Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application
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A technique for porcine epidemic diarrhea and salmonella
Active Publication Date: 2013-04-03
HUAZHONG AGRI UNIV +1
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Problems solved by technology
There are many defects in the traditional construction method of recombinant Salmonella vaccine. For example, the previously used expression plasmid carrying the resistance gene is not accepted by people because of its biosafety problems.
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Embodiment 1
[0086] Embodiment 1: Prepare the gene fragment of the porcine epidemic diarrhea protective antigen COE and SD protein capable of prokaryotic expression
[0087] (1) Design of primers required for preparation of prokaryotic expression plasmids pET-32a-COE and pET-32a-SD, the sequences of which are described in Table 1.
[0088] Table 1 The primer sequences required for the preparation of prokaryotic expression plasmids pET-32a-COE and pET-32a-SD
[0089]
[0090]
[0091] The underlined parts of the primers in Table 1 are restriction sites.
[0092] (2) Preparation of COE and SD fragments capable of prokaryotic expression
[0093] Using the multiplex RT-PCR detection method of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine group A rotavirus reported by Zhang Kun (Zhang Kun, 2010), the clinical disease materials submitted for inspection were detected. Genomic RNA was extracted from porcine epidemic diarrhea virus-infected positiv...
Embodiment 2
[0097] Example 2: Construction and Identification of Recombinant Plasmids pYA-COE and pYA-SD
[0098] Recombinant plasmid pET-32a-COE with EcoR I and HindIII restriction sites and pET-32a-SD with ECOR I and Sal I restriction sites were subjected to double digestion and recovery, and then subjected to the same double digestion Recovered shuttle plasmid pYA3493 (plasmid construction map see image 3 ) ligation, the ligation product was transformed into Escherichia coli x6097 with asd gene deletion (source: a gift from Dr. Roy Curtiss III, Washington University, USA), positive clones were screened, the plasmid was extracted, and the recombinant plasmid pYA with the correct construction was obtained after identification by PCR and double enzyme digestion -COE and pYA-SD (eg Figure 4 and Figure 5 shown). Wherein, the PCR amplification system and amplification conditions are as described in Example 1, (2).
Embodiment 3
[0099] Example 3: Construction and identification of recombinant Salmonella strains C501-COE and C501-SD expressing porcine epidemic diarrhea virus COE and SD fusion protein
[0100] (1) identify the design of the primers required for the recombined Salmonella C501-COE and C501-SD, its DNA sequence is as shown in table 2:
[0101] Table 2 identifies the DNA sequence of the primers required for the recombined Salmonella C501-COE and C501-SD
[0102]
[0103] (2) Construction and identification method of recombinant Salmonella C501-COE and C501-SD
[0104] The correctly identified recombinant plasmids pYA-COE and pYA-SD (see Example 2 for sources) were electrotransformed into asd-deleted C500-competent cells respectively, and the parameters of the electroporator (Bio-Rad GenePulserII) were set to: voltage 2.2Kv, Capacitance 25μF, pulse resistance 200Ω and time 4ms, construction flow chart see Figure 6 . On the DAP negative plate, pick a single colony and culture it in TSB...
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Abstract
The invention belongs to the technical field of animal bacterial genetic engineering, and concretely relates to a construction of recombinant Salmonella choleraesuis strains C501-Coe and C501-SD with no resistance marker and expressing main antigenic sites of porcine epidemic diarrhea virus, a preparation of a vaccine and an application. The invention obtains the recombinant Salmonella choleraesuis strains C501-Coe and C501-SD with no resistance marker and expressing the main antigenic sites of the porcine epidemic diarrhea virus, and the strains have the following accession number respectively: CCTCC NO: M2011296 and CCTCC NO: M2011297. The two recombinant strains are deleted with an asd gene necessary for growth of the S. choleraesuis, and contain plasmids which can express the asd gene, as well as a COE gene fragment and a SD gene fragment of the Porcine epidemic diarrhea virus in the strains. <{EN3}>The invention further discloses a method and an application by using the recombinant strain to prepare the vaccine of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus. <{EN4}>The vaccine provided by the invention can stimulate swine to generate a protective immunization reaction for resisting the Salmonella choleraesuis and the Porcine epidemic diarrhea virus, and can effectively prevent infection of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus. The invention further discloses a method and an application by using the recombinant strain to prepare the vaccine of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus. The vaccine provided by the invention can stimulate swine to generate a protective immunization reaction for resisting the Salmonella choleraesuis and the Porcine epidemic diarrhea virus, and can effectively prevent infection of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus.
Description
technical field [0001] The invention belongs to the technical field of animal bacterial genetic engineering, and specifically relates to the construction and application of two recombinant Salmonella choleraesuis live vaccine strains expressing respectively the protective antigen gene of porcine epidemic diarrhea virus-COE gene and SD gene without resistance marker . Background technique [0002] Salmonella (Salmonellu) is an intracellular parasitic Gram-negative intestinal bacilli with invasiveness. The most common Salmonella genera are Salmonella typhimurium, Salmonella choleraesuis, and Salmonella enteritidis. Salmonella has strong pathogenicity to both humans and animals, and can cause gastrointestinal diseases in humans and animals. Salmonella has a strong ability to survive in the external environment. After being weakened by genetic engineering, it still has strong pathogenicity. of invasiveness. [0003] The initial vaccine for the prevention of Salmonella is a who...
Claims
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Application Information
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