Research Of Expression And Fermentation Of Subtilisin QK In Pichia Pastoris And Development And Application Of A Sandwish Enzyme-linked Immunosorbent Assay For Detection Of The Recombinant QK

With the improvement of people’s living standards,the thrombotic disease has became one of the most threatening disease to people’s health.The thrombotic disease has a high mortality and morbidity,and it occurs in a variety of forms.People are increasingly concerned about the prevention and treatment of the disease.At present,the main treatments of thrombotic disease are surgical treatment and drug therapy.Our research focuses on the study of a plasmin of high thrombolytic activity.Subtilisin QK(QK)was a serine protease separated from the fermentation supernatant of the Bacillus subtilis QK02.It is highly homologous to nattokinase(NK),and has comparatively high thrombolytic activity in vivo and in vitro.It can resist degradation of trypsin,and exhibit thrombolytic activity by orally taken.At the same time,it is safe and few side effcts.These advantages make QK to be a promising drug for the therapy of the thrombotic disease.This study was undertaken to produce QK in Pichia pastoris(P.pastoris)and to establish relatively mature techniques for fermentation,purification and lyophilisation.Otherwise a double sandwich ELISA method for detection of recombinant QK was developed and its pharmacokinetics was studied.Our results was presented as folliws:1.The DNA sequence encoding QK was designed and synthesized based on the codon bias of P.pastoris.The optimized QK gene was then inserted into the plasmid pPICZaA and the pPICZαA-QK was transformed into P.pastoris GS115.The constructed GS115/pPPICZaA-QK could secrete the recombinant QK of strong thrombolytic ability into the the supernatant.2.Based on the condition of cultivation in shaking flasks,the condition of fermentation in fermenter was established.In the initial batch and glycerol fed-batch phase,the DO level was maintained at over 20%and the cells were incubated at 28℃and pH 5.0.The WCW was gotten 190-200g/L at the end of glycerol fed-batch phase.In the methanol induction,the DO level was maintained at over 20%and the cells were incubated at 26℃ and pH 5.0.After 92h of induction of methanol,the thrombolytic activity of QK could reach 112000 IU(urokinase unit)per mL,and total protein concentration could reach 7.63 g/L.After purification of hydrophobic interaction chromatography(HIC)and gel filtration chromatography(GFC),91.73%of the thrombolytic activity of the recombinant QK could be recovered,and the purity of QK was over 95%.Various agents were investigated to study their protective effects during freeze drying.The addition of 5%trehalose and 1%skimmed milk powder showed relatively high increase in the thrombolytic activity of dried QK and could separately maintain 94.2%(45812 IU/mL)and 96.2%(46787 IU/mL)thrombolytic activity of the recombinant QK after FD.During the preservation,the activity of recombinant QK freeze-dried powder would have nearly no change at room temperature for a week by addition of During the preservation process,the activity of recombinant QK freeze-dried powder could not be changed at room temperature for a week.3.By using the purified recombinant QK to immunize rabbits and mice,the polyclonal antibody and 4 monoclonal antibodies specific to recombinant QK protein were obtained.Baed on it,the sandwich ELISA method with the monoclonal antibody used as coating antibody and polyclonal antibody used as detecting antibody was established.By the optimization of the condition of the sandwich ELISA,a double sandwich ELISA method for recombinant QK protein of high sensitivity,high accuracy,precision,and good reproducibility was successfully eatablished.The detection sensitivity was up to 1.207 ng/mL,and the coefficient of variation was less than 5%and the recovery was between 95%-105%.Pharmacokinetic of recombinant QK in rats after intravenous was successfully detected by the established ELISA method.The concentration-time curve ofrecombiant QK in mice after intravenous was conformed to two compartment opening modeland the main kinetic parameters were obtained.This data will laid the foundation for further study.In this paper,we use P.pastoris expression system to express recombinant QK of strong thrombolytic ability,and eatablish a reference method for its industrialized production.At the same time,the study on the detection method and pharmacokinetics of QK also laid a foundation for the further promotion and application of the thrombolytic enzyme and the elucidation of its metabolic mechani.