PCV2 Cap VLPs Large-scale Preparation Conditions Optimization

With the continuous scale-up of China's swine industry,the prevention and control of vaccines has become the main method for the prevention and control of swine diseases.Porcine circovirus type 2(PCV2)can cause multiple system failure syndrome in weaned piglets,congenital tremors,porcine dermatitis and nephrotic syndrome,sow reproductive failure,porcine respiratory syndrome and other circovirus related Diseases have caused huge human and material losses to the global pig industry.Vaccine immunity is the main means of preventing and controlling PCV2-related diseases.At present,the commercially available porcine circo vaccine is mainly a whole virus inactivated vaccine or a genetically engineered subunit vaccine based on the PCV2 a subtype.Since the changes in antigenic determinants caused by the presence of viruses in these vaccines during inactivation also affect the immune effect,immune-unrelated proteins may have side effects at the injection site.In addition,PCV2 grows slowly in cells,it is difficult to obtain high titer virus in whole virus culture,and the cost of antigen purification for expression is high.Therefore,there is an urgent need to develop new,efficient,and inexpensive PCV2 vaccines.Virus-like particles(VLPs)are hollow particles containing one or more structural proteins of a virus.No virus nucleic acid cannot self-replicate.Its morphology is the same as or similar to a true virus particle and is often referred to as a pseudovirus.Because of its superficial antigenic epitopes,virus-like structure,and its ability to induce cellular and humoral immunity,it is popular among vaccine developers.Studies have shown that PCV2 Cap proteins can selfassemble into VLPs.There is no difference in antigenicity and structure from native Cap proteins.Therefore,many researchers at home and abroad have tried to express recombinant PCV2 Cap proteins in different expression systems.This study was based on the soluble expression of Cap protein in E.coli BL21(DE3)host bacteria.E.coli high-density fermentation technology was used to optimize the conditions for increasing the production of PCV2 Cap VLPs,which laid the foundation for the development of a novel,highly effective and inexpensive PCV2 vaccine.The research content of this paper is divided into the following two parts:1.Expression of PCV2 Cap protein and opti mization of high d ensity fermentation conditionsThe recombinant plasmid p ET-32a-cap was transformed into the E.coli BL21(DE3)host vector and Cap protein expression was induced using IPTG.After a series of pretreatments,the target protein was subjected to SDS-PAGE analysis,Western-blot detection and solubility analysis.Expression identification.The results showed that the Cap protein was soluble on the host vector.Transmission electron microscopy showed that the Cap protein self-assembled into VLPs with 17 nm to 20 nm in diameter similar to the PCV2 virus particle.The recombinant Escherichia coli p ET-32a-cap glycerol preserved at-70°C was streaked onto an LB solid plate containing ampicillin(Amp)and cultured in a 37°C incubator overnight.The plate was removed the next day and picked.A single colony was placed in 5 ml of fresh LB liquid medium containing Amp,and was used as a first-class strain for shaking in a shaker flask at 37°C for 12 hours.According to 1:1000 in 300 ml LB liquid medium to access the first species,200 rpm,37 °C cultured for 9h,to be OD value to reach 0.8 after the suspension of culture,as a second species.Select the appropriate medium after autoclave sterilization and add the prepared secondary bacteria into the fermenter according to 1:100,add Amp at the same time,optimize the medium type,fermentation temperature,aeration ratio,defoamer type,induction temperature and Time and other conditions to achieve high-density fermentation of recombinant E.coli p ET-32a-cap,and finally make the total amount of wet bacteria of the recombinant bacteria per liter of broth from 6.0g/L of shake flask to 30.0g/L of high-density fermentation,wet The yield of bacteria is 5 times that of shake flasks.2.Optimization of conditions for the preparation of PCV2 Cap VLPsThe high-density fermentation broth of recombinant E.coli p ET-32a-cap was centrifuged at 4000 rpm,and the cells were collected and resuspended in a certain proportion.The yield of PCV2 Cap VLPs was increased by optimizing the lysis method of the recombinant bacteria and the concentration of saturated ammonium sulfate,precipitation temperature,and precipitation time during the purification of the Cap protein,achieving a target protein yield of 56 mg/L from shake flask era to high density.After fermentation and purification conditions were optimized at 340 mg/L,the yield was 6 times that of shake flasks.In other words,the yield of the protein of interest is increased by 20%.