A Recombinant Attenuated Salmonella Enteritidis

A Salmonella Enteritidis, recombinant technology, applied in the direction of viruses/phages, recombinant DNA technology, viruses, etc., can solve the problems of lack of and inability to stably express foreign proteins, and achieve good safety, good growth and reproduction performance, and good genetic stability. Effect

Active Publication Date: 2020-07-24
SHANDONG SINDER TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently problems such as the lack of stable attenuated strains of Salmonella, and the inability to stably express foreign proteins, thereby causing effective immune responses.

Method used

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  • A Recombinant Attenuated Salmonella Enteritidis
  • A Recombinant Attenuated Salmonella Enteritidis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the screening of Salmonella enteritidis attenuated strain

[0018] The applicant identified the suspected Salmonella isolated from the clinic as Salmonella enteritidis after biochemical tests, serological tests and sequencing identification, and named it SD strain. The recombinant strain rSD of Salmonella enteritidis was obtained after subculture screening. Through the determination of the growth curve, it was found that the growth rate of the bacteria was not affected by the deletion of the gene, and it was determined to be an attenuated strain through the chick challenge test. The rSD strain was used as the parent strain for further research.

[0019] The attenuated Salmonella Enteritidis rSD strain of the present invention was preserved on November 28, 2016 in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiolo...

Embodiment 2

[0020] The construction of embodiment 2, VP2 gene transfer vector

[0021] According to the sequence of the Cat gene in the plasmid pkD3 and the multiple cloning restriction site of the cloning vector pBluescript KS (+ / -), the specific primers of the Cat gene were designed using Primer 5.0 software: upstream primer pKD3-II P1:CACGGATCCgtgtaggctggagctgcttc added BamH I Restriction site; downstream primer pKD3-II P2:CAGGAATTCcatatgaatatcctccttag added EcoR I restriction site. The plasmid pkD3 was used as a template for PCR amplification, and the target gene was recovered with a DNA purification and recovery kit. The recovered target gene fragment was subjected to double enzyme digestion reaction with the carrier pBluescript KS (+ / -), followed by nucleic acid electrophoresis. After recovering the target gene with a DNA purification and recovery kit, it was ligated with T4 DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli JM...

Embodiment 3

[0023] Embodiment 3, preparation of targeting fragment

[0024] The primers used for homologous recombination consist of two parts, the uppercase 50nt sequence at the 5' end is homologous to the flanking sequence of the target gene, and the lowercase 20nt sequence at the 3' end is identical to the sequence on both sides of the transfer vector cat resistance gene or the target gene source. Upstream primer: VP2-D1: AGTGGACTAACAACATCGGTGATGCCAACACCATCGGCACCCGTCCGGACgtgtaggctggagctgcttc; Downstream primer: VP2-D2: CAGAGCAAAAAACCCCGCGACGCGGGGTTTTTATCAGACGGAAACTTAAttaccttatggcccggat. Using the recombinant plasmid pBKV as a template, the target fragment was amplified by PCR, and the PCR product was identified by 1% agarose gel electrophoresis, the target band was recovered, and the recovered product was digested with Dpn I. After 1% agarose gel electrophoresis, the target band was recovered by cutting the gel, and the DNA concentration was determined.

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Abstract

The invention provides a rSD strain of attenuated Salmonella enteritidis, and its preservation number is CGMCC NO.13346. The attenuated Salmonella enteritidis provided by the invention is used to prepare recombinant vaccine strains. Another aspect of the present invention provides a recombinant Salmonella enteritidis live vector vaccine strain, whose genome contains the VP2 gene of IBDV. The preservation number of one of the recombinant Salmonella enteritidis live vector vaccine strains is CGMCC NO.13251. The recombinant vaccine strain constructed by the attenuated Salmonella enteritidis provided by the present invention as a host has good genetic stability. It has been continuously passed on for 20 generations in ordinary medium, and the inserted exogenous gene has not undergone any mutation; the recombinant bacterium does not contain any resistance, which is in line with modern The development trend of bacterial live vaccines has good safety and does not need to consider antibiotic residues. The growth and reproduction performance of the recombinant bacteria is good, and the proliferation of the bacteria is not affected by the insertion of the foreign gene.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a recombinant attenuated Salmonella enteritidis. [0002] technical background [0003] Infectious bursal disease (IBD) is a highly contagious disease of chickens and turkeys caused by chicken infectious bursal virus (IBDV). The incidence rate is high and the course of disease is short. The disease mainly affects chicks and young chickens aged 3-12 weeks, destroying B lymphocytes in the bursa of Fabricius, resulting in varying degrees of immunosuppression, and can induce various diseases or make various vaccines immune failure, thereby increasing the number of sick chickens. Susceptibility to concurrent and secondary viral and bacterial infections is one of the important infectious diseases that have seriously threatened the poultry industry in my country in recent years. [0004] At present, in the prevention and treatment of IBD, traditional l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/74C12N1/21A61P31/14C12R1/42
CPCC07K14/005C12N15/74C12N2720/10022C12N1/205C12R2001/42
Inventor 李明义孙化露刘阳毕云英李思菲于泽坤马丽马礼照单学强颜瑞娟胡秀香李朝阳
Owner SHANDONG SINDER TECH
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