Reagents
-
Restriction endonucleases, T4 DNA ligase, Taq DNA polymerase and Agarose Gel DNA Fragment Recovery Kit were obtained form the Takara Company (Dalian, China). Anti-His-Tag monoclonal antibody and goat anti-mouse IgG/HRP were purchased from New England Biolabs. All other chemicals were of analytical reagent grade.
Cloning of vif by PCR
-
For PCR amplification of vif, a forward primer (5'-tgcaccatggaaaacagatggcaggtgat-3') and reverse primer (5'-ccgctcgagggtgtccattcattgtat-3') were used, which overhang the Nco I and Xho I sites (underlined) respectively. In a 50μL reaction system, the template used for vif open reading frame (ORF) amplification by polymerase chain reaction (PCR) was 1 μL plasmid of HIV-1 NL4.3 cDNA. Amplification was accomplished using the following reaction conditions: initial heating at 94℃ for 5 min; then 30 cycles consisting of denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s and elongation at 72℃ for l min; final extension at 72℃ for 10 min. The gene products were ligated to the cloning vector pGEM-T. The DNA sequence was determined using an automated sequencer.
Cloning of APOBEC3G by RT-PCR
-
RNA was extracted from H9 cells using TRIzol reagent according to the manufacturer's specifications. APOBEC3G was amplified using these primers: (F): 5'-ccatggctatgaagcctcact-3', (R): 5'-ctcgagcgttttcctgatt ctg-3'. The first step of RT-PCR to synthese cDNA used the reaction condition: 42℃, 30 min. The second step used the reaction condition: initial heating at 94℃ for 5 min; followed by 35 cycles consisting of denaturation at 94℃ for 50 s, annealing at 55℃ for 1 min and elongation at 72℃ for 3 min; and final extension at 72℃ for 10 min. The gene products were ligated to the cloning vector pGEM-T. The DNA sequence was determined using an automated sequencer.
Construction of expression vector for vif and APOBEC3G
-
The amplified PCR product was digested with Nco I and Xho I followed by subcloning into the prokaryotic expression vector pET-32a. This generated the recombinant plasmid pET-vif and pET-APOB-EC3G respectively, in which the C-terminal domains of vif and APOBEC3G were fused with a His-tag for improved purification and immune identification. E.coli DH5α. competent cells were transformed with the ligation reaction, and the DNA was subsequently purified and checked by 1% agarose-gel electrophoresis after digesting with Nco I and Xho I. The samples were then sequenced to confirm the correct insert by the Shanghai Sangon Biology Technology Limited Company (sequencing primer is T7 terminator primer). The results were compared with the expected sequence using the vector NTI 8.0 software.
Recombinant pET-vif and pET-APOBEC3G expression E.coli
-
E.coli BL21 (DE3) competent cells were transformed with pET-vif and pET-APOBEC3G respectively, and the transformant cells were cultivated at 37℃ with shaking at 190 r/min in 5mL of LB medium supplemented with 100 μg of ampicillin and 34 μg of chloramphenicol per mL. Cultures were induced for expression at an OD600 of 0.6~0.8 with 1 mmol/L IPTG, and continued growth with shaking for 4 h. Cells were then harvested by centrifugation at 12 000 r/min for 1 min. Total cellular pellets were analysed by 12% SDS-PAGE and Western blotting.
Purification of recombinant Vif and APOBEC3G
-
Identified pET-vif / BL21 (DE3) and p ETAPOBE-C3G/ BL21 (DE3) cells were cultivated overnight. 8 mL Cultures were added to 800 mL 2×YT medium supplemented with 100 μg of ampicillin and 34μg of chloramphenicol per mL, and were cultivated at 37℃ with shaking at 190 r/min. Cultures were induced for expression at an OD600 of 0.6~0.8 with 1 mmol/L IPTG, and continued growth with shaking for 4 h. Cells were harvested by centrifugation at 5000 r/min for 20 min at 4℃. Cell pellets were thoroughly suspended in 10 mL of lysis buffer (10 mmol/L Tris-HCl, 100 mmol/L NaH2PO4, pH 8.0) per gram of cell paste. The cells were then sonicated at 50% amplitude on ice for 300 s (30 s on/30 s out) followed by centrifugation at 8 000 r/min for 30 min at 4℃. The pellet washing was repeated for a total of three washes and the final pellet was resuspended in 10 mL extraction buffer (10 mmol/L Tris-HCl, 100mmol/L NaH2PO4, and 7.5 mol/L guanidine-HCl, pH 8.0) per gram of cells, followed by incubation overnight at 15℃ with gentle mixing. Following the step, the sample was centrifuged at 12 000 g for 30 min at 4℃. 2 mL supernatant was then filtered through a 0.45 μm syringe filter and applied to an equilibrated gel filtration chromatography column (superdex75、200) using a 2 mL Superloop at l mL/min on the FPLC, and eluted by buffer B(100 mmol/L NaH2PO4, 10 mmol/L Tris, 8 mol/L Urea, pH 6.2). The purified productions were analyzed by 12% SDS-PAGE and Western blotting.
