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STATEMENT OF WORK

Hybrid Screening

· PURPOSE AND OBJECTIVES OF THE ACQUISITION

The goal of this project is to identify host-proteins that interact with Ebola virus components. Goal 1 is to identify host proteins from a human leukocyte library that specifically interact with VP24. Goal 2 is to identify host proteins from a porcine testicle cell line that interact with the Ebola Reston virus genomic trailer element. Yeast 2-hybrid and Yeast 3-hybrid are chosen due to its high selectivity and low false positive rate when compared to other methodologies such as mass-spectrometry.

· CONTRACTOR REQUIREMENTS:

Yeast Two-Hybrid Screening

· Bait vector construction

· Bait protein toxicity and self-activation test

· Perform large scale library screening

· Perform phenotypic validation and prey vector rescue

Yeast Three-Hybrid Screening

· Create custom cDNA library construction

· Bait RNA hybrid construct generation

· Bait RNA toxicity and self-activation test

· Perform large scale library screening

· Perform phenotypic validation and prey vector rescue

· CONTRACTOR SUPPLIED MATERIALS

The Contractor shall provide all necessary supplies and equipment necessary to perform the services listed above.

· GOVERNMENT FURNISHED MATERIALS:

The Government will supply:

· The bait RNA plasmid

· The RNA sequence

· Total RNA extracted from the porcine cell line for library construction

· The bait protein (VP24) expression plasmid and the protein sequence

· REPORTING REQUIREMENTS AND DELIVERABLES

Vendor will deliver sequence information and the plasmid DNA of the prey proteins; bait plasmid DNA; project report with detailed procedures and results.

· INSPECTION AND ACCEPTANCE:

a) All work will be inspected and approved by NIH.