both...

Embodiment 2

[0086] The construction of embodiment 2 Newcastle disease virus fusion protein expression vector

[0087] 2.1 Construction of pET-28a-T-F1-HN-F2 expression vector

[0088]pET-28a and pMD18-T-F1-HN-F2 were digested with SacI and HindIII respectively, the digested products were subjected to electrophoresis, and the corresponding target bands were recovered using a DNA gel recovery kit. The results showed that after digestion, the pET-28a vector had a band around 5300bp, and the pMD18-T-F1-HN-F2 had a band around 2400bp.

[0089] The target bands were recovered and ligated using a DNA ligation kit. The ligation system was: pET-28a 1 μL, F1-HN-F 25 μL, buffer 2 μL, ligase 2 μL, and sterilized water 10 μL; the ligation conditions were: react overnight at 16°C. The ligation product was transformed into Bal21 competent cells. The transformation process was the same as in 1.3. The transformed bacteria were inoculated on LB agar plates containing 50 μg / mL kanamycin.

[0090] 2.2 Iden...

Embodiment 3

[0093] Expression, identification and purification of embodiment 3 Newcastle disease virus fusion protein

[0094] 3.1 Expression and identification of Newcastle disease virus fusion protein

[0095] Inoculate the Bal21 bacteria containing the pET-28a-T-F1-HN-F2 plasmid into LB liquid medium containing 50 μg / mL kanamycin at a 1% (V / V) inoculum amount, and cultivate at 37°C and 180rpm for 6- 8h, make bacteria OD 600 Between 0.6-1.0, add IPTG to make the final concentration 1mmol / L, continue to culture for 5h, take samples for SDS-PAGE electrophoresis, and use chicken anti-Newcastle disease positive serum for Western blot identification.

[0096] The results showed that compared with the control, the Bal21 bacteria containing the pET-28a-T-F1-HN-F2 plasmid were induced by IPTG for 5 hours, and the corresponding target band appeared around 80KDa, mainly in the inclusion body. The results of Western blot showed that the recombinant protein could specifically bind and react with ...

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Abstract

The invention provides a Newcastle disease virus fusion protein, which includes two protective antigen fragments, an F1 protein and an F2 protein, in an F protein, and includes an HN protein, wherein an amino acid sequence of the F1 protein is represented as the SEQ ID NO.2, the amino acid sequence of the F2 protein is represented as the SEQ ID NO.4, and the amino acid sequence of the HN protein is represented as the SEQ ID NO.6. The invention also discloses a vaccine composition containing the Newcastle disease virus fusion protein in an immunizing dose and an application of the vaccine composition in prevention and / or treatment of Newcastle disease virus in a gene VII type. The Newcastle disease vaccine composition can generate cell immunity and humoral immunity at the same time and also can generate protective immunity to the Newcastle disease virus in the gene VII type. The vaccine composition is broad-spectrum.

Description

technical field [0001] The invention relates to veterinary biological products, in particular to a vaccine composition and its preparation method and application. Background technique [0002] Newcastle Disease (ND), commonly known as Asian chicken plague, is caused by Newcastle Disease Virus (NDV) and can cause acute, highly contagious, and fatal infectious diseases in poultry. Difficulty, diarrhea, neurological disorders, and mucosal and serosal hemorrhage are the main features. It is one of the most serious poultry infectious diseases in the world today and is listed as a Class A infectious disease by the World Organization for Animal Health (OIE). [0003] Since ND was discovered in 1926, there have been four major pandemics in ND. Judging from the genotypes of NDV strains isolated clinically, the current prevalent ND is mainly caused by genotype VII NDV strains, which has caused huge economic losses to the poultry industry in my country. At present, vaccination is sti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/17A61P31/14
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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