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Preparation and application of oral recombinant saccharomyces cerevisiae for expressing porcine epidemic diarrhea virus S protein

A technology for porcine epidemic diarrhea and recombinant Saccharomyces cerevisiae is applied in the field of preparation of oral recombinant Saccharomyces cerevisiae, which can solve the problems of poor vaccine effect and inability to effectively prevent the spread of viremia, and achieves induction of intestinal viscous immune response and good application development. Promising, low-cost effects

Pending Publication Date: 2021-12-03
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems existing in the prior art, the present invention provides the preparation and application of an oral recombinant Saccharomyces cerevisiae expressing the S protein of porcine epidemic diarrhea virus, which solves the problem that the porcine epidemic diarrhea virus vaccine in the prior art is not effective and cannot be effective. Problems in preventing the spread of viremia and PEDV

Method used

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  • Preparation and application of oral recombinant saccharomyces cerevisiae for expressing porcine epidemic diarrhea virus S protein
  • Preparation and application of oral recombinant saccharomyces cerevisiae for expressing porcine epidemic diarrhea virus S protein
  • Preparation and application of oral recombinant saccharomyces cerevisiae for expressing porcine epidemic diarrhea virus S protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030]The construction of embodiment 1.GPD-PEDV-S-TU carrier

[0031] (1) Amplification of PEDV-S gene

[0032] Referring to the gene sequence of the plasmid pMD18-T-S containing the PEDV S gene retained by the laboratory, using the pMD18-T-S plasmid as a template, design and synthesize primers to amplify the preferred S protein truncated gene, PEDV-S-F (SEQ ID NO. 5) and PEDV-S-R (SEQ ID NO. 6).

[0033] The PCR amplification system is:

[0034]

[0035] Amplify using the following PCR program:

[0036] PCR reaction program

[0037] 98°C for 3 minutes;

[0038] 98°C 10sec, 60°C 20sec, 72°C 30sec, 30 cycles;

[0039] 72°C for 5 minutes;

[0040] The size of the PCR product is PEDV-S: 1509bp.

[0041] PCR results such as figure 1 As shown, lane 1 is the amplification result of the S gene truncated body.

[0042] (2) Construction of GPD-PEDV-S-TU vector

[0043] Connect the S gene truncated body to the POT-GPD-TU vector constructed in the laboratory [21] . The POT...

Embodiment 2

[0048] Example 2. Construction and detection of porcine epidemic diarrhea virus yeast recombinant strain ST1814G-PEDV-S

[0049] (1) Construction of Yeast Transformation Fragment

[0050] The vector GPD-PEDV-S-TU was cut with BsaI, and the homology arm plasmids (SUR-TU and SUR-TD) and the selectable marker plasmid (Trp) were cut with BsmB I. Refer to the experimental steps of Dai's research group [22] , the SURs homology arm, Trp selective tag, and GPD-PEDV-S-TU transcription unit were spliced ​​according to the specific prefix and suffix sequences, T4 ligase was ligated overnight at 16°C, and the spliced ​​product was used for yeast transformation.

[0051] (2) Construction of ST1814G-PEDV-S recombinant Saccharomyces cerevisiae strain

[0052] ① Strain activation: Pick a single colony of ST1814G and inoculate it into 3 mL of YPD liquid medium, and cultivate overnight at 30°C and 220 rpm. Transfer the overnight cultured bacterial solution to 10mL of fresh YPD medium at a ra...

Embodiment 3

[0071] Example 3. Evaluation of the yeast recombinant strain of porcine epidemic diarrhea virus

[0072]Sows in Jiafa Ranch in Wuyishan City, Fujian Province were divided into two groups, 11 in each group, and fed with ST1814G-PEDV-S No.4 yeast strain after being infected with PEDV, and ST1814G blank yeast strain was used as control. Detection of porcine epidemic diarrhea virus IgA antibody level in sow milk by indirect ELISA.

[0073] The result is as Figure 8 As shown, the level of specific IgA in the milk of sows fed ST1814G-PEDV-S No.4 recombinant yeast group was significantly increased by about 25% and neatly. This shows that the S1 protein surface-displayed oral recombinant Saccharomyces cerevisiae preparation developed by the present invention can effectively promote the level of maternal antibody, and can provide an effective supplement for the prevention and control of porcine epidemic diarrhea virus.

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Abstract

The invention discloses preparation and application of oral recombinant saccharomyces cerevisiae for expressing porcine epidemic diarrhea virus S protein. The oral recombinant saccharomyces cerevisiae comprises S1 structural domains from 234 site to 633<rd> site of the S protein and partial S2 structural domains from 640 site to 736 site. An S protein truncated body complete transcription unit GPD-PEDV-S-TU constructed in vitro is integrated into a yeast genome through homologous recombination; the S protein truncated body is displayed on the surface of a yeast cell by utilizing an Aga1-Aga2 surface display system to obtain an S protein surface display type recombinant yeast strain ST1814G-PEDV-S; and the oral recombinant yeast is prepared by utilizing the obtained strain. The oral recombinant yeast disclosed by the invention has the advantages of low cost, safety in production and utilization, simplicity and convenience for operation, safety and effectiveness, and has an immune protection effect; and a choice is provided for prevention and control of porcine epidemic diarrhea.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to a preparation method and application of oral recombinant Saccharomyces cerevisiae for preparing porcine epidemic diarrhea virus S protein. Background technique [0002] Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV), characterized by diarrhea, vomiting, and , dehydration and high neonatal mortality [1] . Since it was first reported in the UK and Belgium in 1971, it has spread to China, Canada, Japan, South Korea and other countries. The prevalence and outbreak of PED have caused huge economic losses to the world pig industry. [2,3] . [0003] PEDV is an enveloped single-stranded positive-sense RNA virus with a genome length of about 28 kb, with a 5' cap and a 3' polyadenylated tail, and at least 7 open reading frames (ORF1a, ORF1b, ORF2~ 6), encoding replicase polyprotein ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/50C12N15/81C07K14/165A61K39/215A61P31/14C12R1/865
CPCC07K14/005C12N15/81A61K39/12A61K9/0053A61P31/14C12N2770/20022C12N2770/20034C12N2800/102A61K2039/523A61K2039/542
Inventor 黄金海孙瑞骐郭艳余
Owner TIANJIN UNIV
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