Recombinant staphylococcus protein A and application thereof
A staphylococcal protein and protein detection technology, applied in the field of affinity chromatography media and its preparation, can solve the problems of low purification efficiency and low affinity, and achieve the effects of high purification efficiency, high affinity, and improved dynamic adsorption capacity
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Embodiment 1
[0028] The preparation of embodiment 1 recombinant staphylococcal protein A
[0029] 1. Construction of recombinant staphylococcal protein A gene monomer
[0030] By chemical synthesis, design and synthesize a sequence monomer of a repeat segment of the staphylococcal protein A gene (the sequence of the repeat segment is shown in nucleotides 46-192 as shown in SEQ ID NO: 2), which includes a repeat length of 153bp fragment, and the NcoI restriction site connected to the expression vector, 6 His (used for affinity chromatography, combined with nickel column, reducing purification steps), EK restriction site (used to cut His and DDDDK Go, form the correct ProteinA) and AccI restriction site (gtagac, used to join multiple repeat fragments). In addition, it also includes the stop codon TAA, and a BamHI restriction endonuclease linker for cloning with a total of 9 bp. Labeled "Monomer 1".
[0031] In addition, by means of chemical synthesis, a sequence monomer of a repeat fragme...
Embodiment 2E
[0044] Embodiment 2 ELISA method detects recombinant Staphylococcus protein A and antibody binding activity
[0045] ELISA method was used to detect the proteinA1 and antibody binding activity prepared in the above-mentioned embodiment 1, and the specific process was as follows:
[0046] 1) Coating: Coat each well of a 96-well plate with 100 μl of ProteinA1 sample, 37°C, 1 h;
[0047] 2) Blocking: each well was blocked with 100 μl of 1% BSA at 37° C. for 1 hour;
[0048] 3) Add primary antibody: Add about 100ug of human IgG antibody (purchased from Mlbio) to each well, 37°C, 1h;
[0049] 4) Add secondary antibody: Add about 100 μl, 1:1000 horseradish peroxidase-labeled antibody (purchased from Mlbio) to each well, 37°C, 1h;
[0050] 5) Color development.
[0051] The same method was used to detect the binding activity of commercially available Protein A (purchased from Guduo Biotechnology) and antibody.
[0052] ELISA test results: ProteinA1 of the present invention is com...
Embodiment 3
[0053] Embodiment 3 Preparation of affinity chromatography medium
[0054] Utilize the ProteinA1 of embodiment 1 to prepare agarose gel affinity chromatography medium, comprise the following steps:
[0055] 1. Activation of agarose gel: react in aqueous medium with epichlorohydrin, sodium hydroxide and agarose (5% cross-linked agarose gel), and react 2- After 3 hours, after the reaction, wash with distilled water until neutral and then drain to obtain activated agarose gel;
[0056] 2. Chemical coupling of recombinant staphylococcal protein A to activated agarose gel: react the activated agarose gel obtained in the above step 1 with the Protein A1 prepared in Example 1 at a temperature of 5-25° C. for 15-20 hours, After the reaction, it was washed and dried to obtain staphylococcal protein A sepharose affinity chromatography medium.
[0057] Use the same method to prepare Sephadex affinity chromatography media:
[0058] 1. React with epichlorohydrin, sodium hydroxide and de...
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