By Leyi Wang
This targeted quantity presents diagnosticians and researchers with useful methodologies and techniques to take on animal coronaviruses. It explores traditional immunohistochemistry, virus neutralization, enzyme-linked immunosorbent assays, expression and purification of recombinant viral proteins, and numerous molecular assays, together with traditional and real-time opposite transcription-PCR, opposite genetics technique, and subsequent iteration sequencing and series analyses. As a part of the Springer Protocols Handbooks sequence, chapters include comfortably reproducible laboratory protocols in addition to specialist tips about troubleshooting and fending off identified pitfalls.
useful and authoritative, Animal Coronaviruses serves as an amazing reference for researchers reading a large choice of coronavirus species within the Coronaviridae.
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Extra info for Animal Coronaviruses
Predominant phosphorylated nucleocapsid (N) protein, peplomeric glycoprotein, and spike (S) protein that makes up the large surface projections of the virion, and membrane protein (M) [3, 4]. The N protein is abundantly produced in coronavirus-infected cells and is highly immunogenic. The N protein binds to the viral genomic RNA and composes the structural feature of helical nucleocapsid. The N protein is a preferred choice for developing a groupspecific serologic assay because of highly conserved sequence and antigenicity.
Add 50 to 100 μL of 1:100 FITC-conjugated goat anti-turkey IgG secondary antibody (KPL) diluted with PBS to each section and incubate at RT for 1 h in the dark by covering with aluminum foil or using a covered container. 17. Dip slides into ddH2O and wash slides with PBS with stirring for 15 min. 18. Dip slides into ddH2O and get rid of excess water. Leave the slides to air-dry till almost dry. 19. Place Vectashield® mounting medium (Vector Laboratories) over the sections and cover the slides with cover slips.
The PCR cycling parameters are the same as above for N gene 38 Chien Chang Loa et al. Table 2 Preparation of ligation reaction mixture for recombinant plasmid carrying turkey coronavirus nucleocapsid protein gene Components Volumes (μl) Vector x Insert y Sterile water z Ligation premix (2×) 5 (x + y + z = 5 μl) amplification (step 14). The PCR product of correct clone is about 1500 bp. 23. The selected clone is grown in growth medium. Plasmids are purified by QIAquick mini-prep kit and sequenced to confirm that the inserted TCoV N gene is in frame with the vectordefined open reading frame at both its N- and C-termini.
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