Cloning and expression of HIV 1 Tat Protein and injection of Tat protein in camel to isolation of nanobody

Abstract

Designing a novel therapeutic agent has been a critical a challenge for HIV virus treatment. The aim of the present study was the isolation of VHH (Nanobody) gene from camel antibody library in order to present a therapeutic nanobody against Tat protein of HIV. Human immunodeficiency virus (HIV-1) needs to Tat protein for the replication. The results of recent studies have showed that neutralizing antibodies against Tat protein can inhibit HIV virus replication. At first, a DNA fragment of encoding Tat protein of HIV-1 was synthesized and expressed in E.coli. Next, Tat protein was purified by NTA affinity chromatography. The purified recombinant protein was formulated with Freund's adjuvant and injected to one camel for five times. Then, total RNA was extracted from camel lymphocytes and VHH fragments synthesized and amplified using RT-PCR and Nested- PCR methods.The 17KD Tat protein purification efficiency and its conformation were confirmed by SDS-PAGE and Western blot, respectively. The 600 and 400bp of VHH genes were produced by RT-PCR and Nested- PCR shown by gel electrophoresis, respectively. The nanobody may be a useful a promising drug for treatment of HIV. The small sizes of nanobodies give them the potency of the recognizing the cryptic epitopes of tat and neutralizing the virus.