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Application of cq-ns1abp gene and its protein antiviral activity for inhibiting wssv infection

A cq-ns1abp, gene sequence technology, applied in antiviral agents, applications, genetic engineering and other directions, can solve problems such as unclear, achieve the effect of inhibiting infection replication and strong anti-WSSV infection activity

Active Publication Date: 2021-06-11
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Shrimp can use apoptosis pathway to defend against WSSV infection [9] However, studies on Ns1abp in crustaceans have basically not been reported, whether Ns1abp exists in lower animals, and whether it is involved in WSSV infection is unclear

Method used

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  • Application of cq-ns1abp gene and its protein antiviral activity for inhibiting wssv infection
  • Application of cq-ns1abp gene and its protein antiviral activity for inhibiting wssv infection
  • Application of cq-ns1abp gene and its protein antiviral activity for inhibiting wssv infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of the anti-WSSV infection regulatory factor Cq-Ns1abp prokaryotic expression vector of red claw crayfish

[0043] Design and amplify the protein encoding the two important domains (BTB and Kelch) of the Cq-Ns1abp (cDNA) gene and the full-length protein containing the ORF box: Cq-Ns1abp-BTB specific upstream primer BTB-F1 and downstream primer BTB-R1, Cq-Ns1abp-Kelch specific upstream primer Kelch-F1 and downstream primer Kelch-R1, Cq-Ns1abp-Full-length specific upstream primer Ns1abp-F1 and downstream primer Ns1abp-R1. Add an EcoR I restriction site to the 5′ end of each upstream primer; add a Not I restriction site and a 6*His tag to the 3′ end of the downstream primer, so that the recombinant protein is simultaneously equipped with MBP and 6*His tags, for further purification. The corresponding domain regions were then amplified by PCR.

[0044] Cq-Ns1abp-BTB upstream primer BTB-F1:

[0045] 5'-CCGGAATTCTGCGATGTCATCCTCCAGGT-3',

[0046] Cq-...

Embodiment 2

[0058] Example 2 Induced expression of recombinant expression vector pMal-C2x-Cq-Ns1abp in Escherichia coli BL21 (DE3)

[0059] 1. Induced expression:

[0060] 1) Transform the constructed recombinant expression vector into BL21(DE3), and spread it on the LB plate containing ampicillin (Amp+) resistance.

[0061] 2) Pick a monoclonal colony, inoculate it in 5 ml of LB medium containing Amp+, and culture it on a shaking table at 37° C. at 200 rpm for 12 hours.

[0062] 3) Inoculate into 100ml LB medium containing Amp+ at a ratio of 1:100, culture at 37°C, 200rpm shaker until OD600 is 0.3 to 0.5.

[0063] 4) Add IPTG to a final concentration of 0.1 mM, and induce at 16° C. at 150 rpm for 20 h.

[0064] The results show that see figure 1 (2, 4, 6 swimming lanes), after IPTG induction, obvious induction bands can be detected in the bacteria, and the sizes are about 55kDa, 70kDa, and 130kDa, indicating that a higher proportion of expression products can be obtained under this co...

Embodiment 3

[0075] Example 3 Use of pull down experiments to demonstrate the recognition and binding of rCq-Ns1abp recombinant protein to WSSV

[0076]Dilute 2ug of recombinant protein (MBP, rCq-Ns1abp-BTB, rCq-Ns1abp-Kelch, and rCq-Ns1abp-Full-length, without any protein in the control group) and 5ug of WSSV envelope protein in 1ml of PBS solution, add 20ul of Dextrin beads that can specifically bind to MBP-tagged proteins were incubated at 4°C with rotation for 4h. After incubation, centrifuge at 500 g for 3 min, and carefully remove the supernatant. Resuspend and wash with 1ml pre-cooled PBS, centrifuge at 500g for 3min, repeat 5 times. Finally, the binding protein on the beads was eluted with 10mM maltose, added SDS sample buffer, boiled for 10min to denature, placed in 12% SDS-PAGE gel for electrophoresis, and Western Blot was used to detect recombinant protein and WSSV envelope protein Binding of VP28. The result is as image 3 , in the control group without adding protein and a...

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Abstract

A Cq-Ns1abp gene and its protein antiviral activity application for inhibiting WSSV infection, involving white spot syndrome virus. The Cq‑Ns1abp gene was connected to the prokaryotic expression vector pMal‑C2x to construct the anti-WSSV infection regulatory factor pMal‑C2x‑Cq‑Ns1abp recombinant expression vector; the Cq‑Ns1abp gene product contains the N-terminal BTB domain and the C-terminal Kelch domain protein and full-length protein; the resulting recombinant expression vector transformants were respectively introduced into host cells, and the expression products were induced to express, and then rCq-Ns1abp recombinant protein with higher purity was obtained by affinity chromatography. Since Cq-Ns1abp has an inhibitory effect on WSSV infection and plays an important role in the antiviral immunity of shrimp, the anti-WSSV infection regulatory factor of red crayfish is applied in the preparation of antiviral infection drugs and animal disease-resistant feed additives.

Description

technical field [0001] The invention relates to white spot syndrome virus, in particular to a Cq-Ns1abp gene for inhibiting WSSV infection and the antiviral activity application of its protein. Background technique [0002] White spot syndrome virus (WSSV) is currently the most serious viral disease pathogen in shrimp farming. WSSV has a very wide host range and strong pathogenicity to crustaceans, and can infect prawns, crayfish, crabs and lobsters, etc. [1,2] , has become the main obstacle to aquatic crustacean farming and has caused serious economic losses to the aquatic crustacean farming industry. So far, there is still a lack of effective drugs for the treatment of WSSV diseases. [0003] Influenza A nonstructural protein NS1-A binding protein (NS1ABP) was first reported in humans and was named for its ability to bind the nonstructural protein NS1-A of influenza A virus [3] . The Ns1abp protein reported in Hela cells contains N-terminal BTB (bric-a-brac, tramtrack,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P31/16
CPCA61K38/00A23K20/147A23K50/80C07K14/43509C12N15/70
Inventor 刘海鹏谢晓露王克坚
Owner XIAMEN UNIV
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