The Research On Technical Process Of Bioreactor Production Of HP-PRRSV Antigen

Commonly known as highly pathogenic blue aural disease, the highly pathogenicporcine reproductive and respiratory syndrome (HP-PRRS) is the highly contagiousdisease of pigs. The HP-PRRS is caused by mutated porcine reproductive andrespiratory syndrome virus (PRRSV), and there is no effective drugs or treatmentagainst it. The high recurrence rate during treatment has become an importantproblem. For HP-PRRS, vaccination with the high-quality vaccines, is still one of theimportant methods for its prevention and control. Therefore, the quality of the vaccinewill directly determine the effect of vaccination. As a primary technology in thevaccine manufacturing process, the antigen preparation has undoubtedly been a keyfactor to determine the quality of the vaccine. Currently, the production of themajority of vaccines used for domestic animals including the HP-PRRS vaccine stilluse traditional cell culture process with roller bottle, which is built in1950s or1960s.With too many defects, such as batch differences, unstable quality, adverse reactions,inducing low antibody titers, such a production of vaccine obviously can not meet theneeds for the animal infectious disease prevention. In this study, we used cellbioreactor technology to produce PRRSV and optimized the production process,which provided reliable information for the large-scale cultivation of PRRSV.The contents of this study include the following three aspects. Firstly, thosebiological characteristics were studied on Marc145-LD1cell culture and HP-PRRSVproliferation on normal Marc145-LD1cells was cultured in the flask or3L volumeroller. The growth level of these cells was observed with a microscope. The cells wereseeded according to a certain amount. After incubation for24h and72h, cells werecounted and cell doubling time was also calculated. When HP-PRRSV propagated inthe roller bottle, the relationship between virus titers and culture time was determined, and the best harvesting time for virus was also determined.Secondly, bioreactor technology applied on Marc145-LD1cell culture andHP-PRRSV proliferation was studied. During the cell growth and virus proliferation,the fed-batch process was performed and glucose was supplemented. In the cellgrowth stage, the relationship between glucose consumption and cultivation time wasstudied to determine the best virus inoculation in the bioreactor. Moreover, the bestvirus harvesting time was determined according to the relationship between the virustiter and cultivation time.Finally, PRRSV was detected by RT-PCR. A sequence of PRRSV GP5gene wasamplified by RT-PCR technology, which could confirm the existence of PRRSV fromthe molecular level.