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Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof

A technology for porcine epidemic diarrhea and a construction method, which is applied in the field of construction of infectious cDNA clones of porcine epidemic diarrhea virus, can solve problems such as limited protection, achieve good stability, improve rescue efficiency, and improve efficiency.

Pending Publication Date: 2022-04-26
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, current commercial vaccines offer limited protection, and the development of a new generation of vaccines remains urgent

Method used

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  • Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof
  • Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof
  • Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The construction of embodiment 1 porcine epidemic diarrhea virus HM strain infectious cDNA clone plasmid

[0046] Build strategies such as figure 1 shown. According to the full-length genome sequence of porcine epidemic diarrhea virus genotype II HM strain (Genbank accession number: MZ342899), 7 pairs of specific primers (see Table 1) were designed using Snapgene software to amplify F1, F2, F3, Seven fragments of F4, F5, F6 and F7.

[0047] The viral genome RNA of the PEDVHM strain was extracted according to the instructions of the viral genome DNA / RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). Using viral genome RNA as a template, according to IV First-Strand cDNA Synthesis Reaction (Thermo Fisher Scientific) describes the preparation of cDNA of HM strain of epidemic diarrhea virus. The reaction system is: 1 μL of 2 μM gene-specific reverse primer (PEDV-R7, PEDV-R4 or PEDV-R2), 1 μL of 10 μM dNTP Mix, and 11 μL of template RNA. The reaction proc...

Embodiment 2

[0053] Example 2 Construction of recombinant plasmid pYES1L-PEDV-EGFP based on CRISPR / Cas9 gene editing technology

[0054] Build strategies such as Figure 4 shown. Based on the CRISPR / Cas9 gene editing technology, two guide RNAs, PEDV-sgRNA1 and PEDV-sgRNA2 (synthesized in Nanjing GenScript Biotechnology Co., Ltd., see Table 1), were designed to cut the flanking sequences of ORF3 in pYES1L-PEDV to obtain Porcine epidemic diarrhea virus infectious cDNA cloning vector backbone lacking ORF3. The specific operation is: mix 5 μL of Cas9 Nuclease Buffer, 3 μL each of PEDV-sgRNA 1 (300 nM) and PEDV-sgRNA 2 (300 nM), and 3 μL of Cas9 Nuclease, add 3 μg of recombinant plasmid pYES1L-PEDV after 10 min at 37 °C, recover after 2 h at 37 °C Porcine epidemic diarrhea virus infectious cDNA cloning vector backbone lacking ORF3.

[0055] Using pEGFP-N1 (Clontech) as a template, PCR amplification was performed with primers EGFP-F and EGFP-R (see Table 1) to obtain the EGFP gene fragment. ...

Embodiment 3

[0057] Embodiment 3: Construction of helper plasmid

[0058]For the open reading frame region of the N gene in PEDV, use Snapgene software to design specific primers PEDV-N-F and PEDV-N-R (see Table 1), and use pYES1L-PEDV as a template to perform PCR amplification to obtain the N gene fragment. Insert the recovered and purified N gene fragment between the SacI and XhoI sites of the pCAGGS vector through conventional enzyme digestion ligation test, and then transform the ligation product into DH5α Escherichia coli competent cells, and screen out the correct recombinant plasmid pCAGGS- PEDV-N. Extract the plasmid according to the operating instructions of the endotoxin-free plasmid extraction kit, measure the concentration and store in -20°C.

[0059] Table 1 The primer sequences used in the present invention

[0060]

[0061]

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Abstract

The invention provides a construction method and application of porcine epidemic diarrhea virus infectious cDNA clone, and belongs to the technical field of biology. The construction method comprises the following steps: amplifying F1, F2, F3, F4, F5, F6 and F7 fragments by taking a genome RNA (Ribonucleic Acid) reverse transcription product of the porcine epidemic diarrhea virus as a template; on the basis of a homologous recombination mechanism in yeast, a linearized pYES1L vector and F1-F7 fragments are mixed and transformed into yeast competent cells, so that one-step seamless connection of the virus cDNA fragments and an original vector is realized to obtain porcine epidemic diarrhea virus infectious cDNA clone plasmids; based on a CRISPR / Cas9 gene editing technology, a virus ORF3 coding region is replaced by a green fluorescent protein gene, and the recombinant porcine epidemic diarrhea virus expressing the green fluorescent protein is constructed. The method is quick, simple and convenient, has high success rate, can be used for rescuing the porcine epidemic diarrhea virus and other coronaviruses, and provides a reliable technical platform for researching the replication mechanism and pathogenesis of the virus and developing novel vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of porcine epidemic diarrhea virus infectious cDNA clone. Background technique [0002] Porcine Epidemic Diarrhea (PED) is an acute and highly contagious porcine intestinal disease caused by Porcine Epidemic Diarrhea Virus (PEDV), and its clinical symptoms are similar to porcine transmissible gastroenteritis Virus (Transmissible Gastroenteritis Virus, TGEV) similar, characterized by vomiting, diarrhea and anorexia, and finally death due to emaciation and severe dehydration. All age groups can be infected, and the clinical symptoms of piglets after infection are particularly severe, and the mortality rate is close to 100%. In addition to infecting suckling piglets, PEDV can also reduce growth performance in finishing pigs. In recent years, the emergence and widespread spread of highly pathogenic PEDV variant strains have caused huge ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C12N7/01C12N15/63C12N15/12
CPCC12N7/00C07K14/005C12N15/63C07K14/43595C12N2770/20021
Inventor 李燕华周弇扬李晨曦任次成胡晶博
Owner YANGZHOU UNIV
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