Amplification, Cloning And Expression Of HIV-1 GAG Gene Fragment And The Purification And Identification Of The Expressed Product

Acquired immune deficiency syndrome (AIDS), first reported in 1981, has become an unprecedented threat to global health. The causative agent of AIDS has been clarified as human immunodeficiency virus (HIV). No cure for AIDS available yet, the development of a vaccine will be of great importance to the prevention and treatment of AIDS. P17/P18 and P24/P25 of HIV-1 core proteins are potential candidate vaccines. The two vaccines go-ahead for human tests approved by the Great Britan are P24-VLP and HGP30 (A synthetic peptide derived from P17/P18 with a single amino acid difference), both are of capsid proteins. The objective of this study is to clone and express a polypeptide (called P20) which covers most of P18 and a small part of P25 and carries HGP 30 sequence in the center.To carry out research works on molecular biology of AIDS, we introduced the cDNA clone of a HIV-1 strain ARV-2 from the United States of America in 1988. We subcloned this full-length cDNA into pUC19 at Kpn I site, and four subclones were obtained. They are pJE 332 which contains the whole env gene, pJG423 which contains LTR, gag and part of pol gene, pJP163 and pJP032.Amplification in vitro or polymerase chain reaction (PCR) is a newly developed technique that has exhibited its great power in many research fields. There have been some reports using PCR technique to clone specific genes with advantagies of rapidity and simplicity. In the present paper, the primers PG01 and PG02 were synthesized in which the EcoR I and BamH I recognition sites as well as the initiation and termination codons were designed for convenience of subsequent cloning and expression. Micrograms of target gene seqence was produced by 30 rounds of amplification with pJG 423 as a template. The amplified gene fragment was digested with both EcoR I and BamH I and ligated to pUC19 predigested with the same restriction endonucleases. The recombinant plasmed pCY52 was identified and the cloned P20 gene was inserted into M13mp18...