Western blotting analysis for recombinant protein
-
For Western blotting, samples were separated using 12% SDS-PAGE and then transferred to pre-cut nitrocellulose membrane. The membranes were blocked for l h at ambient temperature in phosphatebuffered saline (PBS) with 5% nonfat dried milk, and then incubated for l h with rat anti-His monoclonal antibody (1:200). The membranes were rinsed thrice with PBST (1×PBS, 0.05% Tween 20, pH 7.5), followed by incubation with goat anti-rat IgG antibodies conjugated to horseradish peroxidase (HRP) (1:400) for l h at ambient temperature. The Vif and APOBE-C3G samples were visualized by incubation with DAB (0.06 mg/mL) and 0.1% H2O2.
Protein concentration detection
-
Concentration of protein was detected by the Bradford method.
Preparation polyclonal antibodies
-
Polyclonal anti-recombinant protein antibodies were raised in two rabbits. Rabbit sera were taken prior to immunization as negative control. Rabbits were immunized on days l and 28 with 500μg recombinant protein in their legs. Each immunization was done with l mL 500 μg/mL recombinant protein, which was diluted to 2 mL with Freund's complete adjuvants. Serum samples were collected two weeks after the last injection and stored at -70℃. Polyclonal antibodies in sera were tested by indirect ELISA.
Indirect ELISA
-
Microtiter plates were coated overnight at 4℃ with 60 ng Vif and 15ng APOBEC3G per well in coating buffer (carbonate-buffer, pH 9.6). Unbound antigen was removed by washing with PBST solution (1×PBS, 0.2%Tween 20). 2% bovine serum albumin (BSA) wan added to block the coated wells for 2 h at 37℃. Serum samples diluted 200-204 800 fold were added to the coated wells and incubated for 90 min at 37℃ in a humidified atmosphere. Wells were washed with PBST solution and incubated with 100 μL of HRP-conjugated goat anti-rabbit immunoglobulins. After 60 min of incubation at 37℃, wells were washed and the antigen-antibody complexes were detected by the addition of 100 μL of A and B solution. After 15 min of incubation at room temperature, the enzymatic reaction was stopped by adding 50 μL of 2 mol/L sulfuric acid to each well. Optical densities (OD) were measurd at 450/630 nm. The cutoff value was set two standard deviations above the average of negative control sample which was taken before the immunization.
Construction of pEGFP-vif and transient cell transfection
-
Expression vectors pEGFP-vif expressing HIV-l Vif. The vif gene sequence was amplified by PCR from the HIV plasmid NL4.3 cDNA. For DNA transfection, 6-well plates were seeded with Hela-CD4-LTR-β-gal-CXCR4 cells at a density of 1×105 cells/well in 2 mL of DMEM cell culture medium and maintained at 37℃ and 5% CO2.The following day, the optimal amount of pEGFP-vif for transfection was determined to be 6 μg. Lipofectamine 2000 (Invitrogen, Carlsbad, California) was used essentially according to the manufacturer's instructions, at 3 μL Lipofectamine in FBS-and antibiotic-free DMEM. At 6 h post-transfection, the medium of the cells was replaced with fresh DMEM containing 10% FBS.
Cell preparation
-
Hela-CD4-LTR-β-gal-CXCR4 cells transfected by pEGFP-vif, H9 cells and MT4 cells were fixed in 4% formalin for 10 min at room temperature.
Immunoenzyme and immunofluorescence assay
-
Cells were incubated with polyclonal antibodies for l hour at 37℃. After washing three times with PBS, cells were incubated with HRP-conjugated or TRITC-conjugated goat anti-rabbit antibodies for 45 minutes at 37℃. Immunostaining was revealed by exposure to fresh DAB solution for 5 min, and rinsed with distilled water. The result of immunofluoresence was observed directly by fluorescent microscope.