The Research Of PCV2 Suspension Culture For Scale Production Technology

Porcine circovirus-related disease?PCVAD?is the general principle of Postweaning Multisystemic Wasting Syndrome?PMWS?,dermatitis and nephrotic syndrome?PNDS?and reproductive disorders?Studies have shown that porcine circovirus type 2?PCV2?is the main pathogen causing PCVAD.Pigs have a strong susceptibility to PCV2 and are transmitted in a variety of ways.The disease is no effective treatment,vaccine immunization is an effective means to effectively prevent PCV2 infection.The existing vaccines are mainly subunit vaccines,chimeric virus inactivated seedlings and whole virus inactivated seedlings.The whole virus inactivated seedlings need to break through the difficult process is the virus culture.Traditional rotary bottle production process,due to cultivated area,nutrient supply,gas exchange and other restrictions,the number of virus proliferation,resulting in downstream processes to achieve the required concentration of a virus with the standard vaccine,however,concentration process will result in hybrid protein enriched,increasing the proportion of immune stress,can not effectively meet the needs of farms in the vaccine.Introducing micro-carrier suspension culture in vaccine research ring can increase the amount of cells and toxin production quantity per unit volume of semi-finished products to enhance vaccine potency,further from the effectiveness,stability and safety and other aspects of the vaccine to enhance the quality.In this study,PCV2 bioreactor micro-carrier suspension culture was studied.The suspension culture of PCV2-proliferated PK-15A cells was studied,optimization of micro-carrier concentration,cell inoculation,initial cell agitation,growth stage agitation speed and medium supply method and other key technical parameters.Optimize the cell digestion parameters within the reactor,explore the establishment of the cell tank digestion process.Optimize the dose of virus inoculation,maintain serum concentration,dissolved oxygen parameters,culture temperature parameters.Cell culture process:carrier concentration of 3 g/L,cell inoculation amount of10cells/micro-carriers,the initial inoculation parameters for access to cells after access to the culture medium to 1/3 culture volume,the initial speed set to 20 r/min,2 h after the make up the culture volume,the rate of stirring was 30r/min and the medium was trained2 days for half of the liquid.The cell growth rate was 1.5×10⁶-2×10⁶ cells/mL.Cell digestion process:PBS washed twice,each 1 L PBS,so that the micro-vector digestion and shedding of cells more than 85%,achieve the desired results.Virus culture process:0.1 MOI?2%serum concentration of the maintenance solution,?40%DO?37?,Continuous harvest to F9 maintenance solution,starting from F2,each of the liquid passages is maintained not less than the viral titer 10⁵.5.5 TCID₅₀/mL,can reach 10^7.5TCID₅₀/mL.PK-15A in the bioreactor micro-carrier suspension culture effect is good,reached the same technology at home and abroad to achieve higher cell culture density.To achieve PK-15A in the Bioengineering reactor step by step amplification?16 L-85 L-500L?.PCV₂ original suspension culture scale?500 L?continuous harvesting process,not only greatly increased the potency of viral culture,but also significant savings in production costs,improve the quality of the vaccine made a great contribution.