Recombinant protein of fish tumor necrosis factor family protein and application thereof

A tumor necrosis factor and recombinant protein technology, which is applied in the field of recombinant proteins of fish tumor necrosis factor family proteins, can solve problems such as lack of functional and applied research, and achieve the effect of improving antibacterial and antiviral capabilities

Inactive Publication Date: 2015-12-23
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, tumor necrosis factor has been found in a variety of fish, but its function and application research is still lacking

Method used

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  • Recombinant protein of fish tumor necrosis factor family protein and application thereof
  • Recombinant protein of fish tumor necrosis factor family protein and application thereof
  • Recombinant protein of fish tumor necrosis factor family protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Preparation of recombinant protein rCsTNF of tumor necrosis factor family protein

[0017] 1) Construction of plasmid pCsTNF expressing tumor necrosis factor family protein recombinant protein rCsTNF:

[0018] The tumor necrosis factor family protein CsTNF of the present invention belongs to TNF-α type, and its sequence has been published (GenBankaccessionnumberXP_008330028.1):

[0019] MDSECRVQLDVTDRDGEVREEPTPGVQARSKLTKAVLVLTVCLAAAAALLLVFYRQHNSSPQEEDSFELHHTLRQMSHVRAAIHLQGVHNPKMTNSVEWQDEVEQTHSQGGLKLDNNEVVIPHHGLYFVYSQVSFQVSCGDADVGAVSSPQVHLSHTVQRWSSSFGSDKVKSYRTILHSARTVCQKTASADSADEGSSFAAVYMGAVFNLLKGDRLRTVMEENMLPFLEDDPGKSFFGVFAL

[0020] The tumor necrosis factor family protein CsTNF is composed of 242 amino acids and contains a tumor necrosis factor TNF domain, which consists of 81-242 amino acid residues. The gene of tumor necrosis factor family protein CsTNF was amplified by PCR with primers TNFF1 and TNFR1 using the cDNA of tongue sole as template. The PCR conditions ...

Embodiment 2

[0026] Antibacterial Effect of Tumor Necrosis Factor Family Protein CsTNF

[0027] Dilute the tumor necrosis factor family protein CsTNF protein purified in Example 1 above to 100 ug / ml in PBS, which is the CsTNF dilution. Cultivate Pseudomonas fluorescens (Pseudomonas fluorescens) in LB medium (preserved in CGMCC, General Microbiology Center of China Committee for Culture Collection of Microorganisms, No. Institute of Microbiology, date of deposit is January 9, 2008) to OD 600 was 0.8, then centrifuged (5000g, 4°C, 10min), collected the cells, and suspended them in PBS to a final concentration of 2×10 7 cfu / ml is the bacterial suspension. The bacterial suspension was mixed with equal volume (1:1) of CsTNF dilution or PBS (control). Twenty tongue soles (about 13.6 g in weight) were randomly divided into two groups, 10 in each group. These two groups are named A and B respectively. Each fish in group A was injected with 100 ul of bacteria+CsTNF dilution, and each fish in g...

Embodiment 3

[0031] Antiviral effect of tumor necrosis factor family protein CsTNF

[0032]The cytomegalocytosis virus RBIV-C1 (see ZhangM, XiaoZ, HuY, SunL. Characterization of amegalocytivirus from cultured rockbream, Oplegnathus fasciatus (Temminck & Schlege), in China. AquacRes. 2012; 43:55664) in PBS was diluted to 10 6 copies / ml is the virus suspension. Dilute the CsTNF protein purified in Example 1 above to 100 ug / ml in PBS, which is the CsTNF dilution. The virus suspension was mixed with equal volume (1:1) of CsTNF diluent or PBS (control). Twenty tongue soles (about 13.6 g in weight) were randomly divided into two groups, 10 in each group. These two groups are named C and D, respectively. Each fish in group C was injected with 100 ul virus+CsTNF dilution, and each fish in group D (control group) was injected with 100 ul virus+PBS. On the 6th and 9th day after the injection, the spleen tissues of the fish were taken. DNA was extracted from the tissue using a DNA extraction kit...

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Abstract

The invention relates to the field of molecular biology, specifically to a recombinant protein of fish (cynoglossus semilaevis) tumor necrosis factor family protein (CsTNF) and an application thereof. The recombinant protein of the fish tumor necrosis factor family protein CsTNF is a recombinant protein of cynoglossus semilaevis tumor necrosis factor family protein (CsTNF). According to the invention, cynoglossus semilaevis cDNA is used as a template and primers TNFF1 and TNFR1 are used to carry out PCR amplification of tumor necrosis factor family protein CsTNF gene; and an amplification product undergoes induced expression and purification so as to obtain the recombinant protein. The tumor necrosis factor family protein can remarkably enhance antibacterial or anti-virus capability of fish.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a recombinant protein of tumor necrosis factor family protein (CsTNF) of fish (semi-smooth tongue sole) and an application thereof. Background technique [0002] Tumor necrosis factor (TNF) is an important cytokine that can regulate many physiological processes such as organ development, tissue repair, hematopoiesis and inflammatory response in vertebrates and invertebrates. Tumor necrosis factor can regulate various immune responses of monocytes, macrophages, T lymphocytes, B lymphocytes and natural killer cells, and induce cell proliferation, apoptosis and necrosis. The tumor necrosis factor family is divided into three classes: TNF-α, TNF-β and LT-β. TNF-α is a 26kDa membrane protein, which is hydrolyzed by metalloproteases to form a free activating protein of about 17kDa. The activated form of TNF-α binds to the tumor necrosis factor receptor on the cell surface and exerts ...

Claims

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Application Information

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IPC IPC(8): C07K14/525A61K38/19A61P31/04A61P31/20
CPCC07K14/525A61K38/00
Inventor 李墨非孙黎
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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