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Method for constructing full-length infectious cDNA [complementary DNA (deoxyribonucleic acid)] of PEDV JS2008 (porcine epidemic diarrhea virus JS2008) strains and application of full-length infectious cDNA

An infectious, full-length technology, applied in the fields of molecular biology and virology, which can solve the problems of lack of full-length infectious cDNA and effective vaccines, and achieve high efficiency

Active Publication Date: 2017-10-20
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In the prior art, there is a lack of full-length infectious cDNA and effective vaccines for studying the pathogenic mechanism of PEDV JS2008 strain

Method used

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  • Method for constructing full-length infectious cDNA [complementary DNA (deoxyribonucleic acid)] of PEDV JS2008 (porcine epidemic diarrhea virus JS2008) strains and application of full-length infectious cDNA
  • Method for constructing full-length infectious cDNA [complementary DNA (deoxyribonucleic acid)] of PEDV JS2008 (porcine epidemic diarrhea virus JS2008) strains and application of full-length infectious cDNA
  • Method for constructing full-length infectious cDNA [complementary DNA (deoxyribonucleic acid)] of PEDV JS2008 (porcine epidemic diarrhea virus JS2008) strains and application of full-length infectious cDNA

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Experimental program
Comparison scheme
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Embodiment 1

[0032] Embodiment 1: the design of porcine epidemic diarrhea virus JS2008 strain genome full-length cDNA construction

[0033] The key point of the method of the present invention is the selection of the enzyme cutting site, then the full-length genome of the porcine epidemic diarrhea virus JS2008 strain is divided into several fragments of appropriate size, and the silent mutation is used to eliminate or introduce type II endonuclease PflmI to digest site, after cloning and preservation of each fragment, use an appropriate endonuclease to digest, and then ligate in vitro to obtain the full-length infectious cDNA of PEDVJS2008 strain.

[0034]Various endonuclease cleavage sites in the full-length genome sequence of PEDV JS2008 strain (Genebank accession number: KC109141.1) were analyzed, and a large number of experiments were carried out. Finally, a class II endonuclease PflmI was found to be suitable for segmenting the full-length genome of JS2008 strain to prepare full-lengt...

Embodiment 2

[0039] Embodiment 2: each fragment of the full-length infectious cDNA of amplification JS2008 strain genome

[0040] (1) Viruses, vectors and main reagents

[0041] The classic PEDV strain JS2008 (GenBank accession number: KC109141) was isolated and preserved by our laboratory; the fragment cloning vector pSMART kit (including 4 × CloneSmart Vector Premix and CloneSmart DNA Ligase) was purchased from Lucigen, USA; the viral RNA extraction kit, DNA gel recovery kit, plasmid small extraction kit and large extraction kit were purchased from QIAGEN; the reverse transcription kit SuperScriptIII First-Strand Synthesis System (including SuperScriptTM IIIReverse Transcriptase and Oligo dT(18)) was purchased from Thermo; High-fidelity DNA polymerase Pfu UltraTM II Fusion HS DNA Polymerase was purchased from Stratagene. Other biochemical reagents such as Marker were purchased from Bao Biological Engineering (Dalian) Co., Ltd.; other conventional reagents were domestic or imported analy...

Embodiment 3

[0057] Example 3: Construction of JS2008 strain genome full-length infectious cDNA

[0058] 1. Materials and methods

[0059] (1) Main reagents

[0060] The DNA gel recovery kit was from QIAGEN; all restriction enzymes and T4 DNA ligase were purchased from NEB; the in vitro transcription kit mMESSAGE T7 Ultra Kit was purchased from Thermo Company. Other biochemical reagents such as Marker were purchased from Bao Biological Engineering (Dalian) Co., Ltd.; other conventional reagents were domestic or imported analytically pure.

[0061] (2) Preparation of enzyme-digested fragments

[0062] by connection strategy figure 1 As shown, the recombinant vector pS-A was double-digested with XbaI and PflmI, pS-mB, pS-C, pS-mD and pS-E were single-digested with PflmI, and pS-mF was double-digested with PflmI and EcoRV. Digested with restriction enzymes, recovered cDNA fragments (Qiagen Gel Recovery Kit), and then used Nanodrop to measure the concentration of each digested fragment....

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Abstract

The invention provides a method for constructing full-length infectious cDNA [complementary DNA (deoxyribonucleic acid)] of PEDV JS2008 (porcine epidemic diarrhea virus JS2008) strains and application of the full-length infectious cDNA, and belongs to the field of molecular biology and virology. The method for constructing the full-length infectious cDNA of the PEDV JS2008 strains includes steps of amplifying 6 fragments from reverse transcription products of total virus RNA (ribonucleic acid) of the PEDV JS2008 strains, inserting the fragments into vectors pSMART and carrying out silent mutation on enzyme digestion sites of incision enzymes PflmI at inappropriate locations; carrying out enzyme digestion on various recombinant vectors, recycling target cDNA fragments and connecting the target cDNA fragments with one another to obtain the full-length infectious cDNA of the PEDV JS2008 strains. The reverse transcription products are used as templates. The method for constructing the full-length infectious cDNA of the PEDV JS2008 strains and the application have the advantages that the method is ingenious, is high in efficiency and can be applied to rescuing porcine epidemic diarrhea viruses, and accordingly research on PEDV pathogenic mechanisms and novel vaccine can be carried out.

Description

technical field [0001] The invention belongs to the fields of molecular biology and virology, and specifically relates to a construction method and application of full-length infectious cDNA of PEDV JS2008 strain. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute, highly transmissible disease, the main pathogen causing the disease is porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV). The main clinical features of the disease are vomiting, watery diarrhea, dehydration and metabolic acidosis. Pigs of all ages are susceptible, but if it affects suckling piglets, the morbidity rate is 100%, and the mortality rate is as high as 80%-100%. The International Committee on Taxonomy of Viruses classified PEDV into the order Nestoviridae, the subfamily Coronaviridae, and the genus alpha-Coronaviridae. Other members of Alphacoronavirus include Alphacoronavirus 1 (including cat coronavirus, TGEV and canine coronavi...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/63C12N7/00
CPCC07K14/005C12N15/63C12N2770/20021
Inventor 范宝超李彬何孔旺焦点郭容利俞正玉朱琳
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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