Preparation method of antibody complex
A complex and antibody technology, applied in the field of nanobody complex preparation, can solve the problems of unstable disulfide bridge bridging method, many and complicated click chemistry methods, etc., and achieve extended blood half-life, high yield, and reaction speed. quick effect
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Embodiment 1
[0041] Example 1 C-terminal site-specific modification of Nanobodies
[0042] The coding gene sequence of Nanobody A was cloned into the expression vector plasmid pET23a, introduced into Escherichia coli T7Shuffle (DE3) for expression, the bacteria were collected, and the cells were broken, and the supernatant from the broken cells was analyzed by metal chelation affinity chromatography HisTrap HP 5mL (GE Healthcare) was used to purify Nanobodies, and the final yield was about 100 mg per liter of fermentation broth. The purified nanobody was concentrated by ultrafiltration and replaced with triethanolamine buffer solution (25mM TEAM, pH9.0, 150mM NaCl, 1mM mercaptoethanol), the nanobody concentration was 5mg / mL, and the final concentration was about ten One-third of the purity of 1mg / mL is >90% FGE enzyme (the molecular weight of FGE enzyme is about 33kDa, and the molecular weight of nanobody is about 18kDa). The reaction is catalyzed by gentle shaking at 18°C for 20 hours, ...
Embodiment 2
[0044] Example 2 Realize the preparation of C-C linked bivalent Nanobodies under frozen conditions
[0045] The Nanobody A (Nanobody A0) modified by site-directed aldehydesylation at the carboxyl terminal prepared in Example 1 was concentrated by ultrafiltration and replaced with an acidic buffer, 0.1M acetate buffer pH 4.0 or 0.1M MES buffer pH 5.5, Or neutral buffer solution 0.2M PBS pH7.4, the concentration of Nanobody A0 is 2 mg / mL, that is, 100 μmol / L. Take a certain amount of configured nanobody A0, add bifunctional linker HZ-PEG-HZ 400 (bishydrazide polyethylene glycol-400) and reducing agent sodium cyanoborohydride, wherein HZ-PEG-HZ 400 and cyano The final concentrations of sodium borohydride in the reaction system were 50 μmol / L and 1 mmol / L, respectively, that is, the molar ratio of PEG to nanobody in the reaction system was 1:2, and the molar ratio of sodium cyanoborohydride to nanobody was 10:1. Put the added reaction mixture into a low-temperature tank to cool ...
Embodiment 3
[0048] Example 3 Preparation of C-C Linked Bispecific Nanobodies by Freezing Method
[0049](1) Cloning the coding gene sequence of Nanobody B into the expression vector plasmid pET23a, introducing it into Escherichia coli T7Shuffle (DE3) for expression, collecting the bacteria, and disrupting the cells. Nanobodies were purified by chromatography on HisTrap HP 5 mL (GE Healthcare) with a final yield of approximately 100 mg per liter of fermentation broth. The purified nanobody was concentrated by ultrafiltration and replaced with triethanolamine buffer solution (25mM TEAM, pH 9.0, 150mM NaCl, 1mM mercaptoethanol), the nanobody concentration was 5mg / mL, and the final concentration was about 10% of the nanobody molar weight. One, that is, the purity of 1mg / mL is >90% FGE enzyme (the molecular weight of FGE enzyme is about 33kDa, and the molecular weight of nanobody is about 18kDa), and the catalytic reaction is gently shaken at 18°C for 20 hours, and the protein precipitate is...
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