Fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and application thereof

A technology for anti-lipopolysaccharide factor and penaeus prawn, applied in the field of genetic engineering, can solve the problems of mutated pathogenic microorganisms, increased resistance of pathogenic organisms, lack of knowledge about drug use by farmers, and achieves strong growth inhibitory effect and obvious sterilization. effect, significant growth inhibitory effect

Inactive Publication Date: 2014-04-16
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The weak foundation of my country's fishery medicine industry, the lagging technology of aquaculture biological disease prevention and control, and the lack of farmers' knowledge of medicines have led most farmers to use low-cost pesticides and chemical raw materials with serious residues, resulting in serious water pollution.
In addition, the application of antibiotics, hormones and other drugs in the aquaculture process has played a certain role in disease control, but it has largely destroyed the micro-ecological balance of the water environment and animals, causing aquatic animals to lose their normal health. Bacteria barrier and biological antagonism, which in turn lead to the proliferation of exogenous effector bacteria and endogenous pathogenic bacteria, resulting in potentially larger diseases
Abuse of drugs will lead to drug resistance of pathogenic strains, and mutation of pathogenic microorganisms that are more pathogenic, more harmful, and more prevalent. Long-term use of chemicals will inevitably lead to increased resistance of pathogenic organisms

Method used

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  • Fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and application thereof
  • Fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and application thereof
  • Fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Chinese prawn anti-lipopolysaccharide factor mature peptide FcALF, as shown in the amino acid sequence of SEQ ID NO.1,

[0036] QGWEAVAAAVAVKIVGLWRNEKTELLGHECKFTVKPYIKRFQLYYKGRMWCPGWTAIRGEAKTRSRSGVAGRTAKDFVRKAFQQGLISQQQANQWLNS

[0037] The nucleotide sequence of the gene is shown as SEQ No.2, and its 5' end and 3' end have start codon (ATG) and stop codon (TAG) respectively.

[0038] ATGCGAGTTTCCGTGTTGGCAAGCCTGGTGCTGGTGGTGTCCCTGGTGGCACTCTTCGCCCCGCAGTGCCAGGCTCAAGGGTGGGAGGCTGTGGCAGCGGCCGTCGCCGTCAAGATTGTTGGGCTGTGGAGGAACGAGAAAACCGAACTCCTCGGCCACGAGTGCAAGTTCACCGTCAAGCCTTACATTAAGAGGTTCCAGTTGTACTACAAGGGGAGGATGTGGTGCCCAGGCTGGACGGCCATCAGAGGAGAAGCCAAAACACGCAGTCGGTCCGGGGTGGCTGGAAGGACAGCCAAAGACTTCGTCCGGAAAGCTTTCCAGCAAGGTCTCATCTCTCAACAGCAGGCTAACCAGTGGCTTAACTCATAG

[0039] (a) Sequential features:

[0040] ●Length: 372bp, of which the effective length is 76-369bp

[0041] ●Type: base sequence

[0042] ●Chain type: single chain

[0043] ●Topological structure: linear

[0044] (b) Mo...

Embodiment 2

[0050] 1) Construction of the recombinant expression vector of Chinese prawn anti-lipopolysaccharide factor FcALF

[0051] Based on the sequence information of the mature peptide encoded by the cDNA sequence of ALF of Penaeus prawn (NCBI sequence registration number: AY859500) and the characteristics of the multiple cloning site of the secreted expression vector pPIC9k, primers were designed, and the upstream primer PRI-A1 contained an EcoR I restriction site ; Downstream primer PRI-A2 contains Not I restriction site.

[0052] PRI-A1:

[0053] GC GAATT CCAAGGGTGGGAGGCTGTGGCA (underlined is the EcoR I restriction site)

[0054] PRI-A2:

[0055] TA G+GGCCGC CTATGAGTTAAGCCACTGG (the underline is the Not I restriction site)

[0056] The PCR reaction system is 20ul, as follows:

[0057] Ex Taq buffer 2μl

[0058] dNTPs (10mM) 0.4μl

[0059] PRI-A1 (10μM) 0.4μl

[0060] PRI-A2 (10μM) 0.4μl

[0061] Ex Taq 0.2μl

[0062] 1 μl of blood cell cDNA of Penaeus chinensis

[0...

Embodiment 3

[0082] Example 3 Antibacterial Activity Identification of Recombinant Protein rFcALF

[0083] (1) Detection of inhibition zone by Oxford cup method

[0084] The cell concentration was 10 8 cell / mL of Escherichia coli (E.coli) and Staphylococcus aureus (Staphylococcus aureus) were uniformly coated on the LB solid plate, and placed in a 37°C incubator for 30-60min. After the surface of the plate was completely dry, place the Oxford cup, and then 200 μL of the above purified 50 mg / L recombinant protein solution was added thereto, and the same volume of 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7.2 was used as a negative control. Then place it in a 37°C incubator for 16-18 hours to observe the results, and then measure the size of the inhibition zone with a vernier caliper (see Figure 7 ), the results showed that 50mg / L recombinant FcALF could effectively inhibit the growth of Gram-negative bacteria E.coli.

[0085] (2) Use a microplat...

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Abstract

The invention relates to genetic engineering and particularly to a fenneropenaeus chinensiss anti-lipopolysaccharide factor as well as preparation and an application thereof. A gene of the FcALF (fenneropenaeus chinensiss anti-lipopolysaccharide factor) is (a) protein represented by an amino acid sequence in SEQ ID NO.1 or (b) protein which is derived from (a) through substitution, deletion or addition of one or more amino acids in the amino acid sequence limited by (a) and has the anti-lipopolysaccharide factor activity. According to the factor, pichia pastoris is used as a eukaryon expression system, and recombinant expression is performed on the FcALF. In addition, the antibacterial activity of the FcALF is analyzed, so that guidance is provided for screening of antibacterial agents and immunopotentiators . Specifically, recombinant protein of the FcALF has obvious growth-inhibition and bactericidal effects on Gram-negative bacterium escherichia coli.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a Chinese prawn anti-lipopolysaccharide factor and its preparation and application. Background technique [0002] Entering the 21st century, the aquaculture industry is rapidly developing towards multi-species, high-input, high-density, intensive, and factory-oriented. However, frequent outbreaks of infectious diseases are always threatening the healthy and sustainable development of the industry. At present, the main methods of aquaculture biological disease prevention and control include drug control, immune control and ecological control, and are gradually developing in the direction of high efficiency, low toxicity, no residue and no pollution. However, chemical drugs are still the most direct and effective means for the prevention and treatment of aquatic animal diseases. The weak foundation of my country's fishery medicine industry, the lagging technology of aquaculture biological di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/81A61K38/17A61P31/04A61P37/04A23L1/29A23K1/16C12R1/84A23L33/00
CPCA23K20/147A23V2002/00A61K38/00C07K14/43509C12N15/815A23V2250/542Y02A50/30
Inventor 张继泉桂天书王婧相建海
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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