Expression and Purification of Glycoprotein G1 of Hantaan Virus in E. coli System

ABSTRACT

Envelope glycoprotein 1 (G1) and glycoprotein 2 (G2) of Hantaan (HTN) virus are believed to be major viral antigens that can induce neutralizing immunity against HTN virus infection. The purpose of this study is to clone and express G1 gene in an E. coli expression system. The truncated G1 gene (amino acid residues 35 to 123) of the HTN virus strain 76-118 was amplified by polymerase chain reaction (PCR). The 0.28 kb PCR product was cloned into pCR2.1 vector and named as pCGS1. The truncated G1 gene was excised from the pCGS1 and subcloned into the BamHI and SalI sites of pGEX-4T-2 and named pGGS1. The nucleotide sequence of the 0.28 kb truncated G1 gene was determined. It is revealed four non-silent nucleotide substitutions between the published sequence of strain HTN virus strain 76-118 and our stock of HTN virus strain 76-118 (passaged several times in our laboratory). The first G1 mutation was found to constitute an A to G nucleotide substitution, giving raise to an asparagine to serine mutation at residue 64. The second G1 mutation was found to constitute an A to C nucleotide substitution, giving raise to an lysine to threonine mutation at residue 112. The third G1 mutation was found to constitute an A to C nucleotide substitution, giving raise to an lysine to threonine mutation at residue 112. The fourth G1 mutation was found to constitute an G to A nucleotide substitution, giving raise to an glutamic acid to lysine mutation at residue 117. The truncated G1 gene was expressed as a 37 kDa protein fused to glutathione-S-transferase (GST). The GST fusion protein was purified by Glutathione Sepharose 4B affinity chromatography and reacted with the sera from patients of hemorrhage fever with renal syndrome (HFRS). One of 12 serum samples from HFRS patients was reactive with the 37 kDa fusion protein strongly. Three sera reacted moderately with the fusion protein. Six sera reacted only weakly with the protein, while remaing two were non-reactive. Control sera from patients with scrub typhus leptospirosis, or negative HFRS did not react with the recombinant fusion protein.