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Recombinant protein of cynoglossus semilaevis complement component C5a and application thereof

A semi-smooth tongue sole and recombinant protein technology, applied in the field of molecular biology, can solve the problems of lack of fish applied research

Inactive Publication Date: 2016-05-04
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, C5 and C5a have been reported in fish such as rainbow trout and carp, but their application research in fish is still lacking

Method used

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  • Recombinant protein of cynoglossus semilaevis complement component C5a and application thereof
  • Recombinant protein of cynoglossus semilaevis complement component C5a and application thereof
  • Recombinant protein of cynoglossus semilaevis complement component C5a and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Preparation of Complement Component C5a Recombinant Protein rCsC5a

[0020] 1) Construction of plasmid pCsC5a expressing the recombinant protein rCsC5a of complement component C5a:

[0021] The complement component rCsC5a of the present invention is a part of the complement component C5 (amino acid 685-751) of half-smooth tongue sole, and the sequence of C5 has been published (GenBankaccessionnumberXP_008334663.1).

[0022] CsC5a consists of 67 amino acids and has a typical anaphylatoxin structure.

[0023] The CsC5a gene was amplified by PCR with primers C5F1 and C5R1 using the cDNA of half-smooth tongue sole as a template. The PCR conditions were: 94°C for 60s to pre-denature the template DNA, then 94°C for 40s, 62°C for 60s, 72°C for 65s, 30 cycles, and then extend the reaction at 72°C for 7-10min. PCR products were purified with corresponding kits from Tiangen. The expression vector pET259 (for the construction process, see HuYH, ZhengWW, SunL. Identification and...

Embodiment 2

[0029] Phagocytosis-promoting effect of rCsC5a

[0030] Vibrio harveyi (Vibrioharveyi) (preserved in CGMCC, number CGMCC1985) was cultivated in LB medium to OD 600 0.8, then centrifuged (5000g, 40C, 10min), collected the bacteria, and suspended it in 100ug / mlfluoresceinisothiocyanate (FITC) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.") to a final concentration of 1 × 10 8 cfu / ml, incubate at 37°C for 2 hours, and then collect the cells by centrifugation using the above method. The bacteria were suspended in L-15-medium (purchased from Thermo Scientific HyClone, Beijing) to a final concentration of 1×10 8cfu / ml, that is, FITC-labeled bacteria. Percoll method was used to prepare peripheral blood lymphocytes (peripheral bloodleukocytes, PBL) of semi-smooth tongue sole. For details, see the literature ZhouZX, ZhangJ, SunL. The cells were placed in a 1.5ml centrifuge tube containing L-15-medium (107 cells per tube), and 100ul of the above-mentioned FITC-la...

Embodiment 3

[0033] Antibacterial effect of rCsC5a

[0034] Dilute the rCsC5a protein purified in Example 1 above to 100 ug / ml in PBS, which is the rCsC5a dilution. Culture Vibrio harveyi to OD in LB medium 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 6 cfu / ml is the bacterial suspension. The bacterial suspension was mixed with equal volume (1:1) of rCsC5a dilution or PBS (control). Twenty tongue soles (about 12.7g in weight) were randomly divided into 2 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+rCsC5a dilution, and each fish in group B (control group) was injected with 100 ul of bacteria+PBS. At 24h after the injection, the spleen tissues of group A and group B were taken, homogenized in 1ml PBS, and after dilution, 50ul of the homogenate was spread on the LB plate. The plates were incubated at 28°C for 48 ho...

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Abstract

The invention relates to the field of molecular biology, in particular to recombinant protein of a cynoglossus semilaevis complement component C5a (CsC5a) and application thereof. According to the recombinant protein of the cynoglossus semilaevis complement component C5a, cynoglossus semilaevis cDNA serves as a template, a primer C5F1 and a primer C5R1 are used for PCR amplification of a complement component CsC5 gene, and the recombinant protein is obtained through induced expression and purification of an amplified product. The recombinant protein of the complement component CsC5a can remarkably promote immune cell cytophagy, and the antibacterial or anti-virus capacity of fish is improved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a recombinant protein of the complement component C5a (CsC5a) of tongue sole and its application. Background technique [0002] Complement factor 5 (Complementfactor5, C5) is a component of the complement system. Human C5 protein is composed of α and β polypeptide chains through disulfide bonds, with a molecular weight of 190kDa. The 74th-75th arginine-leucine bond near the N-terminus of C5 is the action site of C5 convertase. After the complement system is activated through three pathways (classical pathway, alternative pathway and lectin pathway), all generate C5 convertase. The C5 protein is cleaved into two fragments, C5a and C5b, under the action of C5 convertase. C5a is a peptide segment of about 8-10kD at the N-terminal of the α chain of C5, which is an important mediator and chemokine of inflammatory response. C5a activates the corresponding signaling pathway by bindi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46A61K38/17A61P31/04A61P31/12
CPCA61K38/00C07K14/461
Inventor 李墨非孙黎
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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