Cq-Ns1abp gene for inhibiting WSSV infection and application of protein thereof in resistance of virus activity

A technology of cq-ns1abp and pmal-c2x-cq-ns1abp, applied in the field of white spot syndrome virus, can solve unclear problems and achieve the effect of strong anti-WSSV infection activity

Active Publication Date: 2018-09-21
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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  • Cq-Ns1abp gene for inhibiting WSSV infection and application of protein thereof in resistance of virus activity
  • Cq-Ns1abp gene for inhibiting WSSV infection and application of protein thereof in resistance of virus activity
  • Cq-Ns1abp gene for inhibiting WSSV infection and application of protein thereof in resistance of virus activity

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

The invention provides a Cq-Ns1abp gene for inhibiting WSSV infection and application of protein thereof in resistance of virus activity and relates to white spot syndrome viruses. The Cq-Ns1abp geneis ligated into a prokaryotic expression vector pMal-C2x to construct an anti-WSSV infection regulatory factor pMal-C2x-Cq-Ns1abp recombinant expression vector of chrtax quadricarinatus. A Cq-Ns1abp gene product contains an N-end BTB structural domain and C-end Kelch structural domain protein and full-length protein; obtained recombinant expression vector transformants are introduced into host cells respectively for inducing expression to obtain expressed products, and rCq-Ns1abp recombinant protein with higher purity is obtained through affinity chromatography. Since Cq-Ns1abp has an inhibitory effect on WSSV infection, Cq-Ns1abp has an important effect on antiviral immunity of shrimps. Therefore, an anti-WSSV infection regulatory factor of chrtax quadricarinatus is applied in preparationof 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 Applications(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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