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Recombinant plasmid and engineering bacterium containing grass carp interferon gene and their application

A recombinant plasmid and interferon technology, applied in genetic engineering, medical preparations containing active ingredients, applications, etc., can solve the problems of high purity requirements, difficult and inappropriate natural extraction methods, etc., to enhance disease resistance, promote The effect of healthy and sustainable development and low price

Inactive Publication Date: 2007-06-06
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In medicine, natural interferon can be isolated from human white blood cells, and recombinant interferon can also be prepared through genetic engineering. Due to the particularity of aquatic animals' water life, it is not suitable to use individual injections for interferon. Feeding with feed additives is an ideal and convenient way. At the same time, in order to reduce costs, natural extraction methods are generally difficult to achieve. Therefore, recombinant expression technology is more Appropriate way

Method used

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  • Recombinant plasmid and engineering bacterium containing grass carp interferon gene and their application
  • Recombinant plasmid and engineering bacterium containing grass carp interferon gene and their application
  • Recombinant plasmid and engineering bacterium containing grass carp interferon gene and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, grass carp interferon gene cloning

[0027] Subcutaneously inject 0.5ml of poly I:C (purchased from Tianjin Pharmaceutical Factory) from the dorsal fin of grass carp, induce another injection at 26°C for 12 hours, take about 10 grams of head kidney after 12 hours, and extract total RNA with TRizol (Gibco BRL, USA) , the extraction method was carried out according to the instructions. Then reverse transcription and PCR amplification were performed with the reverse transcription kit RNA PCR kit (AMV) Ver3.0 (TaKaRa, USA). Primer sequences are shown in Table 1.

[0028] After the amplified product was purified (using the PCR product purification kit DNA Gel Extraction Kit, V-gene), it was inserted into a T vector (TA cloning kit was purchased from Shanghai Sangong). The operation steps are all in accordance with the manual. The ligation product was transformed into Escherichia coli TOP10, and positive recombinants were screened for sequencing identificatio...

Embodiment 2

[0030] Embodiment 2, grass carp interferon gene sequence determination and analysis

[0031] Using NCBI ORF Finder to analyze the cDNA sequence obtained in Example 1, it can be seen that the open reading frame ORF is 543 bp long and encodes 180 amino acids. The rest of the 5'UTR is 34bp long, and the 3'UTR is 614bp long; the potential functional sites were analyzed with PROSITE database and the signal state analysis was carried out with the signal peptide analysis software SignalPprogram (version 3.0), and the conclusion was that the isoelectric point of the protein was 9.73 , and the gene has a signal peptide with a length of 22 amino acids, indicating that its mature peptide is 159 amino acids; using Prosite for site analysis, it is found that the gene has two N-terminal 14 acylation sites: GqcsAC and GTkvSF; 2 protein kinase II phosphorylation sites: SacE ShkE; one N-terminal glycosylation site: NESL; one protein kinase C phosphorylation site: ShK; two chromosomal nuclear l...

Embodiment 3

[0032] Embodiment 3, the construction of grass carp interferon expression plasmid

[0033] The interferon gene sequence design expression vector that obtains according to embodiment 1 constructs primer, and sequence is as follows:

[0034] The upstream primer starts from GcIFN ATG, and the 5' end contains Hind III restriction site and Kozak sequence ACC

[0035] gc-IFN-F3: 5'-CCC AAG CTT GGG ACC ATG GAA ACT CAA ATGTGG-3'

[0036] The downstream primer starts from the stop codon TAA, and an Xho I restriction site is added to the 5' end

[0037] gc-IFN-R3: 5'-GGCGAGCTCGCCTTATCGTCTGTTGGCAATGC-3'

[0038] Carry out PCR amplification according to the method in Example 1, carry out double digestion with Hind III and Xho I (TAKARA company) on the amplified product, insert into the expression vector pYES2, transform Escherichia coli TOP10, according to the "Molecular Cloning Experiment Guide" The recombinant expression plasmid was extracted by the alkaline lysis method and sequence...

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Abstract

The present invention discloses one kind of recombinant plasmid containing grass carp interferon gene with the nucleotide sequence as shown in SEQ ID No. 1. The present invention also discloses engineering bacterium INVSC1-pYES2-IFN as one kind of Saccharomyces cerevisiae in the preservation number of CGMCC1847. The engineering bacterium is used for aquatic animals to resist viruses. The present invention also discloses the application of protein coded by the recombinant plasmid containing grass carp interferon gene with the nucleotide sequence as shown in SEQ ID No. 1 for aquatic animals to resist viruses.

Description

technical field [0001] The invention relates to the technical fields of aquatic organism technology and aquaculture disease prevention and control. In particular, it relates to the use of molecular biology techniques to clone the grass carp interferon gene, construct a recombinant yeast expression plasmid containing the gene, and obtain high-efficiency expression in Saccharomyces cerevisiae, and use the interferon-expressing yeast engineering bacteria to directly develop disease-resistant feed Additive, used in aquatic animal disease control. Background technique [0002] With the rapid development of fishery production, aquatic animal diseases have become increasingly prominent, and have become a worldwide problem that seriously affects the healthy and sustainable development of aquaculture, especially in my country. In recent years, the direct economic loss of aquaculture industry caused by diseases in my country has exceeded 10 billion yuan per year, and it has been incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/20C12N1/19A61K36/064A61P31/12C12R1/865
Inventor 邵健忠胡向东项黎新彭博林慧芳
Owner ZHEJIANG UNIV
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