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Viperin, and preparation and application of viperin

An American red fish and anti-virus technology, applied in the field of molecular biology, can solve the problems of unclear anti-virus effect, destroying the structure of viral lipid rafts, interfering with the interaction between viral proteins and host proteins, and achieving the effect of improving anti-viral ability

Inactive Publication Date: 2014-04-23
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently known antiviral mechanisms of mammalian viperin mainly include (1) interfering with the lipid raft structure necessary for virus budding; (2) interfering with the interaction between viral proteins and host proteins to affect virus assembly
However, fish viperin has less research and its antiviral effect is still unclear

Method used

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  • Viperin, and preparation and application of viperin
  • Viperin, and preparation and application of viperin
  • Viperin, and preparation and application of viperin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The viperin of the present invention is the amino acid sequence in SEQ ID No. 1 of the sequence listing.

[0016] Sequence Listing SEQ ID No.1 is:

[0017] MQLSSIVASPKQLLQLCISTLHCLLASVFSKVSSWTAGTCKESTSPVSPVDGKLDQNKVQNATTPTSVNYHFTRKCNYKCGFCFHTAKTSFVLPLEEAKRGLKLLKESGMEKINFSGGEPFLHDKGEFLGKLVQFCKQDLQLPSVSIVSNGSMIKEKWFQKYGDYLDILAISCDSFDEETNQLIGRAQGRKSHLDNLYKIRNWCQQYKVAFKINSVINTFNVDEDMTESITQLSPVRWKVFQCLLIDGENAGEKALREAERFVISDQLFQEFLDRHSSVSCLVPESNEKMRNSYLILDEYMRFLDCREGRKDPSKSVLDVGVKEAICFSGFDEKMFLKRGGKYVWSKADMKLEW

[0018] (a) Sequence features:

[0019] ●Length: 354 (effective length 354)

[0020] ●Type: amino acid sequence

[0021] ●Chain type: single chain

[0022] ●Topology: Linear

[0023] (b) Molecular type: protein

[0024] (c) Assumption: No

[0025] (d) Antonym: No

[0026] (e) Original Source: American Redfish

[0027] Structural features: The protein contains a SAM family protein domain (amino acids 66-274).

Embodiment 2

[0029] Construction of Viperin expression plasmid pVip:

[0030] Using the redfish cDNA as a template, PCR amplification was performed with primers F1 and R1. The PCR conditions were: 94°C for 60s pre-denaturation of template DNA, then 94°C for 40s, 60°C for 60s, 72°C for 60s, after 5 cycles, it was changed to 94°C for 40s, 65°C for 60s, 72°C for 60s, 30 cycles and then at 72°C ℃ extension reaction 7-10min. The PCR product was purified with the corresponding kit from Tiangen. The expression vector pCN3 (for the construction process, see Jiao XD, Zhang M, Hu YH, Sun L. Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens. Vaccine 2009; 27: 5195–202.) was digested with the restriction enzyme EcoRV. The 5.4kb fragment was recovered, ligated with the above-purified PCR product with T4 DNA ligase, the ligation solution was transformed into E. coli DH5α, cultured on LB medium containing ampicillin (100ug / ml) for 18-24 hours, and the transformants were s...

Embodiment 3

[0033] Application of Viperin Expression Plasmid pVip

[0034] Step 1) Plasmid injection

[0035] Dilute the pVip of Example 1 above to 200ug / ml in PBS, which is the pVip dilution. Twenty American redfish (about 27 g in weight) were randomly divided into 2 groups of 10 each. Name these 2 groups A and B, respectively. Each fish in group A was injected with 100ul of pVip dilution, and each fish in group B (control group) was injected with 100ul of PBS.

[0036] Described PBS composition by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na 2 HPO 4 .12H 2 O, 0.024%NaH 2 PO 4 , the balance is water.

[0037] Step 2) Virus Suspension Preparation

[0038] With cytomegalovirus RBIV-C1 (concrete preparation method sees Zhang M, Xiao Z, Hu Y, Sun L.Characterization of a megalocytivirus from cultured rock bream, Oplegnathus fasciatus (Temminck&Schlege), in China.Aquac Res.2012;43: 556–64) in PBS to 106copies / ml, which is the virus suspension.

[0039] Step 3) Challenge infection...

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PUM

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Abstract

The invention relates to the field of molecular biology, in particular to viperin, and preparation and an application of the viperin. A sequence of the viperin is shown as amino acid SEQ ID No.1 (sequence identifier number 1). The application is an application of a plasmid pVip expressing the viperin with amino acid SEQ ID No.1 in antiviral infection. The antiviral capacity of fish can be improved significantly after the viperin expression plasmid is injected into the fish.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a viperin of American redfish and its preparation and application. Background technique [0002] Viperin is an endoplasmic reticulum-associated, interferon-induced antiviral protein with a molecular weight of about 40 kDa. Viperins from lower vertebrates to mammals show a high degree of amino acid sequence conservation, and their protein structures all contain a SAM (S-adenosyl methionine-dependent radical enzymes) domain and a highly conserved C-terminal region. Viperin is anchored to the endoplasmic reticulum membrane or liposome through its N-terminal alpha helix structure. Since the endoplasmic reticulum and liposomes are necessary sites for viral protein synthesis and viral amplification, their localization on these two organelles is critical for viperin to perform its antiviral function. Under normal circumstances, the expression level of viperin is very low, but when sti...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/70A61K38/43A61P31/12
CPCA61K38/00C12N9/00
Inventor 孙黎张宝存
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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