Pcdnatm3.1 myc his a vector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PcDNATM3.1/myc-His(-) A vector is a mammalian expression vector used for the expression of recombinant proteins. The vector contains a CMV promoter, a myc-His tag sequence, and a multiple cloning site for the insertion of the gene of interest. It also includes an ampicillin resistance gene for selection in E. coli.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Mouse monoclonal anti flag antibody, by Merck Group (1 mentions) Mouse anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions) Mouse anti myc monoclonal antibody, by Santa Cruz Biotechnology (1 mentions) Rabbit anti, by Abcam (1 mentions) Mouse monoclonal anti hemagglutinin ha, by BioLegend (1 mentions) Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions) Rabbit polyclonal anti lc3b, by Merck Group (1 mentions) Mouse monoclonal anti myc, by Santa Cruz Biotechnology (1 mentions) Mouse monoclonal anti flag, by Merck Group (1 mentions) Anti flag monoclonal antibody, by Santa Cruz Biotechnology (1 mentions) Anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions) Monoclonal anti myc antibody, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

PcDNATM3.1(+)

6 protocols using pcdnatm3.1 myc his a vector

Porcine Innate Immune Molecules Cloning

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The coding sequence (CDS) fragments of porcine RIG-I (GenBank: EU126659), melanoma differentiation–associated protein 5 (MDA5) (GenBank: EU006039), mitochondrial antiviral-signaling protein (MAVS) (GenBank: EU082069), TBK1 (GenBank: EU091339), IFN regulatory factor 3 (IRF3) (GenBank: KC860781), and IFN regulatory factor 7 (IRF7) (GenBank: EU294309) with an additional hemagglutinin (HA) tag at the C terminus of each CDS were obtained using the gene synthesis method and cloned into pcDNATM3.1/myc-His(-)A vector (Invitrogen) to yield the HA-tagged expression plasmids of these innate immune molecules. The CDS for HA tag was as follows: 5′-TACCCATACGACGTCCCAGACTACGCT-3′. All the constructed expressing plasmids were analyzed and verified by DNA sequencing. The porcine IFN-β–promoter-luciferase reporter plasmid was generated by our laboratory previously (24 ). The pRL-TK Renilla luciferase reporter plasmid was kindly provided by Professor Hongbing Shu (Wuhan University, China) (25 (link)).

Zhang X., Yang F., Li K., Cao W., Ru Y., Chen S., Li S., Liu X., Zhu Z, & Zheng H. (2021). The Insufficient Activation of RIG-I–Like Signaling Pathway Contributes to Highly Efficient Replication of Porcine Picornaviruses in IBRS-2 Cells. Molecular & Cellular Proteomics : MCP, 20, 100147.

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Preparation of Antibodies and Plasmid Constructs

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Mouse anti-Myc monoclonal antibody, rabbit anti-Flag monoclonal antibody and mouse anti-β-actin monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-Flag monoclonal antibody was purchased from Sigma–Aldrich (St. Louis, MO, USA). rabbit anti-LGP2 polyclonal antibody and rabbit anti-eukaryotic translation initiation factor 4 gamma (eIF4G) polyclonal antibody were purchased from Abcam(Cambridge, MA, USA). Anti-VP1 polyclonal antibody was prepared by our laboratory (unpublished data). The full-length cDNA of LGP2 was amplified from PK-15 cells and cloned into pcDNA^TM3.1/myc-His(-)A vector (Invitrogen, Carlsbad, CA, USA) to generate the Myc-tagged expressing plasmid (Myc-LGP2). Various Flag-tagged viral protein expressing plasmids were constructed by our lab previously as described.^{3 (link)} A series of Myc-tagged LGP2 mutant constructs were generated by site-directed mutagenesis PCR.⁵⁹ All the generated expressing plasmids were analysed and verified by DNA sequencing.

Zhu Z., Li C., Du X., Wang G., Cao W., Yang F., Feng H., Zhang X., Shi Z., Liu H., Tian H., Li D., Zhang K., Liu X, & Zheng H. (2017). Foot-and-mouth disease virus infection inhibits LGP2 protein expression to exaggerate inflammatory response and promote viral replication. Cell Death & Disease, 8(4), e2747-.

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Plasmid Construction and Transfection of Myc-Tagged Proteins

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The full-length porcine RPSA, VIM, ESD, and TPM4 cDNAs were amplified from PK-15 cells and constructed into pcDNATM3.1/myc-His(–)A vector (Invitrogen, Carlsbad, CA) to generate plasmids expressing Myc-tagged RPSA (Myc-RPSA), VIM (Myc-VIM), ESD (Myc-ESD), and TPM4 (Myc-TPM4), respectively. The Flag-VP1 expressing plasmid was constructed previously by our laboratory (3 (link)). A series of Flag-tagged truncated VP1 constructs (Flag-VP1-Δ1-35, Flag-VP1-Δ30-65, Flag-VP1-Δ60-95, Flag-VP1-Δ90-125, Flag-VP1-Δ120-155, Flag-VP1-Δ150-185, Flag-VP1-Δ180-214, Flag-VP1-1-115, Flag-VP1-115-214, and Flag-VP1-37-188) were generated by site-directed mutagenesis PCR, as described previously (41 (link)). All of the constructed plasmids were sequenced and analyzed to ensure the accurate insertion of the target genes into the vector plasmids. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent. All of the transfection experiments were performed according to the manufacturer’s instructions.

