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Avian Escherichia coli vaccine strain

An avian Escherichia coli and Escherichia coli technology, applied in vaccines, veterinary vaccines, bacteria, etc., can solve the problem of unsatisfactory use of Escherichia coli inactivated vaccines, poor immune effect of aluminum hydroxide glue vaccines, and large side effects of oil-emulsion vaccines. question

Active Publication Date: 2021-03-05
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current E. coli vaccines are inactivated vaccines, which are prepared into oil emulsion vaccines and aluminum hydroxide gel vaccines. The side effects of oil emulsion vaccines are large, and the immune effect of aluminum hydroxide gel vaccines is poor. The use effect of E. coli inactivated vaccines in the market None ideal

Method used

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  • Avian Escherichia coli vaccine strain
  • Avian Escherichia coli vaccine strain
  • Avian Escherichia coli vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Separation and purification of bacterial strains and analysis of their physical and chemical properties

[0017] 1) In June 2018, a serious Escherichia coli outbreak occurred in a chicken farm in Shandong. The heart, liver, spleen, etc. of dead chickens were collected, sealed in sampling bags, and brought back to the laboratory and placed in a 4°C ice box. Save for later.

[0018] 2) Grind the collected chicken organs and apply them on the chromogenic medium to display different colored strains. Every 3 bacterial strains showing different colors are inoculated into LB liquid medium as a group for expanded culture, and the thalline is collected by centrifugation at 10000rpm. Then the bacterium was resuspended with 0.5% sterile PBS, and the bacterial concentration of the bacterial strain was determined as 1×10 by turbidimetric method. 7 CFU / ml.

[0019] 3) Strong toxicity separation experiment:

[0020] The isolated bacterial strains were respectively cultur...

Embodiment 2

[0032] Example 2: Knockout and detection of the aroA gene of avian Escherichia coli YBO78

[0033] 1) According to the aroA gene sequence of avian Escherichia coli O78 on NCBI GenBank, design sequencing primers (Table 3), amplify the aroA gene of clinical isolate YBO78 by PCR, and sequence and identify it.

[0034] Table 3: Sequence Information Table of Sequencing Primers

[0035] Upstream sequencing primers Downstream sequencing primers O78-seq-F1 O78-seq-R1 O78-seq-F2 O78-seq-R2 O78-seq-F3 O78-seq-R3

[0036] 2) Design primers for aroA gene knockout according to the sequencing results (Table 4).

[0037] Table 4: Primer sequence list for aroA gene knockout

[0038]

[0039] 3) Construct the recombinant plasmid pSC101-BAD-gbaA-cm, and electroporate into Escherichia coli YBO78 bacteria after the sequencing is correct.

[0040] 4) For arabinose induction, the kanamycin gene cassette loxp-kan-loxp was electrotransferred into Escherichia...

Embodiment 4

[0046] Embodiment 4: Biosafety evaluation of YBO78-1 strain

[0047] First, inoculate the Escherichia coli YBO78-1 strain on the LB solid plate medium without anti-antibody, culture overnight at 37°C, then pick a single colony and inoculate it into 50ml of anti-LB liquid medium, place it in a small shaker, 37 After culturing overnight at 200 rpm at ℃, centrifuge at 10,000 rpm for 1 min to collect the precipitate, resuspend the precipitate with sterile PBS, count the viable bacteria of the bacterial suspension, and prepare for subsequent animal injection infection experiments.

[0048] Twenty-five 30-day-old SPF chickens were selected and randomly divided into 5 groups with 5 chickens in each group. Dilute the YBO78-1 bacterial solution to 1.0×10 8 CFU / ml, 2.0×10 8 CFU / ml, 4.0×10 8 CFU / ml, 8.0×10 8 Four different gradients of CFU / ml were used to inoculate 5 experimental SPF 1.0ml each, and the other group was injected with 1.0ml isolated wild YBO78 strain as a control.

[...

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Abstract

The invention provides an avian Escherichia coli vaccine strain. The avian Escherichia coli vaccine strain is a low virulent strain prepared by removing aroA genes on the basis of screening to obtainnovel avian pathogenic Escherichia coli O78. The avian Escherichia coli vaccine strain provided by the invention is prepared by deleting the aroA genes of an avian pathogenic Escherichia coli YBO78 strain with the preservation number of CCTCC M 2020382. The Escherichia coli vaccine YBO78-1 strain provided by the invention can be used by a conventional method, for example, the strain is applied ona large scale by economical and easy-to-operate spray and drinking water, and can also be applied by intramuscular injection or other modes.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to an avian Escherichia coli vaccine strain. Background technique [0002] Avian colibacillosis is an economically important infectious disease of poultry, the pathogen is avian pathogenic Escherichia coli (Avian Pathogenic Escherichia coli, APEC). APEC usually exists on the surface of intestinal tract and other mucous membranes of poultry and wild birds, and contains serotypes such as O1, O2, O78, O8, and O35, among which serotype O78 is the most common serotype in clinical isolation. The most serious manifestation of E. coli infection in poultry is septicemia, which usually begins with an upper respiratory tract infection following primary mycoplasma or viral infection, leading to infiltration into the blood and viscera, eventually causing pericarditis, hepatitis, air sacs, and salpingitis, etc. disease. [0003] The onset time of poultry colib...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K39/108A61P31/04C12R1/19
CPCC07K14/245A61K39/0258A61P31/04A61K2039/522A61K2039/552Y02A50/30
Inventor 李振楚电峰孙化露孙鹏侯玉超于晓璐张友明范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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