| Porcine Circovirus disease(PCVD)is one of the major diseases affecting the pig industry in the world and the healthy development of Pig industry in China.Porcine Circovirus Type2(PCV2)is the main pathogen of multisystem failure syndrome,dermatitis and nephrotic syndrome(PNDS)in weaned piglets.PMWS was first discovered in Canada.Acute infection can damage the immune system of pigs,lead to immune system disorder,and finally exacerbate the sharp rise in mortality,which can occasionally lead to huge economic losses of pig industry,the virus is also mixed with a variety of pathogens infection.Currently there is no effective control of the disease drugs,so the current prevention and control of the disease is still vaccination.Antigen preparation is the most important step in vaccine preparation process,which also directly affects the quality of vaccine.At present,most of the biological products enterprises now adopt the method of rotating bottle culture or suspension culture of microcarrier virus culture.The results showed that the virus titer was low,the difference between batches was large,the quality was unstable,and the production cost was high.Microcarrier technology also has disadvantages such as low tolerance of shear force,high serum dosage,high cost and complex ball transfer process.Considering the industry development and industrialization background,the application of suspension culture cell lines in the bioreactor for pure suspension virus culture will become the mainstream mode of animal vaccine production in the future.In this study,porcine circovirus type 2 antigen was produced by cell bioreactor and pure suspension culture cell technology,and the key quality control points involved in pure suspension culture were explored and optimized,and the antigen purification and antigen inactivation processes were studied to provide reliable data support for large-scale production of antigen.At the same time,the quality,safety and efficacy of vaccine products prepared by this process were evaluated.(1)Total suspension culture of PK15 cellsPK15 cells were cultured in total suspension with a 150 ml parallel bioreactor system.Various parameters suitable for cell culture were explored,including the optimization of initial cell inoculation density,the determination of dissolved oxygen parameters,the determination of pH parameters,the selection of stirring speed and the optimization of liquid replenishment mode.The results showed that the optimal culture conditions were as follows:cell inoculation density was 5×10~5cells/m L;The dissolved oxygen parameter is 40-50%.When pH was 7.0～7.4,cell growth was the best when pH was 7.2.Stirring speed is 100-120 r/min.Step by step to replenish liquid.(2)Study on suspension culture parameters of porcine circovirus type 2(LG strain)A parallel bioreactor was used to study the suspension culture parameters of porcine circovirus type 2(LG strain)by using the total suspension culture parameters of PK15 cells,including the optimization of cell concentration,the optimization of dose,the optimization of time of receiving the virus,and the comparison of total suspension and flask culture methods.The results showed that the optimal cell concentration was 5×10~5cells/m L.The optimal dose of inoculation was 5%synchronous inoculation.The optimal harvest time is determined to be 120hours;The virus titer obtained by total suspension culture was better than that obtained by rotating bottle culture,and the semi-finished products produced by total suspension culture had the characteristics of high stability between batches,low contamination rate and high production efficiency.(3)Purification of porcine circovirus type 2(LG strain)antigenThe cell fragmentation optimization and antigen purification of porcine circovirus type 2(LG strain)anti-stock harvested by pure suspension culture were studied.Cell fragmentation was compared with traditional freezing and thawing methods using low temperature ultra-high pressure cell fragmentation instrument.Hollow fiber column filtration and membrane filtration were used for antigen purification.The changes of protein content and virus titer were compared before and after purification.The results showed that the cell fragmentation apparatus with low temperature and ultra-high pressure could be used to break the suspended cultured cells well.Compared with the traditional method of repeated freezing and thawing,the cell fragmentation effect was better and the production effect was higher.The 0.45μm and 100k D hollow fiber filtration technology can be used in the purification of porcine circovirus type 2(LG strain)vaccine,which can effectively improve the quality of the vaccine and has a good industrial prospect.(4)Study on inactivation technology of porcine circovirus type 2(LG strain)antigenThe inactivation effect of BEI and formaldehyde on porcine circovirus type 2(LG strain)was compared,and inactivation test was performed.At the same time,safety test and immune efficacy test were carried out for the new inactivated agent.The results showed that the BEI with 1.0‰concentration could completely inactivate porcine circovirus type 2(LG strain)at 32℃for 48h,and the safety and efficacy tests of the inactivated porcine circovirus type 2 were qualified.The production time and peak titer of vaccine antibody prepared by this method are better than that of traditional formaldehyde inactivated vaccine.