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Siniperca chuatsi IFN-alpha 3 (interferon alpha-3) gene, recombinant protein, preparation method and application thereof

A technology of recombinant protein and squid mandarin fish, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems such as difficult to use immunization methods, so as to improve anti-virus ability, reduce mortality, express The effect of volume increase

Active Publication Date: 2018-10-26
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the mandarin fish feeds on live fish for life, oral or injection immunization methods are difficult to use in the culture of mandarin fish

Method used

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  • Siniperca chuatsi IFN-alpha 3 (interferon alpha-3) gene, recombinant protein, preparation method and application thereof
  • Siniperca chuatsi IFN-alpha 3 (interferon alpha-3) gene, recombinant protein, preparation method and application thereof
  • Siniperca chuatsi IFN-alpha 3 (interferon alpha-3) gene, recombinant protein, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Cloning of the full-length cDNA sequence of the mandarin fish IFN-α3 in embodiment 1:

[0048] 1. Synthesis of primers

[0049] The partial cDNA sequence of the IFN-α3 gene of Siniperca sinensis was searched from the sequencing results of the transcriptome of Siniperca sinensis. The cDNA sequence is shown in SEQ ID NO: 1, and the expressed IFN-α3 recombinant protein is shown in SEQ ID NO: 2 Show.

[0050] 2. Extraction of sample RNA

[0051] The RNA in the collected Siniperca sinensis spleen was extracted by Trizol method. Use NANODROP 2000 (Thermoscientific, USA) ultra-micro ultraviolet spectrophotometer to detect the quality and concentration of RNA, and perform agarose electrophoresis to check the integrity of the extracted RNA. If the quality of the extracted RNA is not up to standard, repeat this step to extract again until a qualified RNA sample is obtained.

[0052] 3. Amplification of Siniperca sinensis IFN-α3 5' end sequence

[0053] (1) Use SUPERSCRIPT II...

Embodiment 2

[0071] The construction of embodiment 2 prokaryotic expression vector

[0072] 1. Molecular cloning of IFN-α3 of Siniperca sinensis

[0073] (1) Design primers IFN-α3-EcoRI-F and IFN-α3-XhoI-R with restriction sites according to the cDNA sequence of IFN-α3 after removing the signal peptide:

[0074] IFN-α3-EcoRI-F: 5'-TCCGAATTCTGTGATTGGCTCA-3'

[0075] IFN-α3-XhoI-R: 5'-GCTCGAGTCAGTGTTGGTGA-3'.

[0076] (2) The PCR reaction system is as follows:

[0077]

[0078] (3) The PCR reaction procedure is as follows:

[0079]

[0080] 2. The target fragment is connected to the T carrier: observe the target band by gel electrophoresis of the product obtained by PCR, and then recover it with a gel kit and connect it to the T carrier.

[0081] During the above experiment, figure 1 Swimming lane 2 of A shows the IFN-α3 ORF sequence obtained by PCR, 496bp; after it is connected with pMD19T and sequenced correctly, it is double digested with EcoR I and Xho I, and the target fragme...

Embodiment 3

[0084] Example 3 Prokaryotic expression of recombinant IFN-α3 of Siniperca sinensis

[0085] 1. Construction of recombinant expression plasmids: double enzyme digestion of the pMD19-T-IFN-α3 plasmid and the expression vector pET32a(+) plasmid, the enzyme digestion system is as follows:

[0086]

[0087] 2. Agarose gel electrophoresis, and tap the gel to recover the target fragment after digestion, such as figure 1 As shown in B, the IFN-α3 fragment was ligated with the vector pET32a(+) at a molar ratio of 5:1, and the expression vector was obtained overnight at 4°C.

[0088] 3. Transfer the above-mentioned ligation product into DH5α competent cells, pick positive clones, identify them by bacterial liquid PCR, and select positive clones to extract plasmids.

[0089] 4. Transform the correct recombinant plasmid into the expression strain E.coliBL21(DE3).

[0090] 5. Induction of fusion protein expression

[0091] Pipette 5 μL of pET-32a(+)-IFN-α3 / BL21 bacterial solution an...

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Abstract

The invention discloses a siniperca chuatsi IFN-alpha 3 (interferon alpha-3) gene, recombinant protein, a preparation method and application thereof. The preparation method of the siniperca chuatsi IFN-alpha 3 recombinant protein includes the steps of: cloning the full-length cDNA (complementary deoxyribonucleic acid) sequence of the siniperca chuatsi IFN-alpha 3, as is shown in SEQ ID NO:1 by using rapid amplification RACE (rapid amplification of cDNA end) technology of cDNA ends; cloning the ORF (open reading frame) sequence of the cDNA sequence by using a primer with a restriction site; cloning the ORF sequence into a pMD19T vector and then conducting double enzyme digestion with EcoR I and Xho I to obtain a target fragment; performing double enzyme digestion of an pET32a(+) vector with EcoR I and Xho I, connecting the pET32a(+) vector going through the double enzyme digestion with the target segment to obtain a recombinant expression vector pET32a(+)-IFN-alpha 3 to be transferredto DH5 (competent cells) alpha competent cells, selecting positive clones, and extracting preservation plasmids; transferring the plasmids into an expression bacterium BL21 (electroporation) and adopting an IPTG (isopropyl-beta-d-thiogalactopyranoside) induced expression to obtain IFN-alpha 3 recombinant protein, as is shown in SEQ ID NO. 2. The IFN-alpha 3 recombinant protein has the advantages of being capable of being used for immersion immunity to prevent viral diseases of the siniperca chuatsi, effectively activating the siniperca chuatsi into an anti-virus state, and reducing mortality.

Description

technical field [0001] The invention belongs to the fields of aquaculture, biotechnology and medical bioengineering, and in particular relates to a method for preparing IFN-α3 gene, IFN-α3 recombinant protein, IFN-α3 recombinant protein and IFN-α3 recombinant protein in soaking immune prevention Application of Siniperca sinensis virus disease. Background technique [0002] In fish farming, viral diseases are often an important factor causing large-scale loss and production reduction. Its onset is rapid and there is no effective treatment drug and method. Blind drug use will also cause drug residues and environmental pollution. The use of vaccines can achieve safe and efficient The control effect is the development direction of fish virus disease control in the future. [0003] Interferon (Interferons, IFNs) is a multifunctional, secreted protein that can induce a series of humoral immunity produced by lymphocytes and monocytes in vertebrates when the body and cells are stim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C12N15/21C12N15/70A61K38/21A61P31/20
CPCA61K38/00A61P31/20C07K14/56C12N15/70
Inventor 黄鹤忠肖攀路瑶
Owner SUZHOU UNIV
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