Expression and application of thymosin-repeated protein Cq-TRP1 resisting WSSV (White Spot Syndrome Virus) infection in pichia pastoris

A technology of thymosin and protein, applied in the direction of thymopoietin, application, animal/human protein, etc., can solve problems such as obstacles to prevention and control work

Inactive Publication Date: 2018-04-13
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no cell lines available for research in shrimp, which hinders the resear

Method used

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  • Expression and application of thymosin-repeated protein Cq-TRP1 resisting WSSV (White Spot Syndrome Virus) infection in pichia pastoris
  • Expression and application of thymosin-repeated protein Cq-TRP1 resisting WSSV (White Spot Syndrome Virus) infection in pichia pastoris
  • Expression and application of thymosin-repeated protein Cq-TRP1 resisting WSSV (White Spot Syndrome Virus) infection in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The construction of embodiment 1 red claw crayfish thymosin Cq-TRP1 eukaryotic expression vector

[0050] According to the multiple cloning site of the pPICZaA vector, a specific upstream primer F1 and a downstream primer R1 for amplifying the ORF of the Cq-TRP1 (cDNA) gene encoding the red-clawed crayfish were designed. An EcoR I restriction site was added to the 5' end of the upstream primer F1; an XbaI restriction site, a stop codon and a base encoding His-tag were added to the 5' end of the downstream primer R1.

[0051] Upstream primer F: 5'-CCGGAATTCAGCACCGAATCCTCACTCA-3', downstream primer R: 5'-GCTCTAGATTAATGATGATGATGATGGTGGGCTTTCTTCTCCTGCTCAATCT-3'.

[0052] The PCR reaction conditions were: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s; annealing at 60°C for 30s; extension at 72°C for 30s; repeat 30 cycles; extension at 72°C for 10min.

[0053] The PCR product was recovered using an agarose gel purification kit, and the recovered PCR product...

Embodiment 2

[0055] Example 2 Induced expression of pPICZaA-Cq-TRP1 recombinant plasmid in Pichia pastoris GS115

[0056] The correctly sequenced pPICZaA-Cq-TRP1 plasmid was linearized by BamHI digestion, transformed into Pichia pastoris GS115 competent cells by electric shock method, and the expression was induced by methanol.

[0057] The results showed that compared with before induction, Pichia pastoris GS115 transformed with pPICZaA-Cq-TRP1 recombinant plasmid could express recombinant protein after induction, the size was about 18kDa, and the protein expression level of methanol induction 24h was significantly higher than that of 12h (see figure 2 ).

Embodiment 3

[0058] Example 3 Purification of the expression product of pPICZaA-Cq-TRP1 recombinant plasmid after methanol induction in Pichia pastoris GS115

[0059] Purify the Cq-TRP1 recombinant protein by affinity chromatography, induce the expression of a large number of positive recombinant Pichia pastoris GS115 strains, remove the bacteria by centrifugation (4°C, 12000rpm centrifugation for 30min), and collect 1L of the medium supernatant in dialysate (50mM Phosphate buffered saline, 50mM NaCl) in dialysis three times (dialysis 12h each time), obtain the column sample. Then, the dialyzed protein was subjected to affinity chromatography using a metal chromatographic column. Collect the elution peak components and analyze by SDS-PAGE electrophoresis (see image 3 ) shows two bands, one is about 18kDa and the other is about 16kDa. According to mass spectrometry, the two bands of 18kDa and 16kDa are the rCq-TRP1 protein of the red claw crayfish.

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Abstract

The invention discloses expression and application of thymosin-repeated protein Cq-TRP1 resisting WSSV (White Spot Syndrome Virus) infection in pichia pastoris, relating to cheraxquadricarinatus thymosin-repeated protein. Cheraxquadricarinatus thymosin is named Cq-TRP1. According to multiple clone sites of a pPICZaA carrier, a specific upstream primer F1 and a downstream primer R1 for amplifying and encoding a Cq-TRP1 gene ORF are designed, and an EcoR I restriction enzyme cutting site is added at a 5 end of the upstream primer F1; an XbaI restriction enzyme cutting site, a termination codon and a basic group encoding His-tag are added at a 5 end of the downstream primer R1. The Cq-TRP1 gene is connected to an eukaryotic expression vector pPICZaA, so as to build a pPICZaA-Cq-TRP1 eukaryotic expression vector; the obtained eukaryotic recombined expression vector is imported into the pichia pastoris for induction expression, so as to acquire an expression product; and then dialysis and affinity chromatography are performed on the obtained expression product in sequence.

Description

technical field [0001] The present invention relates to red-clawed crayfish thymosin protein, in particular to the thymosin protein having the function of inhibiting the replication of white spot syndrome virus (WSSV), utilizing genetic engineering technology to construct a red-clawed crayfish thymosin protein gene expression vector, and establishing a Expression of a thymosin protein Cq-TRP1 with anti-WSSV infection activity in Pichia pastoris and its application method. Background technique [0002] White spot syndrome virus (White spot syndrome virus, WSSV) is one of the pathogenic sources that cause shrimp farming, especially prawn farming. This virus has caused a great threat to the economic farming of aquatic crustaceans. Ability is the focus and difficulty of current research. At present, there are no cell lines available for research in shrimp, which hinders the research on the molecular mechanism of shrimp resistance to WSSV infection and the later prevention and c...

Claims

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Application Information

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IPC IPC(8): C07K14/66C07K14/435C12N15/81C12N15/66A61P31/20A23K20/184
CPCA23K20/184C07K14/43504C07K14/66C12N15/66C12N15/815
Inventor 刘海鹏李东利张秋霞王克坚
Owner XIAMEN UNIV
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