Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm

A technology of lysozyme and silkworm, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, animal husbandry, etc., can solve the problems of low benefit, high cost, complicated production process, etc., to improve production efficiency, simplify purification methods, The effect of improving economic efficiency

Inactive Publication Date: 2011-10-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The production process is complicated,

Method used

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  • Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm
  • Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm

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Embodiment 1

[0019] The pBSer1hLYZ-A3EGFP plasmid ( figure 1 ) and a helper plasmid capable of providing transposase ( figure 2 ) were mixed at a ratio of 5:1, the total concentration of the two plasmids was 0.3 μg / μl, the plasmids were dissolved in 0.1 mM phosphate buffer containing 4 mM sodium chloride, and then introduced into silkworms within 8 hours after spawning by microinjection Into the fertilized eggs, the total volume of introduction is 30nl. The microinjected silkworm eggs were reared to adults at 23°C, 75% humidity, and 8 hours of light, and selfed for successive generations (G1 generation). At the G1 generation silkworm stage of the transgenic experiment, the transgenic silkworm moth 1 expressing the EGFP marker gene was observed and obtained by a fluorescence microscope (Olympus, SZX12, Japan). . The silkworms were reared until the adult and sericin silkworms were crossed to lay eggs for the next generation (G2). The transgenic silkworms after the G2 generation were all...

Embodiment 2

[0022] The pBSer1hLYZ-A3EGFP plasmid ( figure 1 ) and a helper plasmid capable of providing transposase ( figure 2 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 1 μg / μl, the plasmids were dissolved in 2 mM phosphate buffer containing 1 mM sodium chloride, and then introduced into silkworm fertilization within 6 hours after egg laying by microinjection In ovo, the total volume of introduction was 5nl. The microinjected silkworm eggs were reared to adults at 28°C, 90% humidity, and 12 hours of light, and the transgenic silkworms were self-bred for successive generations (G1 generation). At the G1 generation silkworm stage of the transgenic experiment, the transgenic silkworm moth 1 expressing the EGFP marker gene was observed by a fluorescence microscope (Olympus, SZX12, Japan). . The silkworms were reared until the adult and sericin silkworms were crossed to lay eggs for the next generation (G2). The transgenic silkworms after the G2 gener...

Embodiment 3

[0025] The pBSer1hLYZ-A3EGFP plasmid ( figure 1 ) and a helper plasmid capable of providing transposase ( figure 2) were mixed at a 2:1 ratio, the total concentration of the two plasmids was 0.5 μg / μl, the plasmids were dissolved in 1 mM phosphate buffer containing 2 mM sodium chloride, and then introduced into silkworms within 4 hours after spawning by microinjection In fertilized eggs, the total volume of introduction is 15nl. The microinjected silkworm eggs were reared to adults at 25°C, 80% humidity, and 12 hours of light, and then mated with non-transgenic silkworms (G1 generation). At the G1 generation silkworm stage of the transgenic experiment, the transgenic silkworm moth 1 expressing the EGFP marker gene was observed by a fluorescence microscope (Olympus, SZX12, Japan). . The silkworms were reared until the adult and sericin silkworms were crossed to lay eggs for the next generation (G2). The transgenic silkworms after the G2 generation were all bred by single m...

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Abstract

The invention discloses a method for synthetizing secretion lysozyme by middle silkgland cell of silkworm. The method comprises the following steps: building a pBSer1hLYZ (lysozyme)-A3EGFP (Enhanced Green Fluorescent Protein) plasmid for synthetizing secretion lysozyme by the silkworm; then, introducing the plasmid and an assistant plasmid capable of providing transposase into a silkworm germ cell according to the microinjection transgenosis silkworm technology; according to the transposition characteristics of a piggyBac transposon, introducing a green fluorescent protein gene and a lysozymegene into a silkworm gene group, to obtain stable heredity and expression so as to create the transgenosis silkworm capable of synthetizing secretion lysozyme by middle silkgland cell of silkworm by specificity; further, hybridizing the transgenosis silkworm with sericin silkworm; carrying out back crossing on the hybridized descendant with the sericin silkworm for 3-5 generations; and finally, carrying out selfing on the obtained product to carry out homozygosis on the lysozyme gene so as to obtain the new species of the transgenosis silkworm of the secretion lysozyme. According to the method, a basis for improving lysozyme production efficiency and lowering production cost is laid.

Description

technical field [0001] The invention relates to a method for synthesizing and secreting lysozyme by using transgenic technology to construct a silk gland cell in the middle of silkworm. Background technique [0002] Lysozyme (Lysozyme), also known as muramidase, has a molecule composed of 129 amino acids, a molecular weight of about 15 KDa, and an isoelectric point of about 10.8. Lysozyme is widely found in the egg whites of poultry and birds, in the tears, saliva, heart, lung, liver, kidney and other fluids and organs of mammals, and in plants such as barley, turnip, and fig. Among them, egg white has the highest content, about 0.3%, while human milk, tears and saliva have the highest activity, which is 3 times higher than the enzyme activity in egg white. [0003] Lysozyme is a hydrolase that specifically acts on the cell wall of microorganisms, it destroys the cell wall of microorganisms, causes bacterial lysis, and has antiseptic and bactericidal effects. Human and ani...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/04
Inventor 钟伯雄危浩庄兰芳
Owner ZHEJIANG UNIV
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