This system is used for antibody gene expression and preparation of recombinant antibody. The host strain TG1 was replicated and expressed by plasmid vector pCANTAB5e, and was cultured on 2 *YT medium at 37 C. The vector pCANTAB5e is used to construct recombinant scFv. It has ampicillin resistance and the size is 4522 bp. The auxiliary phage M13K07 is used to rescue the phage particles. It is Kana-resistant and can be replicated by the auxiliary phage and expressed on the phage surface in the form of fusion. There is a sequence encoding Tag tail peptide (E-Tag) behind the scFv gene. There is an amber termination codon behind the tail peptide. It is located between the scFv gene and the cpIII gene. In the inhibitory bacteria TG1, only 20% of the amber codon is effective, so it can be read through the protein translation process to form scFv-cp. In non-inhibitory strains, such as HB2151, the terminator is recognized, and the scFv gene terminates before the cpIII gene in the translation process, forming an independent antibody protein that remains in the cell membrane gap, and leaks into the culture medium after a long period of culture to form soluble expression.
Summary
Function E.coli Cloning plasmid
Promoter: Lac/lac promoter
Plasmid size: 5216bp
Plasmid tagging: N-g3 signal, C-E tag
Prokaryotic resistance: ampicillin Amp
Clone strain: Escherichia coli DH5α
Culture conditions: 37 C, aerobic, LB
Vector sequence
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
301 ctgaagatca gttgggtgct cgagtgggtt acatcgaact ggatctcaac agcggtaaga
361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
1561 cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
2221 accatgatta cgccaagctt tggagccttt tttttggaga ttttcaacgt gaaaaaatta
2281 ttattcgcaa ttcctttagt tgttcctttc tatgcggccc agccggccat ggcccaggtg
2341 aagctgcagc agtcaggacc tggcctggtg gcgccctcac agagcctgtc catcacatgc
2401 accgtctcag ggttttcatt aaccagctat ggtgtacact gggttcgcca gcctccagga
2461 aagggtctgg agtggctggg agtaatatgg gctggtggaa gcacaaacta taattcagct
2521 ctcaaatcca gactgaacat cagcaaggac aactccaaga gccaagtttt cttaaaaatg
2581 aacagtctcc aaactgatga cacagccatg tactactgtg ccagaaactg gggcagctac
2641 tggtacttcg atgtctgggg ccaaggccac ggtcaccgtc tcctcagtgg aggcggttca
2701 ggcggaggtg gcggaggtgg ctctggcggt ggcggatcgg acattgagct cacccagtct
2761 ccagcaatca tgtctgcatc tccaggggaa aaggtcacca tgacctgcag ggccagctca
2821 agtataagtt ccagttactt gcactggtac cagcagaagt caggcgcttc ccccaaaccc
2881 ttgattcata ggacatccaa cctggcttct ggagtcccag ctcgcttcag tggcagtggg
2941 tctgggacct cttactctct cacaatcagc agcgtggagg ctgaagatga tgcaacttat
3001 tactgccagc agtggagtgg ttacccattc acgttcggtg ctgggaccaa gctcgagatc
3061 aaacgggcgg ccgcaggtgc gccggtgccg tatccggatc cgctggaacc gcgtgccgca
3121 tagactgttg aaagttgttt agcaaaacct catacagaaa attcatttac taacgtctgg
3181 aaagacgaca aaactttaga tcgttacgct aactatgagg gctgtctgtg gaatgctaca
3241 ggcgttgtgg tttgtactgg tgacgaaact cagtgttacg gtacatgggt tcctattggg
3301 cttgctatcc ctgaaaatga gggtggtggc tctgagggtg gcggttctga gggtggcggt
3361 tctgagggtg gcggtactaa acctcctgag tacggtgata cacctattcc gggctatact
3421 tatatcaacc ctctcgacgg cacttatccg cctggtactg agcaaaaccc cgctaatcct
3481 aatccttctc ttgaggagtc tcagcctctt aatactttca tgtttcagaa taataggttc
3541 cgaaataggc agggtgcatt aactgtttat acgggcactg ttactcaagg cactgacccc
3601 gttaaaactt attaccagta cactcctgta tcatcaaaag ccatgtatga cgcttactgg
3661 aacggtaaat tcagagactg cgctttccat tctggcttta atgaggatcc attcgtttgt
3721 gaatatcaag gccaatcgtc tgacctgcct caacctcctg tcaatgctgg cggcggctct
3781 ggtggtggtt ctggtggcgg ctctgagggt ggcggctctg agggtggcgg ttctgagggt
3841 ggcggctctg agggtggcgg ttccggtggc ggctccggtt ccggtgattt tgattatgaa
3901 aaaatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag
3961 tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt
4021 ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc
4081 tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc
4141 cgtcaatatt taccttcttt gcctcagtcg gttgaatgtc gcccttatgt ctttggcgct
4201 ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt
4261 gcgtttcttt tatatgttgc cacctttatg tatgtatttt cgacgtttgc taacatactg
4321 cgtaataagg agtcttaata agaattcact ggccgtcgtt ttacaacgtc gtgactggga
4381 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
4441 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
4501 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata
4561 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
4621 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat
4681 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
4741 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccaa
4801 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
4861 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
4921 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
4981 acccgccgcg cttaatgcgc cgctacaggg cgcgtactat ggttgctttg acgggtgcag
5041 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
5101 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
5161 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcga
//
