pCANTAB-5E

$215.00

  • Model: PVT10573
  • 43 Units in Stock
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pCANTAB-5E


Catalog No. PVT10573                                                    
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pCANTAB-5E Information

Plasmid size: 4517bp

Plasmid Tags: n-g3 signal, C-E tag

Prokaryotic resistance: Amp

Clone strain: TG1

Culture conditions: 37℃

Plasmid host: Escherichia coli

Plasmid use: gene template

Fragment type:

Prokaryotic resistance: Amp

 

pCANTAB-5E Description

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 4517 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.

 

[Operation method]

This method comes from the network. This platform has not been confirmed by experiments, for reference only.

 

1. preparation of auxiliary phage virus species

 

1) the auxiliary bacteriophage M13K07 was crossed on the 2 * YT agar plate.

 

2) Prepare 2 *YT semi-solid agar (0.7% agar) as top Agar, cool to 50 ~C, take 4 mL Top Agar and add 0.5 mL overnight culture of fresh TG1 bacteria (OD660 up to 0.8), fully mix, along the line concentration from low to high direction, pour Top Agar;

 

3) Cultured at 37 C for 6-12 hours, single plaque was selected by inoculation needle, inoculated at 30-200 mL 2 YT-K (kanamycin 70 UG / mL), and cultured at 37 C for 10-14 hours.

 

4) 8000 rpm centrifugation 15 min, 4 C;

 

5) Take the supernatant carefully, and suggest that 0.45 micron filter membrane be used to filter, then pack aseptic tubules and store them at 4 C for at least half a year.

 

2. titration of bacteriophages

 

1) prepare 2 x YT culture plates without any antibiotics; 5-6.

2) 0.7% agar plate was prepared with 2 *YT medium, namely Top Agar, which was cooled to 42 C and stored at 42 C.

3) the above bacteriophage solution was diluted with 10-6, 10-7, 10-8, 10-9 and 10-10 with the medium.

4) Take the overnight culture of TG1 bacterial solution (OD660 = 1 or so), pack small test tube, 500 mu L / small test tube, marked 10-6 to 10-10 dilution;

Remarks: preparation of host bacteria should be carried out according to the instructions of TG1 strain.

5) Add 100 mu L of the phage diluted successively in step 3 above to each standard dilution tube, mix well, and oscillate gently at 37 C for 30 min.

6) Add Top Agar 3 mL to each tube, pour the prepared dishes immediately, and incubate at 37 C overnight.

7) calculate the number of plaque on the plate, multiplied by the corresponding dilution multiple, and the general concentration can reach 1012pfu/mL.

[note]

1. bacteriophage virus species should be titrated according to the recommended method before using.

2. phage virus species can be stored at 4 degrees centigrade, not frozen at -20 or lower temperatures.



pCANTAB-5E Sequence

GACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT
TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA
ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTT
TTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCT
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT
GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATAC
ACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA
TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAAC
CGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG
ACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG
GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT
CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC
TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT
TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGT
CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA
CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC
GCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACC
TCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG
TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCATACAGCCCAGCTTGGAGCGAACGACCTACACCGA
ACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG
GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC
CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTT
TTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTA
TTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA
GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG
GAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTC
CGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTT
TGGAGCCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTATGCGGCCC
AGCCGGCCATGGCCCAGGTCCAACTGCAGGAGCTCGAGATCAAACGGGCGGCCGCAGGTGCGCCGGTGCCGTATCCGGAT
CCGCTGGAACCGCGTGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTG
GAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTG
GTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGT
GGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATAC
TTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGT
CTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACT
GTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTG
GAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCCATTCGTTTGTGAATATCAAGGCCAATCGT
CTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGCGGCTCT
GAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGTGGCGGTTCCGGTGGCGGCTCCGGTTCCGGTGATTTTGATTATGA
AAAAATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAAC
TTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGT
GCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTT
CCGTCAATATTTACCTTCTTTGCCTCAGTCGGTTGAATGTCGCCCTTATGTCTTTGGCGCTGGTAAACCATATGAATTTT
CTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTT
TCGACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAATAAGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG
AAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGC
ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTG
CGGTATTTCACACCGCATATAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCAT
TTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCA
GTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC
ACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCC
CCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGG
GCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTACTA
TGGTTGCTTTGACGGGTGCAGTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACAC
CCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG
TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGA

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