Antibody cloning and expression

Ab cloning and expression were performed as described with modification (23 (link)). V(D)J gene regions from Iga HCs and VJ regions from kappa or lambda light chains were synthesized (Integrated DNA Technologies) and inserted into pFUSE-CHIg-hG1 (IgG1) and pFUSE2-CLIg-hK (IgK) or pFUSE2-CLIg-hL2 (IgL) expression vectors (InvivoGen) using the Cold Fusion Cloning Kit (SBI). Plasmids encoding HC and LC V(D)J and VJ inserts were cotransfected into Expi293T cells (Life Technologies).

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Nair N., Feng N., Blum L.K., Sanyal M., Ding S., Jiang B., Sen A., Morton J.M., He X.S., Robinson W.H, & Greenberg H.B. (2017). VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans. Science translational medicine, 9(395), eaam5434.

Publication 2017

Cold Fusion Cloning Kit Expi293T cells

Cells Cold Gene Igg1 Plasmids Vectors

Corresponding Organization : VA Palo Alto Health Care System

Other organizations : National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention

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Variable analysis

independent variables

Ab cloning and expression protocol
V(D)J gene regions from Iga HCs and VJ regions from kappa or lambda light chains

dependent variables

Protein expression in Expi293T cells

control variables

PFUSE-CHIg-hG1 (IgG1) and pFUSE2-CLIg-hK (IgK) or pFUSE2-CLIg-hL2 (IgL) expression vectors

controls

Positive control: None specified
Negative control: None specified

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