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Production of rough-type salmonella enteritidis and genetic modifications thereof for use as avian vaccine

A Salmonella Enteritidis, gene technology, applied in veterinary vaccines, vaccines, genetic engineering and other directions, can solve problems such as decreased immune response

Pending Publication Date: 2020-05-01
兽医制药股份公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the case of supply of an attenuated vaccine strain of the pneumococcal antigen PspA, all mutations tested resulted in reduced immune responses against both PspA and Salmonella antigens

Method used

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  • Production of rough-type salmonella enteritidis and genetic modifications thereof for use as avian vaccine
  • Production of rough-type salmonella enteritidis and genetic modifications thereof for use as avian vaccine
  • Production of rough-type salmonella enteritidis and genetic modifications thereof for use as avian vaccine

Examples

Experimental program
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Effect test

Embodiment approach

[0119] Preferred Embodiments of the Invention: Examples

[0120] The following examples are provided herein to illustrate the nature of the invention. These examples are for illustrative purposes only and should not be construed as limitations of the invention.

example 1

[0121] Example 1: Expression of fiber antigens in Salmonella Enteritidis 3934Vac strain by immunoblotting.

[0122] protein extraction

[0123] We placed 1 colony in 5 mL of LB medium for ClyA fibers with an insertion in the chromosome. Incubate overnight at 37 °C at 200 rpm.

[0124] We centrifuged at 110000rpm for 5 minutes, then added lysis buffer 1 (Tris HCL 10Mm, EDTA 5mM, NaCl 50mM) + protease inhibitors 40μL (3g / mL) + 50μL lysozyme (10mg / mL), and SDS of each sample Buffer (50 μL Easy Buffer 2x + 50 μL Urea 8M + 5 μL B-Mercaptoethanol 50 μL sample), homogenize and heat at 100°C for 5 minutes, then place on ice for 5 minutes, take 20 μL for loading. Use 0.1Amps for each gel at 1:30.

[0125] A clyA-fiberFAdV-His protein band was observed in the results of Western blot. Markers used; ladder P7709V (175KDa) on the left, ab 116029 (245KDa) on the right ( Figure 10 ).

[0126] Also, when using two positive His+ controls, but with the initials A and B by weight, A is ab...

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Abstract

The present invention relates to a strain of salmonella enteritidis 3934vac from which the waaL gene has been deleted in order to obtain a rough phenotype (3934vac DwaaL), as well as to the method forobtaining the same and to the oligos used, with a view to reducing toxicity and maintaining immunogenicity for the use of the same as a vaccine. The present invention also relates to a strain of thesalmonella enteritidis 3934vac DwaaL, i.e. of the rough type, which has been modified to express the fibre gene of type-1 avian adenovirus, as well as to the method for obtaining the strain of the salmonella enteritidis 3034 vac DwaaL which expresses a fibre gene of Ava-I. The invention further comprises the development of a novel avian vaccine against the AvA-I virus, which is live, recombinant,effective and harmless and was developed by using a process involving inserting and integrating AVa-I fibre genes into the chromosome of a non-pathogenic attenuated strain of the salmonella enteritidis bacteria.

Description

technical field [0001] The present invention belongs to the field of animal health. In particular, the present invention relates to the development of attenuated and rough-surfaced Salmonella Enteritidis Enteritidis (S. Enteritidis or SE) strains obtained by deletion of genes involved in lipopolysaccharide synthesis. In this context, it also includes the use of the strain as a vaccine vector against the Ava-I virus by the process of insertion and integration into the chromosome encoding the Ava-I fiber antigen. The SE vector that has been used has been genetically modified as a vector for the expression of immunodominant genes of AvA-I viral fibers with the aim of stimulating an efficient and long-lasting immune response against HCI (viral hepatitis via inclusion bodies) in avians. The resulting strain is completely safe as it does not contain the twelve genes encoding the proteins of the signaling pathway of the second cyclic di-GMP message, the sigma RpoS factor and the Waa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/11
CPCC12N9/93A61K2039/523A61K2039/552A61K2039/522A61K39/12C12N2710/10234C12N1/20C12N15/102C12N15/902C12N2500/34
Inventor 克里斯蒂娜·拉塔萨·奥斯塔
Owner 兽医制药股份公司
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