A Recombinant Attenuated Salmonella Enteritidis Vaccine

A Salmonella Enteritidis and vaccine technology, applied in the directions of viruses/phages, viruses, bacteria, etc., can solve the problems of lack of and inability to stably express foreign proteins, save labor costs, prevent and control Salmonella infection, and have a simple and convenient production process. Effect

Active Publication Date: 2020-03-20
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 Vaccine
  • A Recombinant Attenuated Salmonella Enteritidis Vaccine
  • A Recombinant Attenuated Salmonella Enteritidis Vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0018] After conducting biochemical tests, serological tests and sequencing identification on the suspected Salmonella isolated clinically, the applicant identified it as Salmonella Enteritidis 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 o...

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, and 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 present invention provides an attenuated Salmonella Enteritidis vaccine, wherein the antigen is the recombinant Salmonella Enteritidis vaccine strain with the deposit number of CGMCC NO.13251. The vaccine provided by the invention is safe and reliable, and does not cause the risk of dispersing; the conventional injection of the vaccine will cause stress and the absorption effect of the vaccine is not good, but the vaccine can be immunized by oral administration, and the possibility of the immunogen being degraded before reaching the intestinal mucosa is avoided. ; This vaccine integrates the carrier and the adjuvant, which can not only induce the body to produce humoral immunity, but also produce cellular immunity and mucosal immunity, which can not only prevent and control IBDV, but also prevent and control Salmonella infection to a certain extent; The production process is simple and convenient, the production cost is extremely low, and no protein purification is required, and the vaccine bacteria only need to be expanded and lyophilized, and then oral immunization can save labor costs.

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 vaccine. [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, tradi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/36C12N1/21A61K39/39A61K39/112A61P31/04A61P31/14C12R1/42
CPCA61K39/0275A61K39/39A61K2039/542A61K2039/55594A61K2039/57A61K2039/575C07K14/005C12N1/36C12N2720/10022C12N1/205C12R2001/42
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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