scholarly journals RG203KR mutations in SARS-CoV-2 Nucleocapsid: Assessing the impact using Virus-like particle model system

2022 ◽  
Author(s):  
Harsha Raheja ◽  
Soma Das ◽  
Anindita Banerjee ◽  
Dikshaya P ◽  
Deepika C ◽  
...  
Keyword(s):  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Structural Proteins ◽  
Particle Model ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Baculovirus Expression ◽  
Virus Like Particle ◽  
The Impact

The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on Spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of the mutations at the virion level, recombinant baculovirus expression system based VLPs were generated for the prototype Wuhan sequence along with Spike mutants like D614G, G1124V and the significant RG203KR mutation in Nucleocapsid. All the four structural proteins assembled in a particle wherein the morphology and size of the particle confirmed by TEM closely resembles the native virion. The VLP harbouring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by the immunized mice sera. Results demonstrate a non-infectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant.

scholarly journals Pigs Immunized with the Virus-like Particle Vaccine Are Protected against the Hepatitis E-3 Virus

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1265
Author(s):  
Hyeon-Jeong Go ◽  
Byung-Joo Park ◽  
Hee-Seop Ahn ◽  
Dong-Hwi Kim ◽  
Da-Yoon Kim ◽  
...  
Keyword(s):  
Expression System ◽  
Vaccine Dose ◽  
Baculovirus Expression System ◽  
Hepatitis E ◽  
Positive Control ◽  
Negative Control ◽  
Fecal Shedding ◽  
Viral Shedding ◽  
Baculovirus Expression ◽  
Virus Like Particle

In this study, we generated the HEV virus-like particle (VLP) vaccine expressing 239 amino acids (367–605 aa) of the HEV-3 ORF2 using the baculovirus expression system. The HEV-3-239-VLP vaccine efficacy was evaluated by dividing 12 pathogen-free pigs into four groups: negative control, positive control, 100 μg VLP-, and 200 μg VLP-vaccinated groups for 10 weeks. The pigs in either of the vaccinated groups were administered the corresponding first and booster doses on weeks 0 and 2. At week 4, the positive control and two vaccinated groups were challenged with 106 HEV-3 genomic equivalent copies; viremia and fecal shedding of the virus were identified in pigs in the positive control and 100 μg VLP-vaccinated pigs showed transient viremia and fecal viral shedding. However, no viruses were detected in the serum or fecal samples of the 200 μg VLP-vaccinated pigs. The 100 and 200 μg VLP-vaccinated pigs had significantly higher (p < 0.01) anti-HEV antibodies than the negative control pigs from weeks 6–10 with normal levels of liver enzymes. The 200 μg VLP-vaccinated pigs showed statistically less liver tissue fibrosis (p < 0.05) than that of the positive control pigs. Thus, the novel baculovirus expression system-generated VLP vaccine dose-dependently protects against HEV-3 challenge and may be useful in other animal species, including humans.

scholarly journals Expression, Purification and Sublocalization of SARS-CoV Nucleocapsid Protein in Insect Cells

2004 ◽  
Vol 36 (11) ◽  
pp. 754-758 ◽  
Author(s):  
Ai-Xia Ren ◽  
You-Hua Xie ◽  
Yu-Ying Kong ◽  
Guan-Zhen Yang ◽  
Yao-Zhou Zhang ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Trichoplusia Ni ◽  
Insect Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Functional Study ◽  
Expression And Purification ◽  
N Protein ◽  
Baculovirus Expression

Abstract The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1–4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.

scholarly journals Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

2013 ◽  
Vol 19 (4) ◽  
pp. 508-515 ◽  
Author(s):  
Denise M. Lowe ◽  
Michelle Gee ◽  
Carl Haslam ◽  
Bill Leavens ◽  
Erica Christodoulou ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Solid Phase ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Lead Discovery ◽  
Human Form ◽  
Baculovirus Expression ◽  
Substrate Conversion ◽  
Assay Interference

Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z′ value 0.8) and provided several tractable hit series for further investigation.

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