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Recombinant bacteria expressing avian Escherichia coli type 1 pilin, and construction method and use thereof

A technology of chicken Escherichia coli and fimbriae protein, which is applied in the field of bioengineering, can solve the problems of small expression amount, increased vaccine cost, complicated fimbriae extraction, etc., and achieves the effects of strong immunogenicity, low cost and high yield

Inactive Publication Date: 2009-05-20
JINLING INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the vaccine made of natural pili has high protection, the cost of the vaccine is increased due to the low expression level and the complicated extraction of pili

Method used

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  • Recombinant bacteria expressing avian Escherichia coli type 1 pilin, and construction method and use thereof
  • Recombinant bacteria expressing avian Escherichia coli type 1 pilin, and construction method and use thereof
  • Recombinant bacteria expressing avian Escherichia coli type 1 pilin, and construction method and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the segmentation PCR amplification of chicken E. coli type 1 pili operon gene

[0040] 1. Primer design

[0041] According to the sequence published on GenBank, use Primer 5.0 to design four pairs of primers and send them to Invitrogen Company for synthesis. The primers were diluted to 20pmol / L with ultrapure water and stored at -20°C. The primer sequences are as follows:

[0042] P1: 5'-GGGAATTCAGTGACCAAAGCCATATTTACC-3'

[0043] P2: 5'-GGGGTACCTGGGTAGGTTATTGATACTG-3'

[0044] P3: 5'-AAGGTACCGGGACGTCATTACGGGCAG-3'

[0045] P4: 5'-GGGCCCCCAATCCGATTCTGTACACTA-3'

[0046] P5: 5'-CTGACGATCCCTCAGGCATTTATGAG-3'

[0047] P6: 5'-AAGTCGACACGCCCCCTTAACGACATTC-3'

[0048]P7: 5'-AAGGTTTGTTTCTCATCACGCCCCC-3'

[0049] P8: 5'-CGTCGATTTAGATAACGCGGTTGCTAACG-3'

[0050] P9: 5'-ATGTCGACTGGCCTACAAAGGGCTAACGTG-3'

[0051] The gene structure of chicken Escherichia coli type 1 pili operon and the relative positions of primers are shown in figure 1 .

[0052] 2. Prepa...

Embodiment 2

[0108] Embodiment 2: the cloning of PCR product

[0109] ① Preparation and transformation of competent cells

[0110] Using CaCl 2 Competent cells TG1 were prepared by the method for transformation of recombinant plasmids, and the experimental operation was as follows.

[0111]The PCR product of the fragment BEA was digested with EcoR I and Kpn I, identified by electrophoresis, recovered by gel cutting, melted in 30 μl TE, and stored at -20°C for future use. pUC18 was digested by EcoR I and Kpn I enzymes, dephosphorylated by CIAP, extracted with phenol / chloroform, dissolved in 20 μl TE, and stored at -20°C for later use. Mix the target fragment with the digested vector at a ratio of 3:1, connect overnight with T4 DNA ligase at 16°C, and transform the competent cell TG1, and analyze by ampicillin resistance and restriction endonuclease digestion Screening identified positive clones.

[0112] ② T / A cloning of fragments IC and D

[0113] 6 μl each of fragments IC and D were ...

Embodiment 3

[0115] Embodiment 3: the construction of the type 1 pilus operon gene expression vector of chicken Escherichia coli

[0116] ①Construction of pBC

[0117] Plasmids pBEA and pUCm-IC were double digested with Kpn1 and BamH1, identified by electrophoresis and recovered by gel cutting, dissolved in 30 μl TE respectively, and stored at -20°C for future use. Take 3 μl of recovered pBEA, 8 μl of recovered fragment IC, add 1 μl of T4 DNALigase, 2.5 μl of T4 DNALigase Buffer, add deionized water to 25 μl, and connect overnight at 16°C. Take 10-15 μl of the ligation product and transform it into competent cell TG1, screen and identify positive clones through ampicillin resistance and restriction endonuclease analysis, and construct vector pBC.

[0118] ②Construction of pBD

[0119] Plasmids pBC and pUCm-D were double digested with BamH1 and Sal1, identified by electrophoresis and recovered by cutting gel, dissolved in 30 μl TE respectively, and stored at -20°C for future use. Take 3 ...

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Abstract

The invention discloses an avian Escherichia coli type 1 pilin expression recombinant bacteria, a construction method and application thereof. The strain is imported with avian Escherichia coli type 1 fimbriae operon gene recombinant plasmids and is constructed with the following steps: 1. PCR augmentation of avian Escherichia coli type 1 fimbriae structure gene and each functional gene; and 2. recombinant plasmid construction. The invention further discloses an avian Escherichia coli type 1 fimbriae engineered vaccine. The avian Escherichia coli type 1 pilin expression recombinant bacteria has expression product with protuberant fimbriae, good immunogenicity, high culture expression yield, low cost and easy implementation of industrial vaccine production.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a recombinant bacterium expressing chicken Escherichia coli type 1 pili protein and its construction method and application. Background technique [0002] Chicken colibacillosis is caused by pathogenic Escherichia coli (Escherichia coli). The disease has many clinical manifestations, the most common manifestations are air sacculitis, pericarditis, perihepatitis and death. With the development of intensive poultry farming, the risk of pathogenic Escherichia coli continues to increase, making the disease difficult to control and causing heavy losses worldwide. Although the use of antibiotics may provide temporary protection, the development of resistance in bacteria has become a potential harm. Therefore, there is an urgent need for a safe and effective immunogen as a vaccine. Escherichia coli type 1 pili is an important pathogenic factor of chicken colibacillosis. It is a filament...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/70A61K39/108A61P31/04C12R1/19
Inventor 戴鼎震陈钟鸣张志成王国相甘黎明方光远张玉红蒋加进
Owner JINLING INST OF TECH
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