The process of secreting SOD outside the cell, such as solid culture of Penicillium, Mucor or Aspergillus, can make SOD secrete outside the cell and obtain SOD. In genetic engineering, foreign countries have also made a lot of progress. For example, in Japan, the suspension culture of genetically engineered bacteria containing SOD recombinant DNA was treated by ultrasonic wave in a buffer containing copper salt and zinc salt for a period of time to obtain Cu, Zn-SOD, while in the United States, the production of SOD by genetic engineering technology was more advanced.
2. Domestic research status
At present, the research on the crude separation and purification process of SOD in China is getting deeper and deeper, but its application is far less than that in foreign countries. Many kinds of medicinal SOD have been used in clinic abroad, and the application of SOD in China is mostly concentrated in the field of food and health care products. For example, Jaco -8 and others used bovine blood as raw material and purified it through a series of steps such as precipitation, thermal transformation, reprecipitation and DEAE-SephadexA-50 column. The specific activity of SOD in bovine blood increased from 65438 0 834 U/mg to 2325U/mg. Cui Maoyao 9 and others used shrimp as raw material to purify SOD by thermal denaturation, ammonium sulfate salting out and layered purification. Cheryl L. Fattman 10 et al. purified the crude enzyme solution of SOD in rat lung cells by agarose chromatography. Zhang Shuwen 1 1 etc. The secondary thermal denaturation method is adopted, which saves the use of organic solvents such as chloroform. Sun Yongjun 12, etc. Using garlic (plant) as raw material, SOD crude enzyme solution was prepared by phosphate buffer extraction and thermal denaturation. In terms of genetic engineering, China Siping Institute of Science and Technology, after years of efforts, has developed the SOD project, which is to extract SOD gene from human body. After PCR amplification, human cDNA was constructed, and then plasmid was constructed. Then the plasmid was transferred to the recipient cell, where it was expressed in large quantities. This hospital is the only manufacturer of recombinant humanized SOD in China. There is still a certain gap in the research on the production of medicinal SOD by bioengineering and genetic engineering at home and abroad.
Third, the research significance
In the existing SOD purification process, the rough classification process is an inevitable link, in which a large number of organic solvents (such as ethanol, chloroform, etc. ) or inorganic salts (such as ammonium sulfate, phosphate, etc. ) must be used to remove most impurities by fractional precipitation of organic solvents and inorganic salts to obtain crude SOD. Then we can use acetone precipitation, ultrafiltration concentration, dialysis, column chromatography and other processes for deep purification. First of all, after the target protein is extracted by fractional precipitation, the waste liquid often contains high concentration of organic solvents or inorganic salts, which is very difficult to recover, and once discharged, it will cause great pressure on environmental protection; Secondly, the fractional precipitation method of organic solvents and inorganic salts has a long operation cycle, and the extraction solution is often left standing at low temperature overnight; Besides, in order to make protein sink.