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Human cytoglobin for preventing and curing cirrhosis and preparation method thereof

A globin and cell technology, applied in the field of biochemistry, can solve the problems of no biological activity, difficulty in ensuring the stability and uniformity of production batches of proteins, etc.

Inactive Publication Date: 2009-11-18
SOUTHERN MEDICAL UNIVERSITY
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Problems solved by technology

However, the rh STAP obtained by the above method is expressed in the form of inclusion bodies, which are misfolded, biologically inactive, and precipitated proteins that may only have partial biological activity even after renaturation. Even without biological activity, it is difficult to ensure the stability and uniformity of each production batch of protein

Method used

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  • Human cytoglobin for preventing and curing cirrhosis and preparation method thereof
  • Human cytoglobin for preventing and curing cirrhosis and preparation method thereof
  • Human cytoglobin for preventing and curing cirrhosis and preparation method thereof

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Embodiment Construction

[0049] In order to better understand the present invention, the following will further illustrate the technical effects of the present invention through specific preparation examples and effect experiments.

[0050] 1. Construction of the expression plasmid of human cytoglobin of the present invention

[0051] (1) Extract the total RNA of Hep G2 cells, and use primer 1 and primer 2 to carry out reverse transcription PCR to obtain a DNA fragment whose sequence is SEQ NO.2;

[0052] The reaction conditions for reverse transcription PCR are as follows: 94°C for 1 minute; 58°C for 1 minute; 72°C for 1 minute; 30 cycles.

[0053] (2) using the DNA fragment whose sequence is SEQ NO.2 as a template to carry out PCR reaction with primer 2 and primer 4 to obtain the DNA fragment whose sequence is SEQ NO.3;

[0054] The PCR reaction conditions are as follows: 94° C. for 1 minute; 58° C. for 1 minute; 72° C. for 1 minute; 30 cycles.

[0055] (3) using the DNA fragment whose sequence is...

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Abstract

The invention provides an expression plasmid of human cytoglobin, the expression plasmid is obtained by the process that firstly the enzyme digestion of the DNA segment shown in SEQ NO.1 with Nco I and Hind III is carried out and then the product of the enzyme digestion is cloned in the representation area of a carrier pET-32a. The expression plasmid of human cytoglobin provided by the invention can be used to express fusion protein in a pronuclei host, the fusion protein is composed successively of Trx tag, His tag, FXa recognition sequence and human cytoglobin which are in connection with each other, clotting factor FXa is used for cutting Trx tag, His tag, FXa recognition sequence by the method of enzyme digestion to obtain recombinant human cytoglobin. The recombinant human cytoglobin has the same biological activity of natural human cytoglobin and can cure and prevent cirrhosis with a significant effect.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a recombinant expression of a eukaryotic protein used for preventing and treating liver cirrhosis. Background technique [0002] my country is a big country with liver disease. According to the statistics of the Liver Disease Research Group of the Ministry of Health in 2002, the number of hepatitis B infection clinic visits in the country was 75 million, ranking first among all kinds of infectious diseases in the country. At the same time, the incidence of other liver diseases such as fatty liver, alcoholic liver, drug liver and cirrhosis increased by 17.8% year-on-year, reaching 50 million, which has become another serious social problem that threatens the life and health of our people. About 25% of chronic hepatitis will turn into liver cirrhosis after 5 to 10 years, and about 20% of liver cirrhosis will turn into primary liver cancer. Another data statistics show that 60% of alcoh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C07K14/435C12P21/02A61P1/16C12R1/19
Inventor 魏威董文其
Owner SOUTHERN MEDICAL UNIVERSITY
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