Application of IL-4-like recombinant protein of fishes

An interleukin and recombinant protein technology, applied in the field of aquatic biotechnology and aquaculture disease prevention and control, can solve the problems of lack of function, unclear immune regulation mechanism, low homology, etc., to improve the survival rate, reduce toxic and side effects, The effect of enhancing the ability to fight pathogens

Inactive Publication Date: 2012-07-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to their low homology (11-15%) with the IL-4 sequence of higher species, and the lack of functional research, the corresponding immune regulation mechanism is not yet clear, and it cannot be truly confirmed that it is IL-4 (currently referred to as IL-4-like factor only)

Method used

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  • Application of IL-4-like recombinant protein of fishes
  • Application of IL-4-like recombinant protein of fishes
  • Application of IL-4-like recombinant protein of fishes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, method for obtaining zebrafish IL-4-like gene (hereinafter referred to as IL-4-L):

[0027]First, pre-inject zebrafish (scientific name Danio rerio, body length 3-4cm, body weight 3-4g) with Escherichia coli endotoxin LPS (English full name, Sigma, O55: B5E. For the induced expression of L mRNA, the experimental fish injected with LPS were reared at 26-28°C water temperature. After 12 hours, the kidney or spleen tissue of the zebrafish was taken, and the total RNA was extracted with TRizol (Invetigen). The extraction method was carried out according to the instructions. Then reverse transcription and PCR amplification were performed with the reverse transcription kit RNA PCR kit (AMV) Ver3.0 (TaKaRa). The primer sequence is F15'-GAAGACACTTCTGCTGCTCGC-3', R1 5'-CAAATTGTCT TCAGGTAGAAC-3', which were synthesized by Shanghai Yingjun Company. The optimized PCR conditions were: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 40 seconds, anneali...

Embodiment 2

[0029] Embodiment 2, the method for obtaining the prokaryotic expression plasmid of zebrafish IL-4-L gene:

[0030] First, BamH I and Xhol I restriction sites were introduced at both ends of the upstream and downstream primers of the IL-4-L gene, and the redesigned sequences of the two primers were F2 5'-CTAGCTAGCGGATCCGGGATGGAACATAAGACTGTATTAAAGG-3', R2 5'-CCGCTCGAGTCACTTGTCGTCGTCGTCCTTGTAGTCACTTGGTCTTGGGCTTTT- 3', the primer was synthesized by Shanghai Yingjun Company. Then, using the T-IL-4-L recombinant plasmid as a template and F2 and R2 as primers, the cDNA of IL-4-L was amplified according to the following PCR cycle program: pre-denaturation at 94°C for 4min, denaturation at 94°C for 40s, annealing at 52°C for 1min , 72°C for 30s, 72°C for 10min, a total of 30 cycles. PCR products were identified by 1.2% agarose gel electrophoresis. Then the PCR product was recovered. The specific method was carried out according to the operation manual provided by Axygen. After doubl...

Embodiment 3

[0031] Embodiment 3, the acquisition of soluble recombinant IL-4-L protein:

[0032] Inoculate PET41a-IL-4-L positive clones in 5ml of LB liquid medium containing ampicillin (50μg / ml), place on a shaker at 37°C, and culture at 250rpm for 5 hours, and then culture the culture at a ratio of 1:100 Add LB liquid medium containing ampicillin (50 μg / ml), place on a shaker at 37° C., and continue culturing at 250 rpm. Determination of bacterial growth curve, wait for OD 600 When the temperature reaches 0.6-1.0, IPTG is added to make the final concentration 0.1-1 mM, and then transferred to a low-temperature incubator at 20° C. at 100 rpm overnight to induce the expression of soluble recombinant protein. Then the bacteria were collected by centrifugation (5000×g, 15 min), the supernatant was discarded, after the bacteria were resuspended with an appropriate amount of PBS, the bacteria were disrupted by ultrasonic waves until the solution became translucent. The supernatant and bacte...

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Abstract

The invention discloses an application of IL-4-like recombinant protein of fishes, which is used for preparing an immune reinforcing agent capable of improving the immune ability of fishes. The IL-4-like recombinant protein of fishes has an amino acid sequence shown as SEQ ID NO: 2. The IL-4-like recombinant protein of fishes can be also used for preparing a preparation for preventing diseases of fishes, for example a preparation for preventing hemorrhagic septicemia of the fishes, and the preparation comprises aeromonas hydrophila whole cell vaccine and the IL-4-like recombinant protein of the fishes, which has the amino acid sequence shown as SEQ ID NO: 2.

Description

technical field [0001] The invention relates to the fields of aquatic biology technology and aquaculture disease prevention and control. In particular, it relates to the new use of a fish interleukin-4-like (IL-4-like, IL-4-L) cytokine expressed recombinantly by molecular biology technology as an immune enhancer in the prevention and treatment of fish diseases. Background technique [0002] With the rapid development of fishery production, aquatic animal diseases have become increasingly prominent, and have become a worldwide problem that seriously affects the healthy and sustainable development of aquaculture, especially in my country. In recent years, the direct economic loss of aquaculture industry caused by diseases in my country has exceeded 10 billion yuan per year, and it has been increasing year by year. But for a long time, there has been a lack of effective disease control methods; the abuse of various antibiotics and pesticides has caused serious pollution to the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K39/02A61K39/39A61P37/04A61P7/00A61P31/04
Inventor 邵建忠项黎新潘萍萍董伟仁朱律韵方玮
Owner ZHEJIANG UNIV
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