Method for purifying anti-S1 protein receptor binding domain polyclonal antibody from plasma of COVID-19 rehabilitative person
A receptor binding domain, polyclonal antibody technology, applied in the field of medicine and biology, can solve the problem that the production of antibodies needs to be improved
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example 1
[0058] Preparation Example 1. Construction of an expression vector having the S1 protein receptor binding domain gene sequence
[0059] 1. The main reagent preparation method
[0060] (1) Plasmid: pGEM-T Easy vector, PET32(a) vector
[0061] (2) Strains: DH5α, BL21
[0062] (3) LB liquid medium: Weigh 5g of Trypton (Trypton), 5g of NaCl, 2.5g of yeast extract, distilled water to a volume of 500mL, autoclave, and store at 4°C for later use.
[0063] (4) LB solid medium: Agar with a final concentration of 20% was added to the above liquid medium.
[0064] (5) Amp / LB medium: When the LB solution is not completely cooled and not hot to the touch, take out the medium and shake it gently to distribute the agar evenly throughout the medium. Subsequently, 250 μl of 1 mg / L Amp was added to 250 mL of culture medium, so that the final concentration of Amp was 100 μg / mL.
[0065] (6) 30% acrylamide storage solution: Weigh 29g of acrylamide and 1g of methylene bisacrylamide, dissolve...
preparation example 2
[0078] Preparation Example 2: Large-scale purification of recombinant protein (S1 protein containing receptor binding domain)
[0079] 1. Preparation of main reagents:
[0080] (1) PMSF (100mmol / L): 17.4mg PMSF dissolved in 1mL isopropanol, stored in -20°C refrigerator;
[0081] (2)NiSO 4 Solution 0.1mol / L 10mL: 0.2629g NiSO 4 Add distilled water to make up to 10mL;
[0082] (3) Equilibrium buffer: 20mmol / L Tris-Cl, pH=7.5, 300mmol / L NaCl;
[0083] (4) 2M imidazole stock solution: add 2.724g imidazole to Tris-Cl solution (20mmol / L, pH=7.5), dilute to 20mL, and store in a refrigerator at 4°C;
[0084] (5) Eluent: ①100mmol / L Tris-Cl (pH7.5) stock solution (1L): dissolve 12.114g Tris in 950mL distilled water, adjust the pH to 7.5 with concentrated HCl, and dilute to 1L; ②2mol / L NaCl 500mL stock solution: dissolve 58.44g NaCl in 450mL distilled water, and dilute to 500mL after complete dissolution;
[0085] (6) Column regeneration solution: ①guanidine hydrochloride solutio...
preparation example 3
[0094] Preparation Example 3: Preparation of S1 Protein Antigen Affinity Chromatography Column
[0095] The invention adopts the purification method of antigen-antibody affinity chromatography to purify the anti-S1 protein receptor binding domain polyclonal antibody. The protein loading capacity of CNBr activated Sepharose™ 4FastFlow filler is 20-60mg / mL. Determine the type of analysis column and the amount of affinity filler according to the amount of protein to be purified.
[0096] Weigh 30 dry powders of CNBr activated Sepharose™ 4 FastFlow filler and dissolve in 100mL of 1mM HCl, then add 500mL of 1mM HCl to rinse the filler until the filler is fully swollen. Rinse the swollen CNBr activated Sepharose™4FastFlow with 500mL Coupling Buffer (0.1MNaHCO3, 0.5M NaCl, pH8.3), and then drain the excess liquid in the filler. First, 1000 mg of S1 protein was dialyzed in Coupling Buffer, then the swollen CNBr activated Sepharose™ 4 FastFlow filler was added to the S1 protein sol...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More