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Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system

An in vitro expression and protein technology, which is applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, etc., to achieve the effect of separation and purification

Inactive Publication Date: 2009-05-06
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the use of silkworm middle silk gland tissue extracts for in vitro expression

Method used

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  • Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system
  • Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system
  • Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0037] 1. Preparation of silk glands of Bombyx mori

[0038] Take the fourth or fifth instar silkworm, immerse the silkworm in 75% ethanol and anesthetize it for 30 seconds, then take out the silkworm, fix the head and tail of the silkworm on the wax plate with a pin, and cut the abdomen with dissecting scissors. The skin is spread left and right and secured with pins. Hold the rectum with an anatomical clip, lift it up toward the head, and cut the trachea and muscles that are clinging to it. After the digestive tract was removed, the silk glands on the left and right were exposed. The silk glands were taken out with tweezers and placed in 0.9℅ normal saline or PBS. The anterior and posterior silk glands were then separated with scissors. -70°C for future use.

[0039]2. Preparation of the extract from the middle silk glands of silkworm

[0040] Take the middle silk glands of 10 silkworms, place them in a homogenizer, add 1 ml of normal saline or PBS, and homogenize them o...

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PUM

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Abstract

The invention discloses a method for synthesizing protein by using an in-vitro expression system of middle silk glands of silkworms. The method comprises the following steps: adding any one of pUC19-Serl-PolyA recombinant plasmids or pUC19-Serl-GFP-PolyA recombinant plasmids or recombinant plasmids inserted with exogenous genes into tissue extract of the middle silk glands of the silkworms; adding a protease inhibitor into the mixture; and treating the mixture in water bath at a temperature of between 25 and 29 DEG C, so as to obtain expresses of target protein. The method has the following advantages: the protein is expressed under a low temperature condition of between 25 and 29 DEG C, so the method is favorable for generating expression protein in a dissolving state and favorable for separation and purification of the protein. At the same time, the method is not limited by cells, and can synthesize the protein in a short time.

Description

technical field [0001] The invention relates to a method for synthesizing protein, which utilizes an in vitro expression system of the silk gland in the middle of the silkworm. Background technique [0002] At present, gene recombination technology has been widely used and expression systems such as yeast live cells and insect cells (hereinafter often referred to as "cell systems") have been widely used as the production method of proteins. However, living cells show a tendency for exogenous proteins to disappear due to the preservation of their functions, and there are many proteins that cannot be easily expressed because the expression of cytotoxic proteins in living cells prevents cell growth. The in vitro expression system is relatively free of the above-mentioned limitations on protein synthesis in cellular systems, and can synthesize proteins without harming the organism. In addition, since the production of protein is not accompanied by operations such as culturing, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/66
Inventor 张耀洲陈健吕正兵陈琴聂作明盛清夏佳音
Owner ZHEJIANG SCI-TECH UNIV
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