Study On Freeze-dried Power Injection Of Recombinant Ganoderma Lucidum Immunoregulatory Protein(rlZ-8)

The instability of protein molecules is one of the problems of the protein drug development process.As a result,it is particularly important to develop a pharmaceutical formulation which is stable,long validity and suitable for the enterprise production.At present,water injection and freeze-dried powder injection of two kinds of formulations of protein drugs are widely used in clinical.Water injection is easy to product without the need of using before the complex solution,while the protein need to be saved at low temperature because of bad stability.However,Freeze-dried powder formulations of protein need to be re-dissolved before use with high stability,as well as transport storage convenience.Fungal immunomodulatory protein(Fip)is a kind of small molecule protein extracted from Ganoderma lucidum,mushroom or other fungus.The recombinant Ganoderma Lucidum Immunomodulatory Protein is a fungal immunoregulatory protein extracted from Ganoderma lucidum,which has the ability of blood cell agglutination,promoting lymphocyte proliferation and the secretion of various cytokines,as well as the anti-tumor and elevating white blood cells,etc.In recent years,Fip has attracted the high attention of researchers as its important research significance,meanwhile it has broad prospects for development.In this study,rLZ-8 was expressed recombinantly by Pichia pastoris.However,it was found that rLZ-8 was instability in water solvent,what is more,it was not suitable for preparation of water injection on account of the state of the liquid making trouble in the process of storage and transportation.As a consequence,the research offreeze-dried powder injection is essential.Based on the above problems,firstly this paper constructed rLZ-8 expression strain,then optimized its fermentation conditions and purification technology,accordingly obtained the target protein to meet the requirements.For another,the accessory choosed by the rLZ-8 freeze-dried powder injection with its prescription combination was carried through screening,furthermore,freeze-dry process was optimized by the technical of vacuum frezze-frying.Summary of the research content is as follows:Firstly,The construction,expression and identification of rLZ-8 expression strainThe recombinant plasmid p GAPZαA-rLZ-8 was constructed based on genetic recombination means.Then,the recombinant plasmid was linearized and integrated into the genome of the X33 yeast strain by the method of electrotransfomation.After the authentication of the Yeast positive transformation,express rLZ-8through the shake-flask cultivation.The study ultimately select the bacterial strain which express the highest quantity under the different measurement conditions of rLZ-8 expression by recombination strain.Secondly,construction and optimization of the rLZ-8 fermentation technologyThe study was carried through the exploration of the optimum fermentation working condition and the purification on the pilot scale with the use of the highest quantity of protein expression after identification.After inoculating the bacterial strain which was screened into the fermentation cylinder to cultivate,detect the biomass and monitor the growth state of the yeast,simultaneously detect the rLZ-8expression quantity in the fermentation broth by ELISA.Then,identify the technological parameters of 7.5 L scale fermentation: cultivate temperature was28 ℃,cultivate time was 60 h,PH was 6.0,air flow rate was 2v/v/m,dissolved oxygen control was 25%,and stirring speed was controlled at 800 rpm.It turned out that the rLZ-8 obtained from the optimized fermentation conditions accounts for11.75% in the bacteria secrete with the protein yield of 528.09 mg/L.Meanwhile,the purity after the separation and purification of the rLZ-8 obtained from the fermentation reached 99.98%.Thirdly,the prescription screening of rLZ-8 freeze-dried powder injection and the study of the freeze-drying processAccording to the physical and chemical properties of rLZ-8 and the preliminary investigation,the phosphate buffer system(p H7.0)was selected as the basic buffer system,at the same time,the trehalose,mannitol and lactose as stabilizers,Tween-80 and polo Sham-188 as a surfactant,to carry out the influence factors test of rLZ-8 accessories.It turned out that the accessories of the rLZ-8 made almost no impact on the content,purity and activity which leaded to the feasibility of the follow-up study.The experiment designed 9 prescription combination to the sample investigation of activity,insoluble particle and purity so as to screen the suitable freeze-dried powder prescription for rLZ-8: trehalose 50 mg,mannitol 50 mg,rLZ-810 mg.After the determination of the prescription,selected the different temperature,vacuum degree,freeze-drying time so as to explore the freeze-drying process of rLZ-8 with the execution of the single-factor experiment which took the properties and moisture as the index of evaluation.The suitable freeze-drying process was determined: sublimation drying conditions was-29 ℃,vacuum degree was 250 m Torr,drying time was 90 h,the drying conditions were temperature 40℃,vacuum degree 250 m Torr and drying time 30 h.