Modified human immunodeficiency virus membrane protein and application thereof
A technology of recombinant protein and furin, which is applied in the direction of antiviral immunoglobulin, virus, viral peptide, etc., can solve the problems of HIV-1 irreversibility, loss of pathogenic infection ability, etc., and achieve uniform composition, three The effect of increasing the polymer content and improving thermal stability
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Embodiment 1
[0204] Embodiment 1: Expression of TSTIP protein of two strains of NL4-3 / BG505
[0205] 4-3 / BG505 TSTIP modification design
[0206]The base and amino acid sequences of the two strains BG505 / pNL4-3-gp160 on NCBI were used as templates for transformation. Taking the transformation of BG505 as an example, according to the three-dimensional cryo-electron microscope structure of BG505-SOSIP (PDB: 4tvp), part of the loop sequence (aa610-616, WNSSWSN) at the C-terminal of β27 of BG505 gp41 was removed, and the C-terminal of gp120 including furin enzyme The sequence of 10 amino acids including the cleavage site (aa501-511, AKRRVVGREKR). The truncated C-terminus of β27 of gp41 was then linked to the N-terminus of gp120, and the truncated C-terminus of gp120 was linked to the N-terminus of the α8 domain of gp41. The amino acid sequence of the modified complete gp140 is as follows: α6 / α7 / β27 (501-606) + part of the loop region between β27 and α8 (607-609) + gp120 (33-500) + the loop...
Embodiment 2
[0216] Embodiment 2: NL4-3 / BG505 TSTIP protein purification
[0217] After 6 days of transient transfection, collect the cell culture medium, centrifuge at 7000g for 10 minutes with JA-14 rotor, take the cell supernatant, centrifuge at 20000g for 10 minutes, take the supernatant and filter it twice with a 0.22um pore size filter membrane, and use this sample for the next step of Ni-excel Column purification.
[0218] Purification by Ni affinity chromatography using the AKTA system;
[0219] Instrument system: AKTA Pure preparative liquid chromatograph;
[0220] Purification medium: Ni Sepharose excel affinity medium; buffer: divided into A and B buffer, A solution is 1×PBS buffer, B pump is 1×PBS+250mmol / L imidazole buffer;
[0221] System sample flow rate: 8mL / min; detection wavelength: UV@280nm
[0222] System elution flow rate: 4ml / min; detection wavelength: UV@280nm
[0223] Elution conditions: 20mM imidazole was used to elute the impurity protein, and the 250mM imidaz...
Embodiment 3
[0224] Example 3: Identification of biochemical properties of NL4-3 / BG505 TSTIP protein
[0225] SDS-PAGE:
[0226] Dilute the concentrated sample and BG505 / 4-3SOSIP protein in Example 2 to 1ug / ul, take two tubes of 50ul samples, add 10ul reduced 6Loading Buffer and 10ul non-reduced 6Loading Buffer respectively, prepare reduced samples and non-reduced samples, reduced samples In a boiling water bath at 100°C for 10 minutes. Take 10ul of the reduced and non-reduced samples and electrophoresis at 80V for 120min in 8% SDS-PAGE. After staining with Coomassie brilliant blue, the electrophoresis bands are displayed. For the electrophoresis results, see Figure 2A-2B . SDS-PAGE analysis shows that after one-step Ni-EXCEL purification, high-purity BG505 / NL4-3TSTIP protein can be obtained, and the TSTIP protein is a complete 140KD band in the presence of DTT, while the BG505 / NL4-3SOSIP protein is under reducing conditions A 120KD band appeared, indicating that the protein designed...
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