Virus-like particle vaccines for poliovirus types 1, 2, and 3 with enhanced thermostability expressed in insect cells

Poliovirus (PV) is a pathogen that causes poliomyelitis, which may lead to paralysis and fatality. Inactivated PV vaccines (IPVs) and live-attenuated oral PV vaccines (OPVs) are currently used to defend against PV worldwide. Vaccines must be developed in a PV-free environment given the biosafety issues associated with OPV and IPV production and to eradicate PV globally. In this study, PV1, PV2, and PV3 virus-like particles with enhanced thermostability (PV-sVLPs) were produced in large quantities by using a baculovirus expression vector system (BEVS). Mice immunized with PV-sVLPs generated antibodies with strong PV-neutralizing response. In addition, splenocytes collected from immunized mice expressed high levels of IFN-γ, IL-2, GM-CSF, IL-5, and IL-10 upon PV-sVLPs stimulation. These data suggest that PV-sVLPs can serve as vaccines against PV infection.     

鲑鱼降钙素重组杆状病毒载体的构建及表达

目的：利用昆虫细胞杆状病毒系统表达重组鲑鱼降钙素（recombinant salmon Calcitonin，rsCT），探索一条真核生物表达生物活性rsCT的新途径。方法：将鲑鱼降钙素基因重组到pFastBac杆状病毒穿梭载体（baculovirus shuttle vector，Bacmid）中，构建重组鲑鱼降钙素杆状病毒表达载体即重组鲑鱼降钙素Bacmid，后将其转染昆虫细胞Sf9，表达目的蛋白，并对表达产物进行分子量及免疫反应性鉴定。结论：证实目的基因在昆虫细胞中获得了表达。结论：成功构建了鲑鱼降钙素重组杆状病毒载体并在昆虫细胞中初步表达。     

Hemagglutinin Displayed Baculovirus Protects Against Highly Pathogenic Influenza

Baculovirus (BV) replicating in insect cells can express a foreign gene product as part of its genome. The influenza hemagglutinin (HA) can be expressed from BV and displayed on the surface of baculovirus (HA-DBV). In this study we first generated six recombinant baculoviruses that expressed chimeric HAs with segments of the BV glycoprotein (gp64). The signal peptide (SP) and cytoplasmic tail (CT) domains of gp64 can enhance the display of HA from A/PR8/34 on BV surface, while the transmembrane (TM) domain of gp64 impairs HA display. Different doses of either live or β-propiolactone (BPL)-inactivated HA-DBV were administered to BALB/c mice. Live HA-DBV elicited higher hemagglutination-inhibition (HAI) titers than BPL-inactivated HA-DBV, and provided sterilizing protection. A second generation recombinant BV simultaneously displaying four HAs derived from four subclades of H5N1 influenza viruses was constructed. This tetravalent H5N1 HA-DBV vaccine elicited HAI titers against all four homologous H5N1 viruses, significantly decreasing viral lung titers of challenged mice and providing 100% protection against lethal doses of homologous H5N1 viruses. Moreover, mice vaccinated with HA-DBV had high levels of IFNγ-secreting and HA-specific CD8+ T cells. Taken together, this study demonstrates that HA-DBV can stimulate strong humoral, as well as cellular immune responses, and is an effective vaccine candidate for influenza.     

杆状病毒蛋白表达系统制备可溶性LRIG1蛋白及其鉴定

目的为进一步研究肿瘤的治疗提供高质量的可溶性多亮氨酸重复区免疫球蛋白样蛋白(sLRIG1)。方法用重组sLRIG1的杆状病毒(Bac-sLRIG1)感染sf9昆虫细胞,表达的蛋白经提纯,获得sLRIG1蛋白,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹进行分析鉴定。结果使用杆状病毒表达载体系统(BEVS)成功表达了重组sLRIG1蛋白,最佳感染复率为5,孵育时间为72 h,经鉴定蛋白表达正确,纯度高达95%。结论用BEVS可成功获得大量高纯度重组sLRIG1蛋白,可满足后续实验的需要。     

Influence of codon usage and translation initiation codon context in theAcNPV-based expression system: Computer analysis using homologous and heterologous genes

Codon usage by all the known gene sequences fromAutographa californica nuclear polyhedrosis virus (AcNPV) was compared with that of firefly luciferase (luc) and the beta subunit of human chorionic gonadotropin (hCG) expressed to contrasting levels in the baculovirus system. The highly expressedluc gene showed a codon usage similar toAcNPV genes, as reflected by a very low D-squared statistic value (0.78) and a similar G/C usage (45%) at wobble positions. However, the underexpressed hCG gene displayed a high D-squared value (7.3) and G/C usage (82.5%) at the wobble base positions. Alignment of the 20 nucleotides around the initiation codon of 23AcNPV genes identified a novel consensus translation initiation sequence aag/ta/tat/aa/cAAaATGaa/ct/ag/aAan, which was quite different from the Kozak consensus sequence (GCC)GCCA/GCCATGG. An extension of these analyses to a sample of other heterologous genes overexpressed and underexpressed in BEVS suggested similar trends. These theoretical analyses have important implications for heterologous gene expression in this system.