Zhu Z., Li W., Zhang X., Wang C., Gao L., Yang F., Cao W., Li K., Tian H., Liu X., Zhang K, & Zheng H. (2020). Foot-and-Mouth Disease Virus Capsid Protein VP1 Interacts with Host Ribosomal Protein SA To Maintain Activation of the MAPK Signal Pathway and Promote Virus Replication. Journal of Virology, 94(3), e01350-19.

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Cloning and Characterization of Myc-Tagged EGR1 and IFN Pathway Plasmids

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The full-length porcine EGR1 cDNA fragment was cloned into a pcDNA^TM3.1/myc-His(-)A vector (Invitrogen) to construct a Myc-tagged EGR1 eukaryotic expressing plasmid (Myc-EGR1, including a C-terminal Myc tag). The constructed plasmid was analyzed and verified by DNA sequencing. A series of plasmids expressing HA-tagged type I IFN pathway-related proteins [including MDA5, RIG-I(CARD), VISA, TBK1, IRF3 and IRF7], and the IFN-β promoter luciferase reporter plasmids and control plasmid Renilla luciferase pRL-TK were kindly provided by Hongbing Shu’s Laboratory (Zhou et al., 2014 (link); Li D. et al., 2016 (link)). The plasmids were transfected into cells using OPTI-MEM medium and the Lipofectamine 2000 (Invitrogen) reagent according to the manufacture’s protocol.

Zhu Z., Du X., Li P., Zhang X., Yang F., Cao W., Tian H., Zhang K., Liu X, & Zheng H. (2018). Early Growth Response Gene-1 Suppresses Foot-and-Mouth Disease Virus Replication by Enhancing Type I Interferon Pathway Signal Transduction. Frontiers in Microbiology, 9, 2326.

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Plasmid Construction and Antibody Generation

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The full-length porcine NME1 cDNA was amplified and inserted into pcDNA^TM3.1/myc-His(-)A vector (Invitrogen) to generate a plasmid expressing Myc-tagged NME1 (Myc-NME1). A series of plasmids expressing Flag-tagged viral proteins were constructed as described previously⁵². HA-tagged p53 plasmid (HA-p53) was constructed by inserting full-length p53 cDNA into pCAGGs vector (including a C-terminal HA tag). Flag-p53, p53-Luc reporter plasmids and control plasmid Renilla luciferase pRL-TK were kindly provided by Zhiyong Ma (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China)^{53 (link)}. All the expressing plasmids used in this study were analyzed and verified by DNA sequencing.
Commercial antibodies included: mouse monoclonal anti-Myc, monoclonal anti-Flag, rabbit polyclonal anti-NME1, and mouse monoclonal anti-β-actin (Santa Cruz Biotechnology); mouse monoclonal anti-hemagglutinin (HA) (BioLegend); mouse monoclonal anti-Flag (Sigma); rabbit polyclonal anti-eukaryotic translation initiation factor 4 gamma (eIF4G) (Abcam); rabbit polyclonal anti-LC3B (Sigma); rabbit monoclonal anti-ATG12 (Cell signaling technology); rabbit polyclonal anti-VP1 antibody was prepared by our laboratory as described previously^{48 (link)}.

Feng H.H., Zhu Z.X., Cao W.J., Yang F., Zhang X.L., Du X.L., Zhang K.S., Liu X.T, & Zheng H.X. (2018). Foot-and-mouth disease virus induces lysosomal degradation of NME1 to impair p53-regulated interferon-inducible antiviral genes expression. Cell Death & Disease, 9(9), 885.

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Myc-tagged DDX1 Expression Construction

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A Myc-tagged expression construct was generated by inserting the cDNA of porcine DDX1 into the pcDNATM3.1/myc-His(–)A vector (Invitrogen, Carlsbad, CA, USA).
The commercial antibodies used in this study include an anti-Myc monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology), an anti-IRF3 monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), an anti-P-IRF3 monoclonal antibody (Cell Signaling Technology), an anti-DDX1 polyclonal antibody (Abcam, Cambridge, MA, USA), and an anti-β-actin monoclonal antibody (Santa Cruz Biotechnology). An anti-VP1 polyclonal antibody was prepared in our laboratory (Li et al.2017 (link)). Anti-3D polyclonal antibody (500 μg/mL) was produced in rabbit by immunization with FMDV 3D protein.

Xue Q., Liu H., Zeng Q., Zheng H., Xue Q, & Cai X. (2019). The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication. Virologica Sinica, 34(6), 610-617.

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