cdna encoding sars cov 2 spike  (Addgene inc)

Journal: Biomedicines

Article Title: Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion

doi: 10.3390/biomedicines12040821

Figure Lengend Snippet: Target CHO-K1 cells co-expressing human encoded ACE2 and NPC1L1 significantly enhances SARS-CoV-2 spike glycoprotein-mediated cell fusion. ( A ) Effector CHO-K1 cells expressing S glycoprotein were co-cultured with the target cells co-expressing ACE2 and NPC1L1 or pCDNA3.1 with the luciferase gene. Membrane fusion as a means of virus cell-to-cell spread was detected by monitoring relative luciferase units (RLUs). ( B ) Cell-to-cell fusion using effector CHO-K1 cells expressing S glycoprotein from three different mutants; wild type: Wuhan-Hu-1, D614G mutant, and N501Y mutant. RLU data in this experiment are expressed without TMPRSS2. Asterisks indicate a significant difference between the groups and ACE2 positive control (* p = 0.0246, ** p = 0.0044, *** p = 0.0006, and **** p < 0.0001).

Article Snippet: The mammalian expression plasmids encoding SARS-CoV-2 spike glycoprotein D614G (Cat # VG40589-UT), N501Y (Cat # VG40771-UT), and human ACE2 receptor (Cat # HG10108-UT) were purchased from Sino Biological (Wayne, PA, USA).

Techniques: Expressing, Cell Culture, Luciferase, Membrane, Virus, Mutagenesis, Positive Control

Journal: Biomedicines

Article Title: Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion

doi: 10.3390/biomedicines12040821

Figure Lengend Snippet: Impact of NPC1L1 expression on syncytia formation between effector and target cells using 3 different variant S glycoproteins; wild type: Wuhan-Hu-1 (panels a–c) ( A ), D614G mutant (panels d–f) ( B ), and N501Y mutant (panels g–i) ( C ). Images were captured under 10× and 40× magnification on EVOS FL auto imaging system after 24 h of co-culturing effector and target cells to highlight differences in size and abundance of syncytia in the presence and absence of NPC1L1.

Article Snippet: The mammalian expression plasmids encoding SARS-CoV-2 spike glycoprotein D614G (Cat # VG40589-UT), N501Y (Cat # VG40771-UT), and human ACE2 receptor (Cat # HG10108-UT) were purchased from Sino Biological (Wayne, PA, USA).

Techniques: Expressing, Variant Assay, Mutagenesis, Imaging

Journal: medRxiv

Article Title: Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life

doi: 10.1101/2023.01.28.23285133

Figure Lengend Snippet: A) Diagram depicting the experiment (n infants and young children: pre=12, acute=12, conv=21; n adults: acute=17). B-G) Spike-specific B cells were sorted using FACS, and the BCR sequence of each clone was determined. B) Frequency of SARS-CoV-2 spike-specific IgG+ memory B cells as a proportion of CD20+ B cells. Samples of the same donor a connected by a gray line. Blue line indicates average values; shaded areas indicate 5th to 95th percentiles. C,D) Frequency of SARS-CoV-2 specific IgG+ memory B cells in C) infants and young children at convalescent phase and D) at acute phase in adults and infants and young children. E) Clonality analysis of sorted SARS-CoV-2 spike-specific IgG+ memory B cells in infants and young children (n = 220). Each clone is represented as a circle. Circle size indicates the number of IGHV sequences in each clone; color represents the mean IGHV somatic hypermutation rate. F) Somatic hypermutation rates of the IGHV genes in single sorted SARS-CoV-2 spike specific IgG+ memory B cells at acute phase. G) Mean somatic hypermutation rate of all cloned IGHV genes in indicated infant samples. H-J) T cells were stimulated with overlapping peptides against WT and Omicron variants. Cytokine production was determined via flow cytometry. H) Box plot showing the fraction of multifunctional T cells (IFNg+, IL-2+, TNFa+) at different infection stages. I) Kinetics of multifunctional CD4+ T cell response. J) Comparison of multifunctional CD4+ T cell response during the acute phase of infection in infants and young children and adults. Statistical comparisons were conducted with Wilcoxon rank sum test. Solid line indicates median healthy response; dashed line indicates 3x median healthy response.

Article Snippet: Briefly, human codon-optimized cDNA encoding SARS-CoV-2 spike glycoprotein of the WA1/2020 and variants was synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamH I and Xho I sites.

Techniques: Sequencing, Clone Assay, Flow Cytometry, Infection, Comparison