HIV-1 10384 200026032 UDC HIV-1 : : : : 2003.7.15 : 2003.7.24 2003 1
HIV-1 2003 7 12 2
HIV-1 HIV-1 PCR SCG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD Synechococcus sp.pcc7942 groesl UB Km r phrf3 rbcs polya ph3eue, Synechococcus sp.pcc7942 hrf3 pprs-1 Synechococcus sp.pcc7942 hrf3 ppreue ph3eue HindIII 5.4kb HindII 1-3kbDNA groesl UB rbcs Km r, ph3eue ppreue Synechococcus sp.pcc7942 Synechocystis sp.pcc6803 DNA PCR DNA 42 SDS-PAGE, Ub-Tep 11KD 3
HIV-1 Western-blot Elisa HIV-1 Synechococcus sp.pcc7942 Synechocystis sp.pcc6803 DNA DNA HIV-1 HIV-1 HIV-1 HIV-1 Synechococcus sp.pcc7942 Synechocystis sp.pcc6803 HIV-1 Neutralizing Epitope Peptides Express in cyanobacteria Abstract Cyanobacteria are a diverse and widely distributed group of prokaryotes, phylogenetically distant from other eubacteria. They are important contributors to photosynthesis, nitrogen fixation and evolution. In recent years it has been rapid progresses on genetic engineering of cyanobacteria with the advance of molecular biology in cyanobacteria. In this study, the nucleotide sequence of HIV-1 three epitope peptides (SCG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) was obtained by PCR. The integrative vector ph3eue, which contained the homologous fragment of Synechococcus sp.pcc7942 hrf3, groesl promoter, target gene(ub-tep), rbcs-polya terminator and reporter gene(nptii gene, Km r ), was constructed. 1~3Kb HindIII DNA fragments of total DNA of Synechococcus sp.pcc7942 and Synechocysis sp.pcc6803 were cloned into HindIII site of ph3eue to replace the hrf3 to make into random integrative vectors. Then Meanwhile using EcoRI-HindIII restriction-digested segment of endogenous small plasmid of pprs-1 to replace the hrf3 to form shuttle vector ppreue. These confirmed plasmids were introduced into cells of Synechococcus sp.pcc7942 and Synechocystis sp.pcc6803 and putative transformants were screened by Kanamycin. Results of PCR showed that the target nucleotide had been genetically integrated into genome DNA of host cell by integration vectors. Further confirmation was made by SDS-PAGE, Elisa and Western Blot. Results indicated special 11kD protein expressed in transformants that was coherent with the aimed protein, Ubiquitin-SCG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD, in both molecular weight and immunological characteristics. In this study the HIV-1 epitope peptides had successfully expressed in the cyanobacteria, which gives the basis for the study of oral hiv-1 vaccine. It s the first time to use cyanobacteria as host cell to express HIV-1 peptides. We hope the strong neutralzing antibody can be induced in mice by oral administration of these transformant components. 4
HIV-1 Keywords: epitope peptide HIV-1 vaccine express Synechocysis sp.pcc6803 Synechococcus sp. PCC7942 Abstract 1 1 1 2 10 3 14 16 1 16 2 19 26 1 26 2 Synechococcus sp.pcc 7942 Synechocytis sp.pcc 6803 36 3 Synechococcus sp.pcc7942 Synechocystis sp.pcc6803 43 45 1 HIV-1 45 2 46 3 DNA 47 4 Synechocystis sp. PCC6803 48 5 49 6 49 5
HIV-1 51 58 6
HIV-1 1 1.1 Human immunodefieiency virus, HIV Acquired immunodefieiency syndrome, AIDS AIDS 1981 HIV HIV-1 HIV-2 HIV-1 M O M A-J HIV 2 RNA 5 HIV 9.8kb 2 Gag Env 3 6 tat,vif,vpr[vpx],nef,rev 1. HIV www.virology.net Figure 1 Genome map of HIV CD4 HIV CD4 HIV CCR-5( ) CXCR-4 T 7