(5)Study on laboratory preparation of porcine circovirus type 2(LG strain)Three batches of inactivated porcine circovirus type 2(LG strain)vaccine were prepared by using optimized suspension cell culture technology,antigen treatment technology and inactivation technology.The results showed that the parameters determined by the new process could meet the production requirements of ring inactivated vaccine(LG strain)by testing the semi-finished product and finished product samples,which indicated that qualified vaccine could be prepared under laboratory conditions.(6)Study on safety of inactivated porcine circovirus type 2(LG strain)vaccineOptimization is adopted to define the whole suspension cell culture technology,inactivated antigen processing technology and process in the production workshop manufacture the 5 batch of porcine circovirus virus type 2(LG)inactivated vaccine,for all the items after finished goods inspection,conducted a single dose of immune experiment,single dose repetitive immune piglets piglets,high-dose immune piglets experiment and sow safety testing security research.The results showed that the inactivated porcine circovirus type 2(LG strain)vaccine produced by the new technology was safe and reliable for piglets and pregnant sows with no side effects.(7)Efficacy of inactivated porcine circovirus type 2(LG strain)vaccineFive batches of inactivated porcine circovirus type 2(LG strain)vaccine were trial-produced in the workshop using the optimized total suspension cell culture process,antigen treatment process and inactivation process.After the inspection of all finished products,the efficacy test of inactivated vaccine was conducted.Including vaccine against toxic effect inspection pig weight gain rate test,using the fluorescent quantitative PCR method evaluates the vaccine efficacy test,the law of immune attack poison virus after detoxification and organization determination of viral load test,the vaccine immune antibody duration and the rules of determination test,the minimum immune dose test and antibody and poison attack protection correlation test,porcine circovirus virus inactivated vaccines(LG)strains preserved pHase I trial and passive immunization of porcine circovirus inactivated vaccine(LG strain)in sows.The results showed that the inactivated porcine circovirus type 2(LG strain)vaccine produced by the new method could effectively resist the decrease of weight gain rate of pigs caused by virulent attack by vaccine immunization.The weight gain rate index was used as an index to evaluate the immune effect of the vaccine,which was simple,intuitive and reliable,and could reflect the real effect of the vaccine.Fluorescence quantitative PCR can quantitatively detect viral load,which can be used as one of the evaluation indexes of vaccine immune effect.The antibody produced by the vaccine immune stimulation can resist the attack of strong poison;The duration of vaccine immunization is at least 6 months,the vaccine has good immunogenicity,stimulate the production of antibodies quickly,long duration,can provide adequate immune protection;The minimum immune dose of the vaccine to piglets was 0.5 ml per hog,and all serum antibodies were positive and the immune protection rate was100%after 28 days of immunization.The higher the antibody titer,the better the immune protection effect,and the serum antibody titer can be used as one of the methods to test the efficacy of PCV2 inactivated vaccine.When the titer of serum antibody is more than 800 times by IPMA method,100%immune protection can be obtained.The protective period of the vaccine was determined to be stored at 2-8℃for 18 months.The vaccine is safe for sows and the passively immunized sows have a good immune protection effect on piglets.The results showed that the total suspension culture technology of PK15 cells and suspension culture technology of porcine circovirus type 2(LG strain)were used to produce porcine circovirus type 2(LG strain)antigen in a bioreactor,and the optimized antigen purification process and the optimized antigen inactivator were used to isolate porcine circovirus type 2(LG strain)antigen.The final preparation of porcine circovirus type 2(LG strains)inactivated vaccine finished products,the test indexes meet the current national standard,against sows and piglets with good safety and immune effect,can completely replace the traditional turn bottle process of preparation of porcine circovirus virus type 2(LG)inactivated vaccine,for the prevention and control of porcine circovirus virus type 2 infection.The vaccine produced by the optimized process has better immune effect,stability between batches,production cost and production efficiency than the vaccine produced by the traditional process,which can improve the technical competitiveness of the enterprise and increase the net profit of the enterprise,and has a good application prospect in the future. |
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