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Recombinant expression method of insect C-type lectin and use

A lectin, insect technology, applied in the field of bioengineering, to achieve the effect of increasing phagocytosis, reducing the number of survival, and reducing the production of drug-resistant bacteria

Inactive Publication Date: 2009-05-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been reports on the cloning and functional studies of lectins from silkworms and other insects, so far there are no reports on the recombinant expression of insect lectins and their use in the prevention and treatment of infectious diseases

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Design 1 pair of primers with restriction sites to amplify the C-type lectin gene mature peptide (84-1013bp), that is, remove the signal peptide part. The primers are as follows: LectEco: 5'-TACTCA GAATTC GCGTTTACATGCGACTAC-3' (EcoR I); LectXho: 5'-TACTCA CTCGAG TTATTCAACTTTGTTATT-3'(Xho I), PCR reaction, the conditions are 94°C for 30sec, 60°C for 45sec, 72°C for 1min, and 72°C for 10min. The PCR fragment and the pET-30a(+) vector were digested with EcoRI and XhoI respectively, and the T 4 DNA ligase connects the purified cDNA fragment with the purified pET-30a(+) vector, transforms the ligated product into E.coli DH5α, screens positive clones, extracts the plasmid, takes 1 μL of the constructed expression plasmid, dilutes it 10 times, and transforms the expression Strain E.coli BL21(DE3) competent cells were cultured in LB / Kan medium, and the expression of the inserted gene was induced by adding IPTG with a final concentration of 0.5mM. The induced bacterial liqui...

Embodiment 2

[0040] Design a pair of primers with restriction sites to amplify rHa-CRD1, the primers are as follows: CRD1Eco: 5'-TACTCA GAATTC TGCGACTACAAATACAGTCTA-3'EcoRI, CDR1Xho5'-TACTCA CTCGAG AAAGCAGATGTAAGGTCTGGG-3'XhoI, the following steps are the same as in Example 1.

Embodiment 3

[0042] Design a pair of primers with restriction sites to amplify rHa-CRD2, the primers are as follows: CRD2Eco5'-TACTCA GAATTC TGTGGTACTCCTGATGATGGA-3'EcoRI, CDR2Xho5'-TACTCA CTCGAG CTCACAAATGAATGGAGCTGG-3'XhoI, the following steps are the same as in Example 1.

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Abstract

The invention belongs to the technical field of bioengineering and provides a biological medicament that has recombinant gene expression of helicoverpa armigera C-type lectin and two carbohydrate recognizing structural domains of Ha-CRD1 and Ha-CRD2 of the gene. The helicoverpa armigera C-type lectin gene and the two carbohydrate recognizing structural domains of Ha-CRD1 and Ha-CRD2 of the gene have recombinant expression in Escherichia coli. The invention further provides a recombinant gene expression method of the helicoverpa armigera C-type lectin and the two carbohydrate recognizing structural domains of Ha-CRD1 and Ha-CRD2 of the gene. The lectin can reduce the insect fatal rate of pathogenic organisms, improve the pathogenic organism phagocytizing efficiency of blood corpuscle, and reduce the survival number of pathogenic organisms in insect bodies. The biological medicament does not generate drug-fast bacteria, does not have adverse effect to the normal vital activity of insects, can reduce the usage of antibiotic, and can reduce the drug-fast bacteria generation in environments.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to DNA recombinant expression technology and application in the technical field of bioengineering, in particular to a creation method and application of recombinant expression of a C-type lectin and its functional domain. Background technique [0002] The animal immune system is divided into two parts, innate and acquired. Innate immunity appeared in the early stage of multicellular organism evolution, not only in vertebrates, invertebrates, but also in plants, while acquired immunity only exists in the vertebrate among animals. Innate immunity is the only way for insects and other invertebrates to resist the invasion and harm of pathogenic organisms. Innate immunity includes two pathways of cellular immunity and humoral immunity. The basic process is: first, foreign bodies are recognized by pattern recognition receptors, which is called non-self recognition of infection, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N15/81C12N15/866C07K14/435A61K38/17A61P31/00A23K1/16A23K20/147
Inventor 赵小凡王金星
Owner SHANDONG UNIV
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