HIV-1 gp120 gp41 HIV 0 6 HIV 7 10 HIV P24 2 4 P24 10 14 IgM HIV 6 16 IgG 3 6 95% HIV IgG HIV env gp160 gp120 gp41 gag 24 pol HIV 6 1 HIV env gag pol HIV p24 HIV p24 AIDS 2 HIV CD4+ HIV RNA [1] Figure 2. Clinical, immunological and virological course of HIV-1 infection HIV T CD4+ 8
HIV-1 [2] 1 HIV Table.1 Pathogenesis of HIV infection T B IL-1 IL-1 CD4+ CD4+ IL-2 CD8 HIV, T 2002 4200 1920 15 320 2002 500 2002 97 720 2001 10% 100 2002 25 [3] 120 2010 126 4500 40% AIDS 1.2 1.2.1 HIV 20 AIDS HIV [4], 9
HIV-1 gp120,gp160,gp41 Gag Ty VLPs HIV V3 CTL/Th DNA DNA 9 11 1 16 12 1 2 2 I I/II II III www.iavi.org Table 2. I I/II II and III trials of HIV-1 vaccines IAVI; University of Oxford; UK Medical Research Council; DNA-Ankara A University of Nairobi; Kenya AIDS HIV DNA I/I Vaccine Initiative; Imperial College + HIV Ankara of Science, Technology and Medicine; Cobra Therapeutics Ltd.; IDT GmbH DNA- B HIV DNA Merck & Co. Inc. + HIV GlaxoSmithKline plc, HIV Vaccine Trials Network Vaccine Research Center, US National Institute of Allergy and Infectious Diseases; Vical Inc. Agence Nationale de Recherche sur le VIH/SIDA (France), Biovector Therapeutics SA VaxGen Inc. VaxGen Inc., Aventis Pasteur SA, US National Institutes of Health, Walter Reed Army Institute of Research, Thailand Ministry of Health gp120, nef-tat B HIVgp120 + nef-tat DNA B HIV DNA B AIDSVAX(B E ) gp120 III ALVAC-AIDSVAX( B E ) II HIV II ALVAC + gp120 AIDSVAX HIV HIV HIV HIV DNA HIV 10
HIV-1 HIV HIV-1 B C E GAG GAG-V3 VLP VLP VLP 1.2.2 HIV HIV HIV T B 8 24 -MHC-TCR CTL HIV HIV T HIV Palker [5] Ahlers [6] Eroshkin [7] T B TBI 1.2.2.1 B HIV HIV env- nef- vpr- reverse 11
HIV-1 transcriptase- tat- gag- V3 PND V3 V1-V2 CD4 C4 HIV V3 T T V3 [8] [9] [10] four- -helix bundle [7] original antigenic sin OAS [11] V3 [12-16] 11 multi-epitope polypeptides,mep TAB9 I [17] [18-22] HIV ELDKWA RILAVERYLKD GPGRAFY 1.2.2.2 B T HIV T B T B (T and B cell epitopes containing immunogen TBI) Eroshkin [7,23] HIV BH10 gag env 4 T 5 B 12
HIV-1 four- -helix bundle HIV-1 TBI HIV-1 TBI 1.2.2.3 T HIV HIV gp120 C4 419 436 -helix, C4 CD4 [24] CD4 MHC I HIV [25,26] CD8 + CTL CTL HLA CTL, HIV [27,28] T HIV gp120 gp41 p17 p24 pol nef CTL CTL MHC-I II HLA-I T AIDS 1.2.3 HIV Th1 Th2 T 13
HIV-1 CTL T CTL CTL CTL [29] IL12 IL1 Th1 [30,31] T B CTL IL-12 1.2.4 HIV HIV CTL gp160 LT R192G CTL Th1 Th2 gp41 gp120 [32] V2-V3 SIV DNA -SIV HIV HIV HIV HIV 3 14
HIV-1 3 www.iavi.org Table 3. IAVI Database of Preventive AIDS Vaccines in Human Trials B HIV-1 gp120 (AVEG HIV-1 C4-V3 C4-V3 Wyeth-Lederle 020) 4, TAB9 HGP-30 SynVac (rgp120/hiv-1mn Monovalent Octameric V3 Peptide Vaccine HIV-1 Peptide Immunogen, Multivalent HIV-1 Peptide Vaccine, Microparticulate Monovalent P3C541b Lipopeptide CLTB-36 (gp24e-v3 MN LIPO-5 LIPO-6T gp120 V3 B HIV-1 CIGB V3 B Aventis HIV-1 Env, Gag Pasteur Nef 6 B HIV-1 gag p17 Cel-Sci B HIV-1 gp120 V3 UBI gp120 V3 8 UBI B HIV-1 gp120 V3 UBI B HIV-1 gag UBI B HIV-1 p24 V3 Aventis Pasteur B HIV-1 gag, nef, pol 5 Aventis Pasteur LIPO-5 Aventis Pasteur B Biovector HIV-1 gag, nef, pol CD8 Aventis Pasteur B HIV-1 V3 (ANRS VAC 10) (ANRS-VAC 10) (ANRS-VAC- 04) (ANRS-VAC 04 bis) I/II, I/II, (UBI V106) I, (AVEG 011) I, I I (AVEG 017) (AVEG 023) I/I, (UBI V106) (AVEG 023) (AVEG 018) (AVEG 021) (ANRS VAC 03) (ANRS-VAC (ANRS-VAC02) 12) 15
HIV-1 1.2.5 HIV 1 HIV / 2 3 4 T MHC-II T 2 cyanophyta blue green algae cyanobacterium DNA G - a b I(PSI) II(PSII) 1996 Synechocystis sp. PCC 6803 2001 Anabaena sp. PCC 7120 2.1 1 DNA DNA DNA 2 DNA 3 CO 2 16
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