Sheep aphthosis virus recombinant subunit vaccine, and production method thereof

A kind of oral ulcer virus and subunit vaccine technology, applied in biochemical equipment and methods, antiviral agents, viruses/bacteriophages, etc., can solve the problems of inability to obtain genetically stable expression cell lines, difficulty in culturing and proliferation, poor immunogenicity, etc. , to achieve the effect of increasing expression and immunogenicity, good genetic stability, and increasing expression

Pending Publication Date: 2019-05-03
YULIN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, like the literature report by Liu Yuan et al., the method of the patent application for this invention can only achieve transient expression in eukaryotic cells, and cannot obtain a stably expressed subunit vaccine, and involves the transformation of multiple plasmids. Plasmid loss is also prone to occur in , which cannot obtain genetically stable expressing cell lines
[0005] Based on the above technical research, it

Method used

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  • Sheep aphthosis virus recombinant subunit vaccine, and production method thereof
  • Sheep aphthosis virus recombinant subunit vaccine, and production method thereof
  • Sheep aphthosis virus recombinant subunit vaccine, and production method thereof

Examples

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Example Embodiment

[0041] Example 1 Construction, expression and identification of the vector of the fusion protein of sheep oral aphtha virus B2L and F1L

[0042] 1. Gene Synthesis

[0043] According to the sequence of the B2L and F1L natural protein genes of the ORFV-Yulin strain of Ovine Oral Virus, after codon optimization, a tandem fusion gene expressing the full-length B2L gene and the F1L truncated gene (deleting amino acids 1-70) was synthesized. Thus obtained the B2L and F1L fusion protein for the ovine aphthous virus. At the same time, the C-terminus of the mutant protein is tagged with 6 histidines. Using chemical synthesis, the gene sequence was synthesized, which contained 1908 nucleotides in total. Among them, the 1-1134th position is the full-length B2L sequence of the ovine aphthous virus, and the 1147-1908th position is the F1L truncated gene (deleting amino acids 1-70). The specific nucleic acid sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No....

Example Embodiment

[0055] Example 2 Preparation of Recombinant Subunit Vaccine of Sheep Oral Virus

[0056] (1) Strain culture: Take the second-level seeds of Autographa californica nuclear polyhedrosis virus (Bac-ORFV-01) strain, inoculate 1% of the total culture medium in a suspension culture tank, and cultivate for 48 hours.

[0057] (3) Purification: using a nickel column method, using His antibody for affinity chromatography, and scanning through grayscale, the rB2LcF1L finally obtained is detected by SDS-PAGE and the band is scanned by grayscale. The protein purity should not be less than 75 %.

[0058] (4) Protein content detection: use BCA method to detect protein content. As a result, the protein content was 0.65 mg / mL. After SDS-PAGE detection and gray-scale scanning of the band, the protein purity was 85%.

[0059] (5) Sterility test: According to the appendix of the current "Chinese Veterinary Pharmacopoeia". The results all grow aseptically.

[0060] (6) Vaccine preparation: introduce the...

Example Embodiment

[0071] Example 3 Toxicity test of the B2L and F1L fusion protein rB2LcF1L of sheep mouth sore virus on mice

[0072] The virulence of the fusion protein rB2LcF1L of the ovine aphthous virus B2L and F1L prepared in Example 1 on mice was tested to verify the actual attenuation effect of the mutant in vivo. The B2L and F1L fusion protein rB2LcF1L of ovine aphthous virus were subcutaneously injected into 6-8 weeks old Balb / c mice at a dose of 300μg / mouse, a total of 10 rats. It was found that 100% of the mice were alive, and after being killed by cervical dislocation, no abnormal organs were found after anatomy. This result indicates that the fusion protein rB2LcF1L is non-toxic in mice.

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Abstract

The invention relates to a sheep aphthosis virus fusion protein rB2LcF1L. An amino acid sequence of the sheep aphthosis virus B2L and F1L fusion protein is SEQ ID No:2; compared with the wild type sheep aphthous virus F1L protein, the sheep aphthosis virus fusion protein rB2LcF1L has amino acid sequences of sites No.1 to 70 deleted to facilitate the expression of the fusion protein and increase the expression level. At the same time, 6 groups of histidine tags are added to the C-terminus of the fusion protein to facilitate purification in subsequent production. In the above amino acid sequence, compared with a B2L amino acid sequence of a conventional isolate, the V/L of the amino acid at a site No.9 is mutated to G, namely ''IPVG or IPLG is mutated to IPGG'', and the LDCF at a site No.35is mutated to LYCF. The above mutation significantly increases the expression level and immunogenicity of the fusion protein.

Description

Technical field: [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a recombinant subunit vaccine of aphthous ulcer virus and a production method thereof. Background technique: [0002] Oral sores, also known as sheep infectious pustule, is a contact and epithelial infectious disease that seriously threatens the sheep industry. It mainly harms 3-6 month-old lambs and some adult sheep. The clinical symptoms of the disease are skin rashes, pustules, ulcers and scabs on the lips and mucous membranes of the mouth. Lambs show ulcers on the teeth and lips and cannot eat. The incidence rate is as high as 100%. If secondary or mixed infection with other pathogens Microbial mortality is high, up to 15%. Mouth occurs worldwide and is widespread. In recent years, with the development of my country's sheep raising industry and the change of feeding methods, the risk of the disease has increased, the affected area and the severity ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N7/01A61K39/275A61P31/20
CPCY02A50/30
Inventor 冯平陈瑞张磊
Owner YULIN UNIV
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