Protein A immunoadsorption medium resistant to irradiation sterilization and efficient disinfection
An immunoadsorption and protein technology, which is applied in the field of genetic engineering technology and antibody adsorption, can solve the problems of not being able to withstand radiation sterilization or efficient disinfection, and high cost of use
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Embodiment 1
[0047] Example 1 Expression and purification of protein and synthesis of adsorbent
[0048] The amino acid sequence of the recombinant protein A is shown in SEQ ID NO.: 1-15, which are respectively denoted as SEQ-1-SEQ-15. The nucleotide sequence of the recombinant protein A can be designed or optimized with reference to the nucleotide sequence of the natural protein A (Uhlen, 1984) or can be designed using other known methods.
[0049] figure 1 It is the comparison result between the natural protein B domain sequence and the mutated sequence.
[0050] Recombinant protein A can be prepared using existing methods. Or prepare as follows:
[0051] Design the expression sequence of recombinant protein A or its concatenate: CATATG-(nucleotide sequence of recombinant protein A) n-GTAGACTGTCTCGAG, the vector is PET30a, using the his tag synthesized by the vector; the expression strain used is Escherichia coli BL21 (DE3), entrusted Shanghai Sangon Bioengineering Co., Ltd. construc...
Embodiment 2
[0052] Example 2 The tolerance of each protein A adsorption medium to irradiation sterilization
[0053] The protein A adsorption medium synthesized by each protein (representative morphological photos are shown in figure 2 shown) respectively take 5mL into EP tubes, and sterilize them by electron beam irradiation ((15KGy)). After sterilization, take 1 mL and use the static adsorption method to measure the adsorption performance of the filler on human IgG solution. Add 80 mg of human IgG solution to 1 mL of filler, place on a room temperature decolorizing shaker at 100 rpm and mix for 1 hour. After the reaction is completed, pour the above reaction solution into a disposable affinity chromatography column, first washed with about 10mL balance solution (PBS), then eluted with 10mL eluent (2.1g / L citric acid, 8.0g / L NaCl), collected the eluted peak, and used UV detection OD 280 The value of adsorption performance (mg / mL) = (OD 280 / 1.38)×10. The results are shown in Table 1...
Embodiment 3
[0057] Example 3 The tolerance of each protein A adsorption medium to strong oxidizing agents
[0058] Take 1 mL of the protein A adsorption medium synthesized by each protein, and use 0.5% CH 3 After COOOH (Huankai, the final concentration of peracetic acid after dilution is 5000mg / L) for 25 hours (calculated according to the disinfection time of 15-30min, it can be reused at least 50 times), wash the medium with purified water until the pH is neutral. Then, the static adsorption method was used to measure the adsorption performance of the filler on the human IgG solution. The determination method was the same as in Example 2, and the results are shown in Table 2.
[0059]
[0060] It can be seen from the results in Table 2 that the protein A adsorption medium synthesized by the proteins of SEQ-7, SEQ-12, SEQ-13, SEQ-14, and SEQ-15 has higher stability after being treated with peracetic acid. (SEQ-12) 4, (SEQ-13) 6, and (SEQ-15) 5 are highly stable in series.
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