Expression And Purification Of RhPH-20 In CHO Expression System

Hyaluronidase?HAase?is widely distributed in nature and is a general term for a class of enzymes that can reduce the molecular weight of hyaluronic acid.HAase can temporarily reduce the adhesion of extracellular matrix,enhance the permeability of the membrane,make the intercellular substance easy to flow and spread,and then make the injected local exudate or leakage liquid easy to diffuse and absorb,and also can change some drugs and physiological activities.The distribution of substances in the human body also promotes the spread of some infectious diseases.PH-20 is a multifunctional protein,besides being a kind of HAase,it is also a receptor that can induce HA-induced signal transduction and a receptor that recognizes zona pellucida.Its role in fertilization is:?1?Degrading the matrix surrounding the egg cell;?2?binding to the zona pellucida;?3?acrosome exocytosis mediated by Ca²⁺signaling.HAase industrial production mainly includes animal tissue extraction and microbial fermentation.Most of these methods are complicated in extraction process.The glycosylation modification of HAase is different from that of human cells,affecting biological properties such as protein biological activity and secretion efficiency.Clinically,it has brought great inconvenience.In this experiment,the CHO expression system and the high-efficiency expression vector pSecTag2 A were combined with genetic engineering technology to carry out recombinant expression of PH-20,and high-purity rhPH-20 was prepared,which solved other preparation methods.problem.In this experiment,the human PH-20 gene was optimized and synthesized,and the recombinant plasmid pSecTag2A-PH20 was constructed.The recombinant plasmid was transiently transfected into CHO-S cells,and the small-scale fermentation was successfully carried out by using the existing conditions in the laboratory.The automatic protein fingerprinting quantitative analysis system was applied for detection,and rhPH-20 was successfully obtained.Subsequent separation and purification by affinity chromatography gave high purity rhPH-20 samples,and the concentration of rhPH-20 was quantitatively detected by Bradford method.Using the standard as a reference,PPSQ-51A protein sequencing aligned the starting sequence by amino acid,which proved the correctness of the sequence.The molecular weight of rhPH-20 determined by MALDI-TOF was consistent with the theoretical sequence.The methods for measuring the activity of rhPH-20 reported in the literature include colorimetric method,ELISA-like method,substrate fluorescent labeling method,etc.,but these methods have low sensitivity and high cost,and are not widely used.The turbidity method used in this experiment has good repeatability and high sensitivity,and the activity of rhPH-20 obtained after purification is better.In summary,rhPH-20 can be successfully expressed in CHO cell expression system,and the experimental procedure is more convenient.The upgrading of genetic engineering drug expression systems is imperative.The development of CHO cell expression system related technologies will greatly promote the development of the entire biomedical industry.The expression system will play an increasingly important role in this field.