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Baculovirus Production Methods for Reliable Isolation of Recombinant Baculovirus Engineered for Modified Actin Expression

出版	University of California, Davis, 2014
ISBN	13212115709781321211573
URL	http://books.google.com.hk/books?id=U9bwrQEACAAJ&hl=&source=gbs_api

註釋The Baculovirus Expression Vector System (BEVS), first developed in the early 1980s, was utilized for the following two decades in research and pharmaceutical industry labs as a means for rapidly generating large numbers of recombinant proteins for structural and functional studies. In the early part of the twenty first century, BEVS was first employed in the production of animal vaccines, and the Food and Drug Administration (FDA) has since approved human vaccines produced using BEVS. FDA approval in 2009 of a bivalent human papillomavirus vaccine against cervical cancer produced using BEVS, was a landmark moment for regulatory acceptance of BEVS and is already leading to expanded use of BEVS for human and animal vaccine production. As baculovirus moves out of the research lab and into bioprocessing streams destined for clinical applications, greater attention must be focused on production of quality baculovirus stocks. Certain procedures in the process of generating recombinant baculovirus stocks have not matured to incorporate modern technology that has the potential to increase assurance of quality. Given the requirement that baculovirus produced for clinical applications be made using Good Manufacturing Practices (GMP), steps must be taken to mitigate risk to the quality of the baculovirus stock wherever possible. Purification of baculovirus stocks is one such procedure that can benefit from adoption of modern technologies. The baculovirus purification methods used today were developed at the conception of BEVS, and lack the stringency necessary for current GMP bioprocessing requirements. I have designed a new baculovirus purification strategy combining aspects of a traditional purification method with incorporation of modern technology to better meet current industry and regulatory standards. The method that I describe herein is a modified End Point Dilution (EPD) purification with a flow cytometric gp64 screen for infection. I provide data demonstrating the sensitivity of the flow cytometric gp64 screen and show that the method can effectively purify one recombinant baculovirus away from contaminating baculovirus. Utilization of this purification technique reduces the risk of cross contamination during the purification procedure, and the sensitivity of the screening method ensures that even low-level baculovirus infections are detected. Increasing the yields of recombinant protein produced with BEVS so as to lower costs associated with protein expression has been an aim of researchers since the technology was first developed. I have worked toward this goal by attempting to prolong the life of baculovirus infected cultures, thereby lengthening the period of recombinant protein expression. Baculovirus has long been known to manipulate the actin cytoskeleton during infection for production of progeny virus, but the exact relationship between baculovirus and actin remains unclear. Here I present evidence that overexpression of exogenous Drosophila 5C actin in insect cells causes a delay in virus induced cell death by approximately 24 hours. No increase in expression of recombinant & beta;-Galactosidase ([beta]-Gal) corresponding to this protracted period of cell viability was observed however. The mechanism by which actin overexpression delays the onset of virus induced cell death remains unclear, but it is likely that nuclear F-actin delays apoptotic cell death in baculovirus infected insect cells. Inhibition of apoptotic cell death may be attributable to sustained nuclear reprogramming by F-actin, or potentially F-actin mediated inhibition of DNase I.I propose that cellular Elongation Factor 1 & alpha; (EF-1[alpha]), a critical factor for protein translation, is bound by the overabundance of exogenous actin, rendering it unavailable for its role in translation. I hypothesize that co-expression of exogenous EF-1[alpha] along with Drosophila 5C actin will delay virus induced cell death and provide additional EF-1[alpha] to support increased expression of recombinant protein during the prolonged life of the culture.