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A kind of giant salamander iridescent virus disease vaccine and its preparation method and application

A giant salamander iridovirus and vaccine technology, applied in the biological field, can solve the problems of high expression of xenogeneic biological cells, difficulty in exogenous genes, etc., and achieve the effects of high expression amount, simple culture and good safety.

Active Publication Date: 2016-09-07
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Codon bias makes it difficult for cloned foreign genes to be expressed efficiently in heterogeneous biological cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Obtaining of truncated optimized recombinant protein JDMCP:

[0057] 1. Truncated region screening

[0058] The antigenic region, hydrophilic region and surface display probability of the giant salamander iridescent virus MCP gene were analyzed, and the hydrophilic sequence with concentrated antigenic determinants and strong antigenicity was selected as the proposed expression gene.

[0059] 2. Codon optimization

[0060] According to the yeast codon preference, codon optimization was performed on the proposed expression gene sequence, and the GC content was kept moderate, and the optimized sequence was synthesized. Compared with the original sequence (MCP), the optimized MCP sequence has 20%-30% bases optimized and the low-frequency codons of Pichia pastoris are all replaced by high-frequency or sub-high-frequency codons.

[0061] 3. Amplification of MCP truncation-optimized genes

[0062] 3.1 Design of PCR primers for MCP truncated optimized gene

[0063] Accordin...

Embodiment 2

[0107] A preparation method of giant salamander iridescent virus disease vaccine, the steps are as follows:

[0108] Pick the Pichia pastoris Km71 / GSIV-MCP strain, inoculate it into 100mL BMGY medium, and culture it at 30°C and 250r / min shaking until OD 600 When the value reaches about 6, centrifuge at room temperature at 1500g for 5min, resuspend the bacteria in 20mL of BMMY medium, transfer the obtained bacterial solution into a 100mL shake flask, seal the bottle, add 100% methanol to a final concentration of 0.5%, and store at 30°C, 250r / The culture was induced under the condition of 1 min, and methanol was added every 24 hours to maintain the concentration of methanol at 0.5%. After continuous induction for 3 days, the yeast in the induction solution was removed by centrifugation (removing the precipitate and leaving the supernatant), and the Bradford protein analysis method was used to calculate the content of the target protein (recombinant protein JDMCP) in the origina...

Embodiment 3

[0112] A kind of application of giant salamander iridescent virus disease vaccine in preparation giant salamander iridescent virus vaccine, its application process is as follows:

[0113] The recombinant protein JDMCP prepared in Example 2 was used to immunize giant salamanders (70 ± 5 grams) with substantially no difference in body weight, good mental condition, and giant salamander iridescent virus negative, and a positive control and a normal saline control (100 microliters of each giant salamander intramuscularly injected, The specific concentration is shown in Table 1), after 15 days, the immunization was boosted with the same dose, and after the antibody was detected by the ELISA kit, the giant salamander iridescent virus strain was used for the challenge experiment, and 500 microliters of 1×10 per fish were injected intraperitoneally. 6.5 TCID 50 / ml giant salamander iridescent virus (CCTCC NO: V201134) to detect whether the antibodies produced have protective power (Ta...

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Abstract

The invention discloses giant salamander iridescent virus vaccine, a preparation method and an application. The giant salamander iridescent virus vaccine is recombinant protein translated after truncating and optimizing a major capsid protein gene of a giant salamander iridescent virus, and has a sequence as shown as SEQIDNO.2 (Sequence Identifier Number 2). A nucleotide sequence corresponding to the protein is optimized according to preference of a yeast codon to synthesize a gene segment which is transferred to a yeast expression vector, and pichiapastoris Km71 / GSIV-MCP (i) Pichiapastoris(1 / i)Km71 / GSIV-MCP (CCTCC (China Center for Type Culture Collection) NO: M2014573) of the high expression recombinant protein is finally obtained via high copy and clone screening. The protein produced by fermentating the yeast has the characteristics of high activity and large yield. An immunoprotection experiment determines that the antigen protein can effectively prevent a giant salamander iridescent virus disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a giant salamander iridescent virus disease vaccine, a preparation method and an application. Background technique [0002] The Chinese giant salamander (Andrias davidianus) is the largest amphibian in existence. Its common name is "salamander". In recent years, giant salamander viral hemorrhagic disease has broken out in the main breeding areas of giant salamanders in Shaanxi, Hubei, Hunan, Zhejiang, Guizhou and other provinces in my country. caused huge economic losses. [0003] The pathogen of giant salamander viral hemorrhagic disease is Chinese giant salamander iridovirus (GSIV), which is a member of the family Iridoviridae and the genus Ranavirus. Iridoviruses are a class of double-stranded DNA viruses with large virion particles and an icosahedral shape. The family Iridoviridae is divided into five genera: Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus and Rana...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C12N1/19C12P21/02A61K39/12A61P31/20C12R1/84
Inventor 周勇曾令兵范玉顶徐进马杰
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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