Process Control And Optimization Of Culture Condition For HPV16 VLPs Production In Recombinant Pichia Pastoris

Human papillomavirus(HPV) was the main pathogenic factor of cervical cancer. Human Papillomavirus virus-like particles (HPV VLPs) as a prophylactic vaccine has been proved to be effective in preventing cervical cancer. But the development of vaccines was limited for its low production, unstable performance and high cost. Therefore, HPV 16 VLPs fermentation condition optimization and process control were researched in this thesis.Initially, an ELISA method was established to semi-quantitatively determine HPV L1. A standard curve with linear coefficient of 0.974 was achieved by optimizing several conditions, andit is the first time that the content of HPV 16/18/52/58 L1 can be determined by ELISA.Shaking flask culture conditions for HPV 16 VLPs production in recombinant Pichia pastor is were optimized. The effect of induction time of methanol, pH value and other factors on HPV 16 VLPs fermentation during induction period was carried out. It showed that protein L1 expression conditions and HPV VLPs assembly condition were different. The induce time of 48 h was benefit to expression and self assembly of HPV 16 L1. Lower concentration of methanol (0.1%～0.3%) promoted the expression of monomer L1, whereas a higher methanol concentration (0.32%～0.6%) was conducive to LI self-assembly into VLPs. Beyond this scope of methanol concentration could result in the degradation of monomer L1. The acidic environment like pH 4.0 was favolable to L1 expression and assembly.Regarding culture temperature,28 ℃ was suitable for monomer L1 expression, and 30 ℃ can promote L1 self-assembling. And the further experiment results showed that the L1 expression was inhibited with the addition of oleic acid at the initial induction period. However, in the middle induction phase,5% oleic acid can not only promote the monomers self-assemble, but also prevented monomer degradation. When methanol concentration was higher than 5%, VLPs depolymerization was easily observed.Based on the shaking flask conditions, fermentation process optimization and control strategy were studied for HPV 16 VLPs improvement. It showed that L1 expression and VLPs assembly was tightly regulated by methanol concentration. Monomer L1 expression was immediately conducted, and then self-assembled into VLPs after methanol addition. However, below a certain threshold of methanol concentration, L1 will no longer assemble into VLPs. Methanol feeding strategy was established according to characteristics of monomer L1 intracellular expression and self-assembly. Besides, the trend of oxygen uptake rate (OUR) can effectively monitor the methanol utilization to control the L1 expression and VLPs assembly.