Fish natural killer (NK)-lysin effective factors and application method thereof

An effector and fish technology, applied in the field of molecular biology, can solve the problems of function and application that are yet to be studied, and achieve the effect of improving the ability of anti-virus and anti-bacterial infection

Inactive Publication Date: 2013-06-12
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

NK-lysin is currently only found in five species of bony f

Method used

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  • Fish natural killer (NK)-lysin effective factors and application method thereof
  • Fish natural killer (NK)-lysin effective factors and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The NK-lysin of the present invention is the amino acid sequence in SEQ ID No.1 of the sequence table.

[0019] The sequence listing SEQ ID No.1 is:

[0020] MNKSPILLFCILAACSVWSVHGKSQEMNIDDEEPAEVELPVEAKPPGLCWGCKWALNKVKKAMTQKETYEKVKAR LIKICNKIGFLKSRCHKFVITHLDELVEELSTTDDVKTICVNVKACNPKEPSHLLFYPNN

[0021] (a) Sequential features:

[0022] ●Length: 135 (effective area 47-121)

[0023] ●Type: amino acid sequence

[0024] ●Chain type: single chain

[0025] ●Topological structure: linear

[0026] (b) Molecule Type: Protein

[0027] (c) Assumption: No

[0028] (d) Antisense: No

[0029] (e) Original source: tongue sole

[0030] Structural features: The protein is expected to contain a Saposin B functional domain (amino acids 47-121).

Embodiment 2

[0032] Construction of NK-lysin expression plasmid pCNK1:

[0033] The cDNA of tongue sole was used as a template and PCR amplification was carried out with primers F1 and R1. The PCR conditions are: 94°C for 60s to pre-denature template DNA, then 94°C for 40s, 50°C for 60s, 72°C for 60s, after 5 cycles, change to 94°C for 40s, 58°C for 60s, 72°C for 60s, after 30 cycles and then repeat at 72°C. ℃ extension reaction 7-10min. PCR products were purified with corresponding kits from Tiangen. The expression vector pIRES2-EGFP (purchased from Clontech, USA) was digested with the restriction endonuclease SmaI to recover a 5kb fragment, which was ligated with the above-mentioned purified PCR product with T4 DNA ligase, and the ligation solution was transformed into Escherichia coli DH5α. Cultured on LB medium containing kanamycin (50ug / ml) for 18-24 hours, screened transformants to extract plasmid, named pCNK1. DNA sequencing analysis proved that pCNK1 is an expression plasmid con...

Embodiment 3

[0036] Application of NK-lys in expression plasmid pCNK1

[0037] Step 1) Plasmid injection

[0038] Dilute the pCNK1 of the above-mentioned Example 2 to 200ug / ml in PBS, which is the pCNK1 dilution. Twenty tongue soles (about 5.1 g in weight) were randomly divided into 4 groups, 5 in each group. These 4 groups are named A, B, C and D respectively. Each fish in groups A and C was injected with 50 ulp of CNK1 dilution, and each fish in groups B and D (control group) was injected with 50 ul of PBS.

[0039] The composition of the PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.

[0040] Step 2) Bacteria and virus suspension preparation

[0041] Cultivation of Vibrio anguillarum C312 to OD in LB medium 600 0.8, then centrifuge (5000g, 4°C, 10min), collect the cells, suspend them in PBS to a final concentration of 1 x 10 7 cfu / ml is Vibrio eelium suspension. The cytomegalovirus RBIV-C1 (see Zha...

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Abstract

The invention relates to the filed of molecular biology, in particular to cynoglossus semilaevis natural killer (NK)-lysin and application method of the cynoglossus semilaevis NK-lysin. NK-lysin is as the display of an amino acid sequence in sequencer (SEQ) ID No.1. The application method is that the cynoglossus semilaevis complementary deoxyribonucleic acid (c DNA) serves as a template, and a primer F1 and a primer R1 are utilized to carry out polymerase chain reaction (PCR) amplification. After a PCR product is connected with an expressive vector, plasmids (p CNK1) are obtained, the plasmids (p CNK1) are injected into fishes and the antiviral capacity and the antibacterial capacity of the fishes can be obviously intensified.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a fish NK-lysin effector and its application. Background technique [0002] Cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTLs) and natural killer cells (natural killer cell, NKC) are the main effector cells of cellular immunity. When stimulated, these cells secrete a series of effectors that cause apoptosis and killing of target cells, one of which is NK-lysin. NK-lysin is a member of the saposin family and has a typical saposin domain. [0003] In mammals, NK-lysin is an antimicrobial peptide with broad-spectrum antibacterial activity. therefore, [0004] NK-lysin has a good application prospect in the prevention and treatment of bacterial diseases. NK-lysin is only found in five species of teleost, but its function and application are still to be studied. Contents of the invention [0005] The purpose of the present invention is to provide an NK-lysin effect factor ...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/70A61K38/17A61K48/00A61P31/04A61P31/12
Inventor 张敏孙黎
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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