| Interferon has important roles in mediating antivirus and involved in the immunomodulatory functions.Interferon has been used in clinic to treat a variety of human diseases,and is widely recognized as one of most effective bio-therapeutic drugs.Porcine epidemic diarrhea virus(PEDV)is an important pathogen that affect the health in animals.PEDV belongs to coronavirus.In 2011,new mutant strains appeared and rapidly caused a national pandemic and lead to a large number of suckling piglets death which caused huge losses to the industry.Due to insufficient protection from classic vaccine against the pandemic,and it takes time to develop new vaccines against the emergence,therefore,it is urgent to develop sufficient steps to prevent such pandemic in clinic.In order to face this challenge,se focus on recombinant porcine interferon-a(rPoIFN-a),and then evaluate its industrial fermentation process,screening of stabilizer,standardization and detecting methods,the vitro inhibition to PEDV,clinical applications and the optimization of clinic usage,and ultimately complete the production of recombinant porcine interferon-a the and clinical validation and the data showed promising application in clinic.My research has four major parts:1 Generation of high-density fermentation craft in Pichia for rPoIFN-a industrializationIn order to establish the fermentation process for Pichia pastoris genetic engineering strain GS115/pPIK9K-PoIFN-a,we use 100 L Fermentation tank(Bioreactor)to test two different ways of production(batch fermentation and fed-batch fermentation)and then evaluate their effects on the growth of engineered bacteria and production of the recombinant protein.The results show that the protein expression peaked at 1.15mg/mL and the protein activity is 8.69×106 U/mL at 16 h in the batch fermentation,the protein expression gradually decreased afterwards;while the fed batch fermentation method,the protein expression reaches the peak at 1.89 mg/mL in 20h and the activity is 5.29×107 U/mL.The results show that the fed-batch fermentation method with dissolved oxygen could significantly increase the density of bacteria and shorten the cycle of the fermentation,suggesting a preferred impact for the practical application.Therefore,we finally select fed-batch fermentation process.2 The study on new stabilizer and freeze-drying technology for rPoIFN-a productIn order to extend the preservation time for rPoIFN-? and increase the stability of the product quality,we analyze the relationships among freeze-dried formula,mixing ratio and freeze-drying curve to optimize the way to freeze-drying rPoIFN-a.The results show that steps including seedlings ratio at 1:1,pre-frozen at-40? for 5 h,sublimation at-40??-10? sublimation for 10 hours,and second sublimation at-10??2? for 5 h,then desorption drying at 2??26? for 6 hours,will produce ideal expectation on the antigen stability,the left water within milk sugar stabilizer,vacuum degree as well as product activity.In addition,analysis on the stability of rPoIFN-a at 2?8? and 37? from different groups show that the protective function of antigen vitality stabilizer is better than milk sugar stabilizer.when it is preserved for 24 months or 37? for 10 d using milk sugar stabilizer,the protein content reduces 3%?6%and the protein activity decreases by 1?2 titer.When it is preserved for 24 months at 2?8?C or 37? for 10 d using antigen vitality stabilizer,the protein content reduces less 1%and activity is of no difference.Thus,the antigen vitality stabilizer is selected as the freeze-dried protective agent for rPoIFN-a.3 Lyophilized preparation of rPoIFN-? product and its effect against porcine epidemic diarrhea virus in vitroWe use three serial batchs of lyophilized products,and then establish a method to detect rPoIFN-? activity using MDBK/VSV system.The activity of rPoIFN-a is about 1×107U/mL.The freezed-dried traits,residual moisture,vacuum,purity,biosafety are all in line with the requirements from "Chinese veterinary Pharmacopoeia".In order to test its effect against PEDV in vitro,the 2015012 batches of rPoIFN-a products were used.Our data showed that(1)rPoIFN-a shows a better inhibitory effect to EBVAC15 when it was incubated in cells for 24 h.One tilter unit(107 in dilution)can completely block the proliferation of viruses.If it was incubated in cells for 1 h,one hundred tilter unit(105 in dilution)can completely block the proliferation of viruses.(2)rPoIFN-a and PEDV are pre-mixed,and then are used to co-infect the cells,one thousand tilter unit(104 in dilution)can completely block the proliferation of viruses.(3)If PEDV was used to transfect the cells for 1 h,the rPoIFN-a shows no inhibitory effect.4 The clinic performance of lyophilized rPoIFN-a productIn clinic,we test the effect of rPoIFN-a in preventing swine epidemic diarrhea,and the results show that rPoIFN-? produced good protection to PED newborn piglets with a survival rate at 50?70%,which was 20?40%higher than that in untreated piglets.Since the clinic follow-up and record,we optimize the use of the product,and we show that(1)rPoIFN-a is ideal to prevent the emerged PEDV;(2)oral treatment shows no significant difference compared to intramuscular injection,which is pretty convenient;(3)Sow and piglet both treated with rPoIFN-a(e.g.sow was given 2.5 dose injection of rPoIFN-a at 10 days and 3 days before delivery,and suckling piglets was injected 1 dose rPoIFN-a at the born day and the second day.)show better protective effect than that sows or piglets alone;(4)rPoIFN-a doesn't show distinct protection for the seriously diseased PED piglets,probably due to large dehydration and acidosis caused by the damaged intestinal tracts.In summary,our study establishes the complete production process and standards of rPoIFN-a.The evidence from the clinic application also show effective protection of rPoIFN-a to PED disease,and we also make the use of the product rPoIFN-a,and the industrialized rPoIFN-a provides a new avenue to prevent and control diarrhea,which is of great practical significance in clinic. |
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