Method for preparing gene serial number of lysozyme of tussah, and expression production

A technology of lysozyme and base sequence, which is applied in the fields of genetic engineering, molecular biology and enzymology, can solve the problems of reports on the preparation of expression products without genetic engineering, application limitations, lack of in-depth research on the gene sequence of tussah silkworm lysozyme, etc.

Inactive Publication Date: 2009-01-14
辽宁省农业科学院大连生物技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitation of raw material sources and complex production process, its application is limited
In particular, there is no in-depth study on the gene sequence of tussah silkworm lysozyme, and there is no report on the preparation of its genetic engineering expression product.

Method used

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  • Method for preparing gene serial number of lysozyme of tussah, and expression production
  • Method for preparing gene serial number of lysozyme of tussah, and expression production
  • Method for preparing gene serial number of lysozyme of tussah, and expression production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Cloning and sequence analysis of tussah silkworm lysozyme gene

[0024] (1) Escherichia coli (E.coli, K12) was used to induce tussah silkworm chrysalis, and after 2 days, the fat body of tussah silkworm chrysalis was taken, and the reagent Reagent (GIBCOLBRL) was used to extract total RNA. With RLM-RACE (FirstChoice TM RLM-RACE Kit, Ambion) method to synthesize 3'RLM-RACE cDNA and 5'RLM-RACE cDNA respectively. According to the known sequences of lysozyme genes of different biological species, degenerate primers were designed, and the sequences were as follows:

[0025] Ply0: 5'-TTCCAGATCAAC G(A) ACA A(G)A(G) TA T(C) TGGTG-3'

[0026] Using the 3' RLM-RACE cDNA synthesized above as a template, the first PCR amplification was performed with primer Ply0 and the 3' RACE Outer primer in the kit. The reaction conditions are: 94°C, 3 minutes; 35 cycles of 94°C, 30 seconds, 55°C, 30 seconds, 72°C, 1 minute; 72°C, 7 minutes.

[0027] Take 1 μl of the firs...

Embodiment 2

[0034] Example 2: Construction and transformation of tussah silkworm lysozyme gene yeast expression vector

[0035] According to the sequence of tussah silkworm lysozyme gene, the following two primers were designed.

[0036] Ply-pic2: 5'-GCTCTCGAGAAAAGAAAATGGTTTACCAAATGTGG-3'

[0037] Ply3': 5'-CTCGAATTCTTAACAGTCGCTAATGTCTG-3'

[0038] Through PCR amplification, the mature peptide (120 amino acids) gene fragment of the tussah silkworm lysozyme coding region is obtained. This gene fragment was digested with restriction endonucleases Xho I and EcoR I, and connected with the yeast expression vector pPIC9K digested with the same enzymes (see appendix Figure 4 ). Screen recombinant plasmids.

[0039] Plasmid DNA was extracted, and the plasmid DNA was digested with Bgl II. Transform the plasmid DNA into yeast GS115 by electroshock method (if the plasmid DNA is digested with Sal I, the plasmid DNA can be transformed into yeast KM71), spread it on an MD solid culture plate, and...

Embodiment 3

[0040] Embodiment 3: Purification of recombinant tussah silkworm lysozyme

[0041] Yeast strains containing tussah lysozyme gene were cultured in BMGY liquid medium at 30°C in shake flasks at 250 rpm. When the OD value is around 9.0, replace the culture medium and use 1 / 10 volume of BMMY medium of the original culture to continue the culture. Methanol was added every 24 hours to maintain the methanol concentration at 0.5%. The culture was terminated after 100 hours, and the culture supernatant was collected for purification and preparation of recombinant tussah silkworm lysozyme.

[0042] Use an ultrafilter with a cut-off molecular weight of 30KD to ultrafilter the supernatant, and the concentrated solution to pass through a CM Sepharose-CL 6B ion-exchange chromatography column. Wash the chromatography column with 50mM sodium phosphate buffer (pH7.2) until no protein can be detected. Continuously elute with 50mM sodium phosphate buffer (pH7.2) / 0.5M sodium chloride solution,...

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Abstract

This invention discloses a method for cloning Antheraea pernyi lysozyme gene and producing its expression product. This coding region of Antheraea pernyi lysozyme gene contains 420 base pairs, while the coding product (with predicted molecular weight of 13986, and isoelectric point of 8.46) contains 140 amino acid residues that include a signal peptide sequence (for 20 amino acid residues) and a mature peptide sequence (120 amino acid residues). The expression product is produced by inserting the gene fragment of the mature peptide into a yeast expression vector, transforming, screening, culturing, separating and purifying with chromatography.

Description

technical field [0001] The invention relates to the technical fields of molecular biology, enzymology and genetic engineering. In particular, the gene sequence of tussah lysozyme and the method for expressing and preparing mature tussah lysozyme polypeptide by using genetic engineering technology. Background technique [0002] Lysozyme (Lysonzyme, EC 3.2.1.17) was first discovered in egg white by British scientist Alexander Fleming in 1922. It is a glycoside hydrolase widely present in various animals and plants. Lysozyme can bind to the bacterial cell wall and act on the β-1,4 bond between N-acetylglucosamine and N-acetylmuramic acid. The cell wall of Gram-positive bacteria is mainly composed of cytoplasmic wall and phosphate, and its cytoplasmic wall is a glycoprotein composed of heteropolysaccharides and polypeptides, and this heteropolysaccharide is composed of N-acetylmuramic Glucosamine is linked by β-1,4 glycosidic bonds, so lysozyme has a strong killing effect on G...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/36
Inventor 范琦张波王林美李树英叶博
Owner 辽宁省农业科学院大连生物技术研究所
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