![]() The PCR product was digested with BamHI and KpnI and inserted into the prokaryotic expression pQE30 digested with the same enzymes to create the p24 expression plasmid pQE30-p24. The HIV-1 p24 open reading frame was amplified from plasmid pHIV which contains the HIV-1 NY5 and LAV strain hybrid genome with the forward primer (5'-GA G GAT CCC CCA TAG TGC AGA ACC TC-3', BamHI site underlined), and the reverse primer (CC G GTA CCT TAG AAA ACT CTT GCT TTA TG-3', KpnI site underlined). Construction of the plasmid expressing the p24 protein Restriction enzymes, Taq DNA polymerase, and T4 ligase were purchased from TaKaRa Biotechnology Co. Plasmid pQE30 (Novagen, Darmstadt, Germany) was used for recombinant protein expression. Both strains were purchased from Invitrogen (Novagen, Shanghai, China). coli strains DH5α and BL21(DE3) were used for cloning experiments and protein expressions, respectively. Enzyme-linked immunosorbant assay (ELISA) and Western blot analysis demonstrated that the recombinant p24 proteins exhibited good immunoreactivity and immunogenicity. The protein was expressed in soluble forms and purified by Ni 2+-NTA affinity chromatography. In this study, a recombinant plasmid was constructed to express the His-tagged p24 protein in Escherichia coli. The p24 protein have been produced in a wide variety of systems, including Escherichia coli, Pichia pastoris, plant-based expression system, baculovirus-insect cell, etc. The p24 protein also can be used as an integral part of any multi-component HIV vaccine.Ī proper recombinant p24 protein with the same antigentic activity as natural p24 protein would be useful for a number of studies. The fourth-generation test assays for HIV infection is established on the basis of the p24 antigen detection and is able to find the HIV-infected at an early stage, resulting in shortened diagnostic windows. P24 antigen detection is also helpful for early diagnosis of HIV-infection. P24 protein is one of the detecting targets of most diagnostic kits. P24 protein is the major core protein of the virus particle and has been suggested as a specific target for antiviral strategies. The Gag protein is cleaved by a viral protease to release p17, p24 and p12 during viral maturation. Gag protein of HIV-1, a polyprotein of 55 kDa, is one of the most conserved viral proteins. Diagnosis of HIV infection, especially early diagnosis, is one of important part of AIDS prevention and control. The human immunodeficiency virus type 1 (HIV-1) is the main cause of the acquired immunodeficiency syndrome (AIDS). This soluble recombinant p24 protein specifically react with HIV infected sera and elicit HIV p24 specific antibodies in mice, indicating this soluble recombinant p24 protein could be a promising reagent for HIV diagnosis. In this work, we report the high level soluble expression of HIV-1 p24 protein in E. Subsequent ELISA and Western-Blot analysis demonstrated that the p24 protein had proper immunogenicity in inducing mice to produce HIV p24 specific antibodies. To study the immunogenicity of this soluble recombinant p24 protein, it was used to immunize mice for the preparation of polyclonal antibody. The results showed that the recombinant p24 protein could specifically react with the HIV infected sera. The recombinant protein was purified by nickel affinity chromatography and used to react with HIV infected sera. SDS-PAGE analysis showed that the His-tagged recombinant p24 protein was highly expressed in soluble form after induction in E. The PCR product was cloned into pQE30 vector, generating the recombinant plasmid pQE30-p24. ResultsĪccording to the sequence of the p24 gene, a pair of primers was designed, and the target sequence of 700 bp was amplified using PCR. The aim of this study was to express and purify the p24 protein in soluble form in E.coli. Recombinant p24 protein with natural antigenic activity would be useful for various studies, such as diagnostic reagents and multi-component HIV vaccine development. P24 protein is the major core protein of HIV virus particle and has been suggested as a specific target for antiviral strategies. ![]()
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