Gene Therapy and Molecular Biology Vol 7, page 37 Gene Ther Mol Biol Vol 7, 37-42, 2003
Antigenicity and immunogenicity of HIV envelope gene expressed in baculovirus expression system Research Article
Alka Arora1, Pradeep Seth2* 1 2
Post Doctoral Fellow, Department of Medical Genetics and Microbiology, University of Toronto, Canada. Professor and Head Department of Microbiology, All India Institute of Medical Sciences, India.
__________________________________________________________________________________ *Correspondence: Dr. Pradeep Seth, Professor and Head, Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India -110029.Tel: 91-11-652 6814; Fax: 91-11-686 2663; E mail: pseth@aiims.aiims.ac.in Received: 28 December 2002; Accepted: 5 February 2003; electronically published: July 2003
Summary Human immunodeficiency virus type I (HIV-1) envelope gene was expressed in Spodoptera frugiperda (Sf21) cells. DNA constructs encoding env-tat-rev genes were cloned into the baculovirus expression vector pBacPAK9. Recombinant baculovirus was prepared by cotransfection with linearized wild type virus DNA. Western blotting of cell extracts containing recombinant HIV-1 proteins demonstrated expression of HIV-1 gp160 and its complete cleavage products gp120 and gp41. A time course experiment suggested that the maximum expression was observed at 48-hrs post infection. In order to measure the biological activity recombinant HIV envelope proteins were used for lymphocyte proliferation assay. The results demonstrated that recombinant gp160 and its cleavage products were antigenically and functionally authentic. tend to decrease with progression of clinical symptoms (Lange et al, 1986; Goudsmit et al, 1987). Recombinant antigen based EIAs have been shown to be more sensitive, especially in detecting early seroconverters and specific than peptide or virus lysate based EIAs (Johnson 1992; Galli et al, 1996). The main objective of this study was to obtain large quantities of purified recombinant protein, suitable to be used as an immunogen and for development of HIV-1 detection kit. We used Baculovirus expression vector system for expressing HIV-1 Gp160 as this system results in efficient processing of the protein, posttranslational modifications and is known to give high yields of expressed protein.
I. Introduction HIV genome, like other retroviruses encode for Gag, Pol and Env. In addition, it also encodes for 6 regulatory and accessory proteins Tat, Rev, Nef, Vif, Vpr and Vpu. The major structural protein encoded by env gene of HIV1 consists of a protein of 850-880 amino acids. Extensive glycosylation of this precursor protein results in the production of Gp160 monomers, which then assemble into oligomers for transport from ER to the plasma membrane (Earl et al, 1991). During transport from Golgi, intracellular cleavage of Gp160 yields an outer envelope glycoprotein Gp120 and trans-membrane glycoprotein Gp41 (Kozarsky et al, 1989). Specifically, the HIV viral envelope protein Gp120 is important for virus-receptor interaction and virus entry (Kowalski et al, 1987, Hill et al, 1997). Gp41 is known to play a central role in the envelope glycoprotein oligomerization and fusion function (Poumbourios et al, 1997). HIV infection results in the production of HIV specific antibodies, therefore detection of these antibodies by ELISA and Western blot assay remains the basis of blood donor and patient screening. Serum specimen from HIV infected people regardless of their clinical stage react efficiently with precursor glycoprotein Gp160 or its cleavage product Gp120 and Gp41 (Lange et al, 1986; Goudsmit et al, 1987). Antibodies to gag protein p24 are the earliest protein detectable by Western blot after infection, however, these
II. Materials and Methods A. Plasmids, cells, reagents and peptides pCR-Script SK (+) cloning vector was purchased from Stratagene, LaJolla, CA, USA. pBRU plasmid containing complete genome of BRU strain of HIV-1 cloned in pUC18 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA. BacPAK, Baculovirus expression system was purchased from Clontech (BD Biosciences Clontech, Palo Alto, CA). Plasmids were grown in DH5! strains of Escherichia coli (Life Technologies, Gaithesburg, MD, USA), and purified using Wizard miniprep columns (Promega Corp, Madison, WI). TNMFH media for insect cell culture was obtained from HyClone (Genetix, New Delhi, India). TNM-FH medium contains Grace's
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