Amplification method of porcine epidemic diarrhea virus S-gene epitope

A porcine epidemic diarrhea and epitope technology, applied in the direction of microbial-based methods, genetic engineering, plant genetic improvement, etc., can solve problems such as unspecified S genes

Active Publication Date: 2013-05-08
GUANGDONG WENS DAHUANONG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, neither of these two amplification methods indicated whether the S gene o

Method used

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  • Amplification method of porcine epidemic diarrhea virus S-gene epitope

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Embodiment 1

[0080] A method for amplifying the porcine epidemic diarrhea virus S gene epitope, comprising the following steps:

[0081] 1) Extraction of viral RNA: centrifuge the water sample feces of pigs at 12000rpm for 5 minutes, take the supernatant or cell culture medium at -20°C for 2-3 times, and then use Ta Ka Ra mini BEST viral RNA / DNA extraction kit Ver .4.0 (DV819A) Extract viral RNA;

[0082] Use TaKaRa mini BEST viral RNA / DNA extraction kit Ver.4.0 (DV819A) to extract viral RNA in sequence as follows:

[0083] ①Put 200μl of disease material treatment solution or frozen-thawed cell culture solution into a 1.5ml centrifuge tube, add 200μl of Solution A, shake vigorously and mix well, and place at room temperature for 5 minutes;

[0084] ② Add 75 μl of Solution B, mix well, and centrifuge at 12,000 rpm for 5 minutes;

[0085] ③ Transfer the supernatant to a new Collection Tube (2ml, provided in the kit), add 250μl of isopropanol (containing 1% glacial acetic acid), and mix up ...

Embodiment 2

[0142] The 15th generation of viral RNA extracted in Example 1 and cultured on Vero cells was amplified according to the same steps as in Example 1 to amplify a 782bp target fragment (comprising the neutralization antigenic epitope sequence between 1495bp-1914bp of the S gene) , the determined sequence is SEQ ID NO:6.

Embodiment 3

[0144] The 30th generation of viral RNA extracted in Example 1 and cultured on Vero cells was amplified according to the same steps as in Example 1 to amplify a 782bp target fragment (comprising the neutralization antigenic epitope sequence between 1495bp-1914bp of the S gene) , the determined sequence is SEQ ID NO:7.

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Abstract

The invention discloses an amplification method of porcine epidemic diarrhea virus S-gene epitope. The amplification method comprises the steps of: (1) extraction of virus RNA (Ribonucleic Acid); (2) amplification of RT-PCR (Reverse Transcription-Polymerase Chain Reaction); (3) amplification of nested PCR (Reverse Transcription-Polymerase); and (4) cloning, sequencing analysis and the like of a PCR product and the like. An S-gene of the porcine epidemic diarrhea virus (PEDV) is amplified, and the amplified fragment is 782bp in length (situated in a range from 1316bp-2097bp of the S-gene) and comprises neutralized antigenic epitope situated in a range from 1495bp-1914bp of the S-gene; and according to the amplification method, different generations of S-genes of PEDV (porcine epidemic diarrhea virus) seed, which are cultured on a vero cell, can be amplified, and the S gene of the porcine epidemic diarrhea virus in a cell culture and passage process, particularly the sequence change of the neutralized antigenic epitope, is captured, thus the relationship between the sequence change of the neutralized antigenic epitope and the immune efficacy change of the PEDV can be known.

Description

[0001] technical field [0002] The invention relates to the technical field of porcine epidemic diarrhea virus, in particular to a method for amplifying antigenic epitope of S gene of porcine epidemic diarrhea virus. [0003] Background technique [0004] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus. It is characterized by vomiting, diarrhea and loss of appetite. Pigs of all ages are prone to feel. The S gene of porcine epidemic diarrhea virus (PEDV) is 4150bp in length, and the S protein encoded by it is located on the surface of the virion. The S gene of PEDV has a neutralizing antigenic epitope between 1495bp-1914bp (420bp) of the S gene. [0005] Liu Deng et al. obtained porcine epidemic diarrhea virus A 966bp (61bp-1026bp) part of the S gene was amplified in the CH / ZJ isolate, but the sequence did not contain the neutralizing epitope located between ...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/10C12N15/70C12R1/19C12R1/93
Inventor 何玲陈瑞爱唐满华梁桂益
Owner GUANGDONG WENS DAHUANONG BIOTECH
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