A genetically engineered immunoglobulin-affinity protein
An immunoglobulin and genetic engineering technology, applied in the field of genetically engineered immunoglobulin-affinity protein, can solve the problems of protein A denaturation, destruction, and loss of antibody binding ability, so as to improve utilization rate, reduce cost, increase Effects of stability and tolerance to adverse conditions
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Embodiment 1
[0041] Primer design:
[0042] The four primers of the primer set were designed according to the gene sequence of the C domain of protein A of Staphylococcus aureus using the LAMP primer design software LampDesigner 1.10 of PremierBiosoft.
Embodiment 2
[0044] Construction of N6T-containing immunoglobulin-affinin monomers
[0045] 1) According to the gene sequence of the C domain of protein A of Staphylococcus aureus, a piece of DNA is synthesized through the full sequence gene, and it is used for future use;
[0046] 2) using the DNA as a template, using C-N6T-F and C-N6T-R in the primer set according to claim 1, and using a PCR point mutation kit to carry out point mutation PCR amplification, the point mutation After PCR amplification, add 0.5 μl of restriction endonuclease DpnI to the PCR product, mix well and react at 37°C for 1 h, the restriction endonuclease is used to digest the template DNA, so as to obtain The digested product of the cloning plasmid;
[0047] 3) Take 1-2 μl of the digested product and mix it with the Escherichia coli DH5 competent cells for routine transformation. After the transformation, spread the transformed Escherichia coli on a resistant LB plate containing ampicillin Cultivate at 36-38°C for...
Embodiment 3
[0051] Construction of Immunoglobulin-Aphilin Monomers Containing N28T
[0052] 1) According to the gene sequence of the C domain of protein A of Staphylococcus aureus, a piece of DNA is synthesized through the full sequence gene, and it is used for future use;
[0053] 2) using the DNA as a template, using C-N28T-F and C-N28T-R in the primer set according to claim 1, and using a PCR point mutation kit to carry out point mutation PCR amplification, the point mutation After PCR amplification, add 0.5 μl of restriction endonuclease DpnI to the PCR product, mix well and react at 37°C for 1 h, the restriction endonuclease is used to digest the template DNA, so as to obtain The digested product of the cloning plasmid;
[0054] 3) Take 1-2 μl of the digested product and mix it with the Escherichia coli DH5 competent cells for routine transformation. After the transformation, spread the transformed Escherichia coli on a resistant LB plate containing ampicillin Cultivate at 36-38°C ...
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