Staphylococcal protein A, preparation method thereof and use thereof
一种葡萄球菌蛋白、亲和的技术,应用在生物领域,能够解决亲和力低、纯化效率低等问题,达到亲和力高、蛋白结合特异性强、动态吸附载量提高的效果
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Embodiment 1
[0025] Example 1 , Synthesis of staphylococcal protein A gene monomer
[0026] Through chemical synthesis, the sequence of a repetitive fragment of staphylococcal protein A gene was designed and synthesized. The sequence includes a length of 174bp, a NcoI restriction site connected to the expression vector, and 6 His (for affinity chromatography, Combine with nickel column to reduce purification steps), EK restriction site (used to cut off His and DDDDK to form the correct Protein A) and AccI restriction site (used to join multiple repetitive fragments). In addition, it also includes the stop codon TAA and the BamH I restriction endonuclease linker for cloning, a total of 9bp.
[0027] Through chemical synthesis, the sequence of a repetitive fragment of the staphylococcal protein A gene was designed and synthesized. The sequence included a length of 174 bp with AccI restriction sites at both ends to construct a sequence containing multiple repetitive fragments.
[0028] The sequenc...
Embodiment 2
[0029] Example 2 , Construction of a pET32a vector containing a staphylococcal protein A gene monomer
[0030] The above-mentioned staphylococcal protein A gene monomer was excised with restriction endonucleases NcoI and BamH I, and connected to the vector pET32a digested with NcoI and BamH I, transformed into E. coli, and screened for ampicillin-resistant transformants. Plasmid extraction, restriction enzyme digestion and identification proved that the staphylococcal protein A gene monomer has been cloned into pET32a.
Embodiment 3
[0031] Example 3 , Construction of pET32a-P vector containing staphylococcal protein A gene
[0032] The above-mentioned pET32a vector containing a staphylococcal protein A gene monomer and staphylococcal protein A gene monomer were digested with AccI, and the corresponding fragments were recovered and ligated, transformed into Escherichia coli, selected transformants with ampicillin resistance, and extracted by plasmid The pET32a-P vector of Staphylococcal protein A containing 3 protein A gene monomers was obtained by restriction digestion and screening. The gene sequence of the pET32a-P vector is as shown in SEQ ID No: 2 after detection.
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