Impact of Molecular Modification on the Efficiency of Recombinant Baculovirus Vector Invasion to Mammalian Cells and Its Immunogenicity in Mice

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells. In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS.
Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed.
Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.

Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1-ZP4). The functions of the three proteins present in mice (ZP1-ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown.
  • Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region.
  • Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity.
  • Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins.
  • These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

Utilizing a Baculovirus/Insect Cell Expression System and Expressed Protein Ligation (EPL) for Protein Semisynthesis

Protein semisynthesis has been used for the chemoselective linking of synthetic peptides and recombinant protein fragments to generate complete native proteins in good yield. The ability to site-selectively incorporate multiple post-translational chemical modifications (PTMs) into proteins via this approach shows great potential for enhancing understanding of the molecular basis of protein function and regulation.
Protein semisynthesis, however, often requires high expression efficiency of the recombinant protein fragments (i.e., high expression yield and ability to preserve protein biological functions), which can be hard to achieve for some human enzymes when using bacterial expression systems. Here, we describe how to use a baculovirus/insect cell expression system and a protein semisynthesis strategy known as expressed protein ligation (EPL) to produce workable levels of proteins of interest containing site-specific chemical modifications.
The protocol provides detailed guidance for generating protein C-terminal thioesters for use with the EPL reaction, performing the EPL reaction, and purifying the protein ligation product. We exemplify the protocols by generating protein kinase Akt1 with site-specific phosphorylations installed into its C-terminal tail, for kinetic kinase assays.
We hope these methods will help increase the use of protein semisynthesis for elucidating the post-translational regulation of human enzymes involved in cell signaling. © 2022 Wiley Periodicals LLC Basic Protocol 1: Generation of the N-terminal protein of interest (POI) fragment containing a C-terminal thioester moiety Basic Protocol 2: Expressed protein ligation (EPL) of the protein thioester with a synthetic peptide and purification of the protein ligation product Basic Protocol 3: Semisynthesis and biochemical analysis of site-specifically phosphorylated Akt1.

Zika virus baculovirus-expressed envelope protein elicited humoral and cellular immunity in immunocompetent mice

Zika virus (ZIKV) is a mosquito-borne virus that has a high risk of inducing Guillain-Barré syndrome and microcephaly in newborns. Because vaccination is considered the most effective strategy against ZIKV infection, we designed a recombinant vaccine utilizing the baculovirus expression system with two strains of ZIKV envelope protein (MR766, Env_M; ZBRX6, Env_Z).
  1. Animals inoculated with Env_M and Env_Z produced ZIKV-specific antibodies and secreted effector cytokines such as interferon-γ, tumor necrosis factor-α, and interleukin-12.
  2. Moreover, the progeny of immunized females had detectable maternal antibodies that protected them against two ZIKV strains (MR766 and PRVABC59) and a Dengue virus strain.
  3. We propose that the baculovirus expression system ZIKV envelope protein recombinant provides a safe and effective vaccine strategy.

An Integrated Platform for Serological Detection and Vaccination of COVID-19

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts.
We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum.
Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines.
Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.
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