Silkworm C-type agglutinin recombination expression method and application thereof

A lectin and silkworm technology applied in the field of bioengineering to reduce the generation of drug-resistant bacteria, reduce the chance of infection, and reduce the fatality rate

Inactive Publication Date: 2010-10-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far there is no research report on the recombinant expression of silkworm lectin and its use in the prevention and treatment of infectious diseases in silkworms

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Design a 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: 5'-TACTCA GAATTC ATGAAAGCGGCACTGGCCAGC-3' (EcoR I); LectXho: 5'-TACTCA CTCGAG TTCATTTGTCGTCGTGCTCTC-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 liquid wa...

Embodiment 2

[0032] Spray the recombinantly expressed silkworm C-type lectin on mulberry leaves or add it to the sterilized feed of artificially cultured silkworms cooled to 37° C., 1 mg / L, to prevent and control the infection of silkworms by microorganisms. Experiments have found that the method can reduce the occurrence of silkworm infectious diseases caused by pathogenic microorganisms in the environment, such as green mold infection and Bacillus thuringiensis infection, and the incidence rate is reduced by 60%.

Embodiment 3

[0034] On the basis of Examples 1 and 2, the gene is mutated to obtain an expression product, which has the same effect of preventing and treating microbial infection. For example: 1) Mutate the third base of any codon encoding an amino acid in the gene without changing the amino acid to obtain a recombinantly expressed silkworm C-type lectin with the same amino acid sequence, such as tgg, cag, gag, gct , cga (respectively encoding W, Q, E, A, R amino acids), mutated into tgg, caa, gaa, gca, cgg (still encoding W, Q, E, A, R amino acids respectively), other sequences in the gene also So; 2) Deleting part of the sequence in the gene to obtain recombinantly expressed silkworm C-type lectin with the same effect, such as deleting the 1-78 bases in the gene coding frame, or deleting the 473-503 bases , or delete bases 926-1010 to obtain recombinantly expressed silkworm C-type lectin with the same efficacy; 3) mutate the amino acid in the gene-encoded protein to obtain recombinantly...

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Abstract

The invention belongs to the bioengineering technical field and provides a biomedicine for expressing silkworm C-type agglutinin by gene recombination, used for expressing silkworm C-type agglutinin recombinant gene into bacillus coli. The invention also provides a method for expressing silkworm C-type agglutinin by gene recombination. The agglutinin of the invention can reduce lethality of disease source organisms to silkworm, increase phagolysis efficiency of blood cell to disease source organisms and reduce survival quantity of disease source organisms in polypide. No drug-fast bacteria is produced, no adverse effect is produced on normal vital movement of silkworm, antibiotic dosage can be reduced in silkworm feeding by using the medicine, and generation of drug-fast bacteria in the environment can be prevented.

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] Bombyx mori is an important breeding insect for the silk industry, an important traditional industry in China, due to its ability to produce silk. The silkworm suffers from various pathogenic biological hazards in the breeding process, causing serious economic losses to sericulture production, and there is an urgent need for efficient and harmless drugs to prevent and control diseases. At present, antibiotics are mainly used to prevent and control microbial infection diseases in silkworm breeding. The result of using antibiotics will lead to the production of drug-resistant bacteria, which will also affect the growth of silkworms....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/435A23K1/16
Inventor 赵小凡宋凯王金星
Owner SHANDONG UNIV
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