2013 생화학분자생물학회 제4회 동계학술대회, 2013, 02, 58─58

The Comparative Evaluations of Quantitative Real-time PCR for Titration of Recombinant Baculovirus Particles

HanSaem Lee, Ho Sun Son, Ho Yeon Lee, Hee-Dong Jung, You-Jin Kim, Jeongsun Yang, Kisoon Kim

Abstract

The baculovirus expression system (BEV) is widely used for the production of large quantities of recombinant protein with post-translational modification. Especially, recombinant baculoviruses are useful to product recombinant proteins as bio-drugs, subunit vaccines, and virus-like particles. Traditionally, plaque assay, end-point dilution, and antibody-based assay have been used for the baculovirus titration. However, previous baculovirus titration methods are labor intensive, time consuming, and require a degree of expertise in cell culture and virus handling.
Nowadays, baculovirus quantitative PCR (Q-PCR) titration methods have offered much faster and simpler way to determine titers based on the intensity of a SYBR-green signal generated during PCR amplification, or the intensity of fluorescent Taqman probe signal. However, they have the limitation to measure low titers under 100-1000 baculovirus particles.
Here, we designed one set of forward and reverse primers for SYBR-green Q-PCR experiment and three sets of forward/reverse/probe primers for Taqman-based Q-PCR experiment. We compare the accurate quantitative PCR to titer recombinant baculovirus using both SYBR-green Q-PCR and Taqman-based Q-PCR. Custom primers and probe were designed to different three regions and used to calculate a standard curve of Q-PCR derived baculovirus genome copies extracted from a purified baculovirus stock by ultracentrifugation method. We also evaluate the consistent and reproducible correlation between the genome copies titrated by Q-PCR and plaque forming units per milliliter. Our work reveals that customer primers designed to IE-1 promoter region have the most accurate and reproducible Q-PCR results. No significant and plaque assay for 8 recombinant viruses, confirming the validity of this Q-PCR technique using our primers as a rapid and accurate method of baculovirus titration. In addition, we compare the baculovirus genome extraction methods for easy baculovirus titration Q-PCR.

• ISBN or ISSN: 0000-0000

• 본 연구는 질병관리본부 연구개발과제(과제번호 2012-N47002-00) 연구비를 지원받아 수행되었습니다.
• This research was supported by a fund(code 2012-N47002-00) by Research of Korea Centers for Disease Control and Prevention.