Preparation method of anti-HER3 single strand antibody
A single-chain antibody and bacterial strain technology, which is applied in the field of preparation of anti-HER3 single-chain antibody, can solve the problems of heavy economic burden for patients, affecting the efficacy and function of monoclonal antibody, and high production cost of monoclonal antibody
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0122] Example 1 Preparation of vector plasmid pGAPZαA-anti-HER3-scFv
[0123] 1. Extraction and purification of vector plasmid pGAPZαA
[0124] (1), the experimental method
[0125] 1. DNA kit to extract pGAPZαA plasmid
[0126] 1) Take 2ml of overnight cultured bacterial solution containing pGAPZαA in a sterile 2ml centrifuge tube, centrifuge at 10000rpm for 1min, and discard the supernatant;
[0127] 2) Take 2 ml of overnight cultured bacterial solution containing pGAPZαA in a sterile 2 ml centrifuge tube, centrifuge at 10,000 rpm for 1 min, and discard the supernatant;
[0128] 3) Add 250 μl Buffer S1 to resuspend the bacterial pellet;
[0129] 4) Add 250 μl Buffer S2, gently and fully turn up and down 6 to 8 times to mix evenly to fully lyse the cells until a translucent solution is formed;
[0130] 5) Add 350 μl of Buffer S3, gently and fully turn up and down to mix 6 to 8 times, and centrifuge at 10,000 rpm for 10 min;
[0131] 6) Aspirate the supernatant and trans...
Embodiment 2
[0213] The acquisition of embodiment 2 highly resistant strains
[0214] 1. Mass extraction, linearization and purification of recombinant plasmids
[0215] (1), the experimental method
[0216] 1) Take the strain prepared in Example 1 that contains the target gene and no base mutation confirmed by sequencing, inoculate 2 ml of low-salt LB medium containing 0.1% Zeocin according to 1% of the inoculum, and cultivate at 37° C. shaker at 170 rpm overnight;
[0217] 2) Inoculate 300 μl of the overnight cultured bacterial liquid with 1% of the inoculum into 30 ml of low-salt LB medium containing 0.1% Zeocin, and culture at 37° C. shaker at 170 rpm overnight;
[0218] 3) 30ml bacterial liquid cultured overnight is used to extract the recombinant plasmid by the kit method;
[0219] 4) Take 1 μl of the purified recombinant plasmid to measure the concentration by 1% agarose gel electrophoresis;
[0220] 5) Prepare a linearized single-enzyme digestion system according to the followin...
Embodiment 3
[0258] Example 3 Expression of anti-HER3 single chain antibody protein
[0259] 1. Experimental method
[0260] 1) Pick the resistant high-copy colony from the high-resistance selection plate and inoculate it in 3ml YPD medium, mark the corresponding number and culture at 30°C and 170rpm for 72h;
[0261] 2) Take 100 μl of bacterial solution and centrifuge at 10,000 rpm for 5 minutes;
[0262] 3) Take 30 μl supernatant, add 10 μl 5×Loading buffer, mix well, cook in boiling water for 10 minutes, and centrifuge at 10,000 rpm for 1 minute;
[0263] 4) Take 10 μl of supernatant for SDS-PAGE electrophoresis identification:
[0264] Prepare the reagents required for gel preparation, assemble the SDS protein electrophoresis glass plate and the gel tank according to the instructions; prepare the separation gel in a 50mL beaker (the concentration of the separation gel depends on the molecular weight of the target protein, and the concentration of the gel in this experiment is 12% );...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More