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Method for synthesizing protein using expression system in vitro of bombyx mori posterior division of silkgland

A posterior silk gland and in vitro expression technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of preventing the disappearance of proteins and foreign proteins, reduce the formation of inclusion bodies, and facilitate separation and purification Effect

Inactive Publication Date: 2009-02-04
ZHEJIANG SCI-TECH UNIV
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Problems solved by technology

However, living cells show a tendency for exogenous proteins to disappear due to their function retention, and there are many proteins that cannot be easily expressed because the expression of cytotoxic proteins in living cells prevents cell growth

Method used

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  • Method for synthesizing protein using expression system in vitro of bombyx mori posterior division of silkgland
  • Method for synthesizing protein using expression system in vitro of bombyx mori posterior division of silkgland
  • Method for synthesizing protein using expression system in vitro of bombyx mori posterior division of silkgland

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Embodiment

[0042] 1. Silkworm silk gland preparation

[0043] Take the fourth or fifth instar silkworm, soak the silkworm in 75% ethanol for 30 seconds and anesthetize it, take it out, fix the head and tail of the silkworm on the wax plate with a pin, and cut the abdomen with dissecting scissors. Spread the skin side to side and secure with pins. Hold the rectum with an anatomical clip, lift it toward the head, and cut off the attached trachea and muscles. After the alimentary canal was removed, the left and right silk glands were exposed, and the silk glands were taken out with tweezers and placed in 0.9% normal saline or PBS. The anterior and posterior silk glands were subsequently separated with scissors. Store at -70°C for later use.

[0044] 2. Preparation of Bombyx mori posterior silk gland extract

[0045] Take the posterior silk glands of 10 silkworms, place them in a homogenizer, add 1ml of normal saline or PBS, and homogenize on ice; draw the homogenate into a 1.5ml EP tube...

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Abstract

The present invention discloses a method which utilizes the in-vitro expression system of the posterior silkgland of Bombyx mori to synthesize a protein. pUC19-FL-GFP-PolyA recombinant plasmid or pUC19-FL-GFP-PolyA recombinant plasmid with an exogenous gene or a pUC19-FL-PolyA recombinant plasmid or a pUC19-FL-PolyA recombinant plasmid with an exogenous gene and protease inhibitor are added into the tissue extract of the posterior silkgland of Bombyx mori, and after water bathing under the temperature between 25 DEG C and 29 DEG C, the expression of the targeted protein is obtained. The method has the following advantages: since the protein is expressed under a temperature between 25 DEG C and 29 DEG C, the low-temperature condition (normally, 37 DEG C) helps to generate the expressed protein in a dissolved state, and the formation of inclusion bodies is reduced, which is favorable for the separation and purification of the protein. Meanwhile, the method is not limited by cells and can synthesize the protein in a short period.

Description

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

Claims

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Application Information

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