Substrate for biological fluid treatment
A liquid processing and biological technology, applied in the field of substrates for biological liquid processing, can solve problems that have not yet been established
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Embodiment 1
[0096] Example 1: Production of Leu3a scFv-SA body
[0097] (Production of the gene of the Leu3a single-chain antibody)
[0098] The nucleic acid sequence of the mouse anti-human CD4 monoclonal antibody Leu3a is disclosed in Genbank Accesion No. M97867.1 for the VH site and Genbank Accession No. M61046.1 for the VL site. In order to make these sequences into single-chain antibodies, a nucleic acid encoding a 15-amino acid sequence (SEQ ID NO: 1 in the Sequence Listing) as a Linker was designed to be linked, and the sequence of VL-linker-VH was completely synthesized. make. In this case, an Nco I site was added directly in front of the VL site so as to include an initiation codon (start codon), and a Not I site was added at the end of the VH site. These nucleic acids were subcloned into pUC57 vector.
[0099] The complete synthesis and subcloning of the above-mentioned nucleic acid sequences utilized the synthesis contract service provided by GenScript Corporation of the Uni...
Embodiment 2
[0110] Example 2: Expression, recovery under guanidine hydrochloride denaturing conditions
[0111] The expression vector constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) strain (NOVAGEN Company) according to the instructions. It was cultured at 37° C. in a 2×YT medium (1.6% Bacto tryptone, 1% Bacto yeast lactate, 0.5% NaCl) containing 34 μg / ml kanamycin and 1% glycerol. The culture was stopped when the absorbance OD at 600 nm reached 0.7-0.8, and glycerol stock (stock) was prepared with a final concentration (final) of 15% glycerol, and stored at -80°C. This preserved 1 ml glycerol stock was mixed with 300 ml of 2×YT medium containing 34 μg / ml kanamycin and 1% glycerol, and cultured at 37°C. When the absorbance OD at 600 nm reached 0.7 to 0.8, IPTG (TAKARA Co.) was added at a final concentration of 1 mM, and cultured at 37° C. for another 4 hours. The Escherichia coli culture suspension was transferred to a 500 ml centrifuge tube, centrifuged at 4,0...
Embodiment 3
[0115] Example 3: Refolding purification under urea stage dilution dialysis
[0116] Use Slide-A-Lyzer (molecular weight cut-off 10kD: PIERCE company) to dilute about 30 ml of the refined liquid of Leu3a scFv-SA protein that is soluble under the denaturation of guanidine hydrochloride as in the first half of Example 2 and dilute with urea 1 L of dialyzed external fluid (50 mM Tris-HCl pH=8.0, 300 mM NaCl, 4 M urea) was dialyzed overnight at room temperature. Then, 1 L of the dialyzed fluid (50 mM Tris-HCl pH=8.0, 300 mM NaCl, 2 M urea, 400 mM L-arginine, 375 μM oxidized glutathione) was diluted with the urea phase of the second stage and dialyzed at room temperature for 24 hours. Then, in order to prevent the formation of a precipitate after the stereostructure refolding of the Leu3a scFv-SA protein, take out 5ml of the refined product of the Leu3a scFv-SA protein in the above-mentioned stage dilution dialysis, and dilute the dialyzed external fluid (50mM Tris-HCl pH=8.0, 300...
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