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Escherichia coli TolC antigen as well as antibody and application thereof

A technology of Escherichia coli and antigens, applied in the fields of application, antibodies, antibacterial drugs, etc., can solve the problems of unclear side reactions and target sequence specificity, and achieve the function of inhibiting drug resistance, overcoming antibody heterophile, and using safe effect

Inactive Publication Date: 2011-02-16
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to address the deficiencies in the prior art, to provide an antibody against the key domain of TolC drug resistance, to overcome the defects of current antibodies with unclear target sequence specificity and unclear side reactions, and to improve the efficacy of antibodies. Effectiveness and Safety

Method used

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  • Escherichia coli TolC antigen as well as antibody and application thereof
  • Escherichia coli TolC antigen as well as antibody and application thereof
  • Escherichia coli TolC antigen as well as antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] TolC gene cloning, prokaryotic expression, protein purification and preparation of rabbit antiserum

[0028] In this embodiment, the full-length E. coli TolC coding sequence or fragment is constructed into the E. coli protein prokaryotic expression vector to achieve expression and purification of the target protein.

[0029] The coding sequence of the TolC gene is shown in SEQ ID NO:3.

[0030] SEQ ID NO: 3:

[0031] 1 atgaagaaat tgctccccat tcttatcggc ctgagccttt ctgggttcag ttcgttgagc

[0032] 61 caggccgaga acctgatgca agtttatcag caagcacgcc ttagtaaccc ggaattgcgt

[0033] 121 aagtctgccg ccgatcgtga tgctgccttt gaaaaaatta atgaagcgcg cagtccatta

[0034] 181 ctgccacagc taggtttagg tgcagattac acctatagca acggctaccg cgacgcgaac

[0035] 241 ggcatcaact ctaacgcgac cagtgcgtcc ttgcagttaa ctcaatccat ttttgatatg

[0036] 301 tcgaaatggc gtgcgttaac gctgcaggaa aaagcagcag ggattcagga cgtcacgtat

[0037] 361 cagaccgatc agcaaacctt gatcctcaac accgcgaccg cttatttcaa cgtgttgaat

[0038] 421 gctattgacg ttctttccta ta...

Embodiment 2

[0077] Cloning of TolC fragment SNGYRDANGI mutation gene (tolC1)

[0078] 1. Design of primers for TolC fragment mutant gene tolC1

[0079] Based on the principle of replacing hydrophobic amino acids with other hydrophobic amino acids and replacing hydrophilic amino acids with other hydrophilic amino acids, the fragment SNGYRDANGI in the TolC amino acid sequence was replaced with amino acids, and the replacement result was: RQCATKFHCL. In the nucleotide sequence of the tolC gene, find the nucleotide sequence corresponding to the amino acid sequence (SEQ ID NO: 7): 5'-AGC AAC GGC TAC CGC GAC GCG AACGGC ATC-3', according to the amino acid codon, The nucleotide sequence was correspondingly replaced with (SEQ ID NO: 8): 5'-CGT CAG TGC GCT ACT AAG TTC CAT TGC CTG-3'. Therefore, according to the nucleotide sequence of the tolC gene, the short-segment mutation tolC gene (named tolC1) was designed with two pairs of primers at the 5'end and the 3'end (Table 1), and at the same time, the 5'...

Embodiment 3

[0093] Construction process of Escherichia coli tolC gene deletion strain and corresponding other strains

[0094] 1. Construction process of E. coli tolC gene deletion strain:

[0095] First, a competent strain of BW25113 strain containing pKD46 plasmid (gifted by Professor Barry L. Wanner of Purdue University) was prepared. Inoculate the BW25113 strain containing pKD46 plasmid in 5mL LB medium, cultivate overnight at 30℃, inoculate 100mL LB medium containing 70mg / mL ampicillin at 1:50 the next day, and when cultivated at 30℃ to 0.25 (OD600), add L -Arabinose to 30mmol / L, induce 1 hour (OD600 can not exceed 0.6), so that the Red recombinase (Exo, Bet and Gam protein) on the pKD46 plasmid can be fully expressed. After pre-cooling on ice for 10 minutes, the precipitate was collected by centrifugation at 4°C, washed with pre-cooled 10% glycerol 3 times, concentrated 100 times into 1 mL competent cells, and stored in a -80°C refrigerator for later use.

[0096] According to the TolC g...

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Abstract

The invention belongs to the biotechnology field, and in particular relates to escherichia coli TolC antigen and antibody. The antibody has the specificity aiming to a sequence shown by SEQ ID NO:1. The invention also provides a gene sequence for encoding the escherichia coli TolC antigen and application of the antibody to preparing a drug for inhibiting the bacterial resistance. The anti-TolC fragment SNGYRDANGI antibody enhances the sensitivity of bacteria to antibiotic and can specifically and efficiently inhibit the functions of drug resistance outer membrane protein of escherichia coli, thereby solving the problem of the bacterial resistance. Compared with the traditional antibody, the antibody has higher safety and operability on the application of the bacterial resistance drug.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an antibody against the TolC fragment of Escherichia coli. Background technique [0002] In recent years, bacterial resistance has become increasingly severe, and it has become a concern in the medical field. The problem of bacterial resistance is particularly prominent due to the abuse of antibiotics. The resistance of some bacteria isolated clinically in my country to certain drugs has ranked first in the world. In addition to penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, enterococcus, fungi and other drug-resistant bacteria, the drug resistance rate of quinolone antibiotics that have entered my country for only 20 years has reached 60%-70% . According to reports, 80% of Staphylococcus aureus infected by people in some large cities in China, including Guangzhou, have developed resistance to penicillin G. The application of thir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12A61K39/40C12N15/31C07K16/06C07K7/06A61P31/04C07K14/795
CPCC07K14/245C07K16/1232A61P31/04
Inventor 彭宣宪李惠
Owner SUN YAT SEN UNIV
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