Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Authors

  • Zackary Tajin Park Boston College

DOI:

https://doi.org/10.6017/eurj.v13i1.9604

Abstract

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

Author Biography

Zackary Tajin Park, Boston College

Zackary Park is from Raleigh, North Carolina, and is currently studying Biochemistry in the Honors Program of the Morrissey College of Arts and Sciences. From a young age, he has always been interested in science, and had a desire to understand how and why things worked. After graduation this year, he will be taking a gap year to research other topics of interest. He will be applying to medical school in the following year in hopes of achieving his dream of becoming a doctor. His other interests include weightlifting, traveling, and eating the best food in the world.

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Published

2017-09-07

How to Cite

Park, Z. T. (2017). Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity. Elements, 13(1). https://doi.org/10.6017/eurj.v13i1.9604

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Articles