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System and method realizing overexpression of target protein

A target protein and overexpression technology, applied in the field of DNA recombination technology, can solve the problems of time-consuming and labor-intensive, and achieve the effect of shortening time, increasing production and improving production

Inactive Publication Date: 2018-10-16
胡育诚
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Problems solved by technology

At present, the CHO cell gene amplification expression system is often used to produce target proteins. Previous literature reports pointed out that when the CHO cell gene amplification expression system is used for gene amplification, if the pressure of drug screening is gradually increased, the number of sets of target gene amplification can be increased. However, due to the need to gradually increase the drug concentration, it is very time-consuming and laborious, and it often takes more than several months to screen high-yield cell lines

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  • System and method realizing overexpression of target protein
  • System and method realizing overexpression of target protein
  • System and method realizing overexpression of target protein

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Embodiment Construction

[0032] "CRISPRi" described in this specification refers to the CRISPR interference (CRISPRi) system, which is the second type of CRISPR / Cas9 system that will be modified from Streptococcus pyogenes - modifying the Cas9 protein so that it loses the nucleic acid Dicer activity (RuvC1 and HNH), known as dCas9 (Cas9 D10A and H841A), works on the same principle by binding to the specified gene sequence through the induction of gRNA or crRNA-trancrRNA complex, but it will not cut the target gene , so it can be used to block RNA polymerase from gene transcription, thereby inhibiting gene expression.

[0033] The present invention is further illustrated with the following specific examples, so that those skilled in the art can fully utilize and practice the present invention without excessive interpretation, and these examples should not be regarded as limiting the scope of the present invention. However, the materials and methods used to illustrate how to practice the invention.

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Abstract

The invention provides a system realizing overexpression of target protein. The system contains dihydrofolate reductase deficiency type CHO cells, a folic acid antagonist, a target protein expressionvector and a CRISPRi expression vector, wherein the target protein expression vector comprises a target protein expression box and a DHFR expression box, and the CRISPRi expression vector comprises agRNA box and a dCas9 expression box. With the system realizing overexpression of target protein, the yield of target protein can be obviously improved, and the time for gene amplification is shortened.

Description

technical field [0001] The invention relates to a DNA recombination technology, in particular to a DNA recombination technology using vectors to introduce foreign genetic materials. Background technique [0002] Since various proteins in an organism control the physiological state, many diseases are associated with the deficiency of certain proteins. Protein drugs are macromolecular drugs that can be defined as formulated proteins administered in vitro for the treatment of human diseases. Protein drug raw materials are mainly natural biological materials, including human body, animals, plants and microorganisms, etc., because of their low toxicity and compatibility with human body, they are the current development trend of new drugs and one of the important projects for the development of my country's biological preparation industry. [0003] In the past, the main sources of protein drugs were extracted from human (blood or urine) or animal organs (such as pancreas). This ...

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N9/003C12N15/85C12N2800/80C12N2810/10C12Y105/01003
Inventor 胡育诚沈志哲
Owner 胡育诚
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