Gold ion adsorption protein and method for enriching and recovering gold ion
A surface display system and gold ion technology, applied in the field of bioengineering, can solve the problems of difficult and efficient selective enrichment and recovery of gold ions, and achieve the effect of convenient operation and low price
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Embodiment 1
[0044] Example 1 Construction and Identification of OmpA-GolB Fusion Protein
[0045] 1. Gene Amplification of Escherichia coli Outer Membrane Protein A (OmpA) Fragment
[0046] According to the desired amplification target DNA sequence and the principle of base pairing, design specific primers Ompa1 (SEQ ID NO: 1) and Ompa2 (SEQ ID NO: 2), from Escherichia coli genomic DNA (reference position in NCBI: NC_000913 ) to amplify the DNA sequence (SEQ ID NO: 3) encoding the first to 159th amino acids of Escherichia coli outer membrane protein A (OmpA).
[0047] The reaction conditions are as follows:
[0048]
[0049]
[0050] PCR conditions:
[0051] Step 1: 95°C for 2 minutes
[0052] Step 2 95°C for 0.5 minutes
[0053] Step 3 63°C for 0.5 minutes
[0054] Step 4 72°C for 0.5 minutes
[0055] Step 5 Repeat steps 2 to 4, 29 cycles
[0056] Step 6 72°C for 10 minutes
[0057] The seventh step is to keep warm at 4°C.
[0058] Recover the PCR product for later use...
Embodiment 2
[0095] Example 2 Construction and identification of Antigen43-gold ion-inducible gene (Gold inductive gene with antigen43) fusion protein expression plasmid
[0096] 1. Gold ion-induced gene amplification
[0097] Using specific primers pGolts1 (SEQ ID NO: 8) and pGolb2 (SEQ ID NO: 9) from Salmonella typhimurium genomic DNA (reference position in NCBI: NC_003197 ) to amplify the DNA sequence (SEQ ID NO: 10) encoding the Gold inductive gene.
[0098] The reaction conditions are as follows:
[0099]
[0100] PCR conditions:
[0101] Step 1: 95°C for 3 minutes
[0102] Step 2 95°C for 1 minute
[0103] Step 3 60°C for 2 minutes
[0104] Step 4 72°C for 4 minutes
[0105] Step 5 Repeat steps 2 to 4, 29 cycles
[0106] Step 6 72°C for 10 minutes
[0107] The seventh step is to keep warm at 4°C
[0108] Recover the PCR product for later use.
[0109] 2. Construction and identification of Antigen43-gold ion-inducible gene (Gold inductive gene with antigen43) fusion p...
Embodiment 3
[0118] Example 3 OmpA-GolB fusion protein, Antigen43-gold ion-induced gene (Gold inductive gene with antigen43) fusion protein transfer into host bacteria and culture of host bacteria
[0119] 1. OmpA-GolB fusion protein into host bacteria
[0120] 1) The plasmid used to express the OmpA-GolB fusion protein was introduced into the Escherichia coli cell DH10b by chemical transformation method, by adding ampicillin (50 μg mL -1 ) on LB solid medium at 37°C for 16 hours, from which positive clones were screened out and preserved.
[0121] The heat shock transformation and dilution coating steps of competent cells are as follows:
[0122] ①Put the product of the ligation reaction in the competent cell (DH10b), and mix it evenly by finger flicking;
[0123] ② After 30 minutes of ice bathing, heat shock at 42°C for 95 seconds, and quickly transfer to ice;
[0124] ③ After 2 minutes in ice bath, add 900 μl of preheated LB medium at 37°C to each tube, and incubate at 37°C, 200 r...
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