HIV mutant type surface glycoprotein and nanometer antigen and preparation method thereof
A surface glycoprotein and mutant technology, which is applied in the field of antigen preparation, can solve the problem that the vaccine cannot be reproduced, achieve good assembly effect and increase the effect of expression
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Embodiment 1
[0025] Example 1 Gene Amplification of HIV Mutant Surface Glycoprotein and Construction of Eukaryotic Expression Plasmid
[0026] 1. PCR amplification
[0027] (1) In this experiment, overlapping PCR was used to amplify the target fragment. Firstly, primers F1 and R2, F3 and R4 (as shown in Table 1) were used to amplify the target gene respectively; finally, primers F1 and R4 were used to amplify. Due to the introduction of two cysteine mutations, the mutations are introduced twice to form a double mutation. The PCR reaction system is the same, and the extension time is 1-2 minutes according to the length of the target fragment.
[0028] Table 1
[0029]
[0030]
[0031] (2) The PCR reaction system of each round of PCR reaction is 50 μL, as shown in Table 2, the reaction conditions are: 95 ° C for 5 min; 32 cycles, the cycle reaction is: 95 ° C for 1 min, 55 ° C for 1 min, 72 ° C for 1- 3 min (depending on the length of the fragment); extend at 72°C for 10 min, and ...
Embodiment 2
[0051] Example 2 Insect Cell Expression System Purification of HIV Surface Glycoprotein
[0052] 1. Bacmid transfection of sf9 cells
[0053] 1-2 hours before transfection, sf9 cells were spread on 6-well cell culture plates, and the cell density grew to 80%. Take Bacmid (about 6-7μg) and 6μL transfection reagent cellfectin Add 100 μL of insect cell culture medium (Sf-900II SFM) to IIreagent, and let stand at room temperature for 5 minutes. Mix the two and let stand at room temperature for 35 minutes. Add 800 μL of medium to each well of a 6-well cell culture plate, then add the bacmid and transfection reagent mixture, and incubate at a constant temperature of 27°C. 6 hours after transfection, the transfection solution was aspirated, 2 mL of medium containing 1% penicillin-streptomycin was added, and the constant temperature culture was continued at 27°C.
[0054] 2. Expression and identification of target protein
[0055] 3-4 days after transfection, draw 1 mL of the vi...
Embodiment 3
[0061] Example 3 Determination of the expression level of HIV mutant surface glycoprotein Western Blot
[0062] 1. Determination of total protein concentration of samples by BCA method: BCA protein concentration determination kit was purchased from Beyontian Company. According to the number of samples, prepare an appropriate amount of BCA working solution with 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), and mix well. Completely dissolve the protein standard, take 10 μL and dilute to 100 μL, so that the final concentration is 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the standard to the standard wells of the 96-well plate, and make up to 20 μL with the solution of the diluted standard. Add an appropriate volume of sample to the sample well of the 96-well plate, and make up to 20 μL with the diluted standard solution. Add 200 μL of BCA working solution to each well, and measure OD after standing at 37°C for 30 minutes 562 nm absorbance value. Afte...
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