The Study Of Secretion Characteristic,drug Suspension And Structure Of Extracellular Protein MrpC From Microcystis Aeruginosa PCC 7806

Previous studies in our laboratory found that M aeruginosa PCC 7806 exocrine protein is the main factor affecting the suspension of algae cells.It has nano-long fibrous reticular structure in algae culture.It can suspend many kinds of algae cells,and also suspend starch and agarose.The filamentous structure and suspension function of MrpC protein have wide application prospects in the development and utilization of pharmaceutical excipients.Because MrpC protein is an extracellular secretory protein of M aeruginosa PCC 7806,four problems need to be faced before applying it to the development of pharmaceutical excipients:1.The amount of extracellular secretion of MrpC is low;2.The influence of microcystins on the extraction of MrpC protein;3.The suspension property of MrpC protein as suspension agent;4.The structure details of MrpC protein.The main research content of this paper is to provide a basis and to find solutions for the problems faced before the development of MrpC protein as a pharmaceutical excipient.The main contents and results of this study are as follows:1.To explore the effects of temperature and light intensity on the expression of MrpC,and to obtain suitable growth conditions for secretion of MrpC protein.The results showed that the increase of temperature and light intensity was beneficial to the increase of the concentration of MrpC protein and the secretion rate of MrpC.The longer the culture time was,the faster the secretion rate of MrpC was.2.By measuring the change trend of microcystins in normal culture of M.aeruginosa PCC 7806 in laboratory and the content of microcystins in protein solution obtained by various protein extraction methods,we can guide how to obtain MrpC protein with low microcystins.The results showed that the initial content of microcystins in algae solution was too high when cultured normally,and the content of microcystins in and out of algae cell increased slowly with the prolongation of culture time.The content of microcystins in protein solution obtained by ammonium sulfate precipitation method was the lowest,and the content of microcystins in other extraction methods was similar to that in algae culture supernatant.3.The mcyB gene of M.aeruginosa PCC 7806 was knocked out by CRISPR-Cas9 and homologous recombination technology to obtain the overexpression of MrpC protein and the non-expression of microcystin mutant strain.Homologous recombinant plasmids and CRISPR-Cas9 plasmids were successfully constructed.4.Nine insoluble or poorly soluble in water drugs were selected to test the suspension f'unction of MrpC protein drugs.Three of them can be suspended in the solution of MrpC protein.Four of' them are can not be suspended,but with chromogenic reaction appealing.Two of them can neither be suspended nor coloured.5.The structure of MrpC protein was analyzed by Cryo electron microscope.The results showed that MrpC protein has hollow tubular fibrous structure with multi-voids on the surface and obvious spiral concave-convex structure on the outside and inside.