Overexpression of recombinant HIV-1 Subtype C Tat and Nef in a Salmonella vaccine vector

Abstract

Tat and Nef are very important regulatory proteins of HIV-1. They enhance viral replication and down-regulate expression of MHC Class I molecules, respectively. The antigens are now considered to be targets for HIV vaccine development. The expression of Tat and Nef in Salmonella vaccines has not previously been investigated. In this study, HIV-1 Subtype C tat and nef genes were cloned into an expression plasmid and their expression investigated in Salmonella. Very high-level expression of the two HIV-1 antigens was demonstrated in the recombinant Salmonella. The antigens were also successfully purified in bulk from the bacterium.Salmonella can therefore potentially be used to overexpress HIV-1 antigens and used as a possible delivery system in HIV-1 vaccine development.

Keywords: HIV; Overexpression; Salmonella; recombinant; regulatory proteins; viral replication.

Figure 1

Expression of HIV-1 Subtype C Tat and Nef in recombinant E. coli and Salmonella bacteria. (A) Coomassie-stained SDS-PAGE showing Tat expression by recombinant E. coli. Lane M: Marker, Lane 1: E. coli expressing Tat, Lane 2: E. coli carrying an empty plasmid (negative control). The position of Tat protein band is indicated by an arrow. (B) Coomassie-stained SDS-PAGE showing Nef expression by recombinant E. coli. Lane M: Marker, Lane 1: E. coli expressing Nef, Lane 2: E. coli carrying an empty plasmid (negative control). The position of Nef protein band is indicated by an arrow. (C) Coomassie-stained SDS-PAGE showing Tat and Nef expression by recombinant Salmonella. Lane M: Marker, Lane 1: Salmonella expressing Tat, Lane 2: Salmonella expressing Nef, Lane 3: Salmonella carrying an empty plasmid (negative control). The positions of Tat and Nef protein bands are indicated by arrows.

Figure 2

Purification of HIV-1 Subtype C Tat and Nef from recombinant Salmonella under denaturing conditions (A) Coomassie-stained SDS-PAGE showing the purification of Tat from recombinant Salmonella. Lane M: Marker, Lane 1: Total bacterial cell lysate, Lane 2: Flow-through, Lane 3: First wash, Lane 4: Second wash, Lanes 5-8: Tat elution fractions. The position of Tat protein band is indicated by an arrow. (B) Coomassie-stained SDS-PAGE showing the purification of Nef from recombinant Salmonella. Lane M: Marker, Lane 1: Total bacterial cell lysate, Lane 2: Flow-through, Lane 3: First wash, Lane 4: Second wash, Lanes 5-8: Nef elution fractions. The position of Nef protein band is indicated by an arrow.