| The baculovirus surface display system is a eukaryotic surface display system that has seen rapid development in recent years. Its core concept involves the fusion of an exogenous gene with an endogenous envelope protein gene, which when introduced into an insect cell for fusion expression, allows for the expression of the exogenous protein on the surface of the cellular—and thus recombinant baculoviral—envelope via envelope glycoprotein-mediated cell surface display processes. The baculorvirus surface display system boasts several advantageous characteristics, among them its high viral titer, generous capacity in accommodating foreign genes, and its ease of post-translational modification. Furthermore, the system has been widely utilized in applications of gene presentation, the targeted attack of cancer cells, monoclonal antibody preparation, and the development of novel multivalent vaccines, among others. However, the baculoviral surface display system employs a Group I type baculovirus protein known as GP64 to mediate the fusion expression of the exogenous protein. GP64 is a polar protein, as evidenced by its receptor sites being localized only within the polar regions of baculoviral envelopes. During the viral replication cycle, wild-type GP64 protein and GP64 fusion protein exhibit strong competitive inhibition, thus leading to low baculoviral surface display efficiencies of the fusion protein.In order to effectively overcome the low baculoviral surface display, this research applied techniques in homologous recombination in bacteria, dual luciferase assays, and confocal laser scanning tomography to better elucidate the construction of the mutant r Sw106-inv( Ac-Zeo-ph\ ? p64) platform, to investigate the recombinant baculoviral replication cycle and to detect the display efficiency of recombinant baculoviruses in the context of mutant r Sw106-inv strains, and to investigate the VSV-G mediated fusion display of product via a unique r Sw106-inv Ac-Zeo-ph\?p64 strain. The following results were thereby obtained:1) The construction of Ac-Zeo-ph\?p64Using overlap extension PCR, homologous recombinant fragment was produced, which were then transformed into a unique invasin-containing, DAP auxotrophic strain of E. coli Ac Multi Bac/?asd Sw106/PGB2?Inv(wild-type strain r Sw106-inv Ac). Upon invasin-mediated transfection, mutant r Sw106-inv strain capable of supporting high-efficiency, multi-gene baculoviral display were ultimately constructed: a gp64-delayed expression mutant r Sw106-inv Ac-Zeo-ph\?p64 strain.2) Investigating the recombinant baculovirus replication cycle using Ac-Zeo-ph\?p64Delayed expression of wild-type GP64 protein was carried out respectively on the Ac-Zeo-ph\?p64. The results indicated that recombinant baculoviruses delayed expression of wild-type GP64 exhibited no significant effect on the recombinant baculovirus replication cycle.3) Detecting the display efficiency of recombinant baculoviruses using Ac-Zeo–ph\ ?p64A renilla luciferase reporter gene was fused with gp64, and a firefly luciferase reporter gene was used as a reference gene. Both products were transfected to Ac-Zeo-ph\?p64, after which dual luciferase assay techniques were applied towards the Ac-Zeo-ph\?p64 to detect the efficacy of recombinant baculovirus surface display. The results indicate that the r Sw106-inv Ac-Zeo-ph\?p64 mutant strain allows the efficacy of recombinant baculovirus surface display to increase approximately 0.8-fold.4) VSV-G-mediated fusion display and Ac-Zeo-ph\?p64 usage applicationsCondon usage frequency analysis of vsv-g codons indicate that sf9 cells exhibit improved expression with the presence of the vsv-g gene. Using the Ac-Zeo-ph\?p64 and VSV-G mediation, recombinant baculovirus demonstrating single gene display(egfp) was constructed. Confocal laser scanning microscopy analysis confirmed the presence VSV-G-mediated exogenous protein on the sf9 cell membranes, and a Western Blot analysis confirmed the successful integration of the exogenous protein in the recombinant baculovirus progeny.The platform successfully constructed in this research are capable of supporting high efficiency, multi-gene presentation in baculoviruses, and as such, have overcome some of the challenges of traditional baculovirus display systems, such as low display efficiency. Research in VSV-G-mediated exogenous gene fusion display and the modification of recombinant baculoviruses provides an important foundation in many fields of study to which recombinant baculoviruses are relevant, including applications in the presentation of multiple genes as well as host cell gene presentation. |
PDF Full Text Request