Recombinant, Expression And Application Of HIV-1 Gag Protein

Acquired immunodeficiency syndrome (AIDS) is one of the most influential and harmful diseases all over the world now and the prophylaxis and treatment for AIDS are extremely urgent. Human immunodeficiency virus (HIV) is the causative agent of AIDS, in which gene the gag gene encoding for core proteins is the most conservative gene sequence. The core proteins, also called Gag proteins mainly including p24 and p17 proteins, are immunogenic and anti-Gag antibodies often are first ones detected in blood after infected, so the Gag proteins run a very important role for AIDS' test.The recombinant, expression and application of HIV Gag protein are studied in this paper. Objective: Lay a foundation for developing diagnostic kit for HIV antibody. Methods: According to the HIV-gag gene sequence gotten from Genbank, the primers are designed respectively: Forward Primer (gagF), 5'-CGGATCCA ATG GGT GCG AGA GCG TCA-3' and Rerverse Primer (gagR), 5'-CGGAATTCGT GTC GTT GCC AAA GAG TGA-3'. The DNA of gag was cloned from HIV gene by polymerase chain reaction (PCR) and was inserted into pTrcHis2 A plasmid by using T4 DNA ligase after cleaved by BamH I and EcoR I , so the gag-pTrcHis2 A recombinant plasmid was constructed. The plasmid was transformed into E.coli BL21(DE3) and induced by IPTG to express. The expressed Gag antigen was detected by SDS-PAGE, Western blot(Wb) and ELISA after purified by Ni2+-NTA affinity chromatography. Result: SDS-PAGE showed the relative molecular weight of the antigen was about 41kD and was good agreement with the prediction value. By Wb, the accuracy rates for the antigen against the positive and negative sera were 100%(10/10) and 100%(40/40) respectively. By ELISA, the identity rates of lOng antigen used for detecting positive and negative sera diluted 100 times were 100%( 10/10) and 100%(80/80) respectively. Conclusion: The prepared recombinant Gag antigen was properly expressed and showed high antigenic specificity and sensitivity. The recombinant antigen was suitable for developing diagnostic kit for HIV-Ab.