Whole genome cloning method of pelteobagrus fulvidraco bacillus-shaped virus
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A technology of whole genome and cloning method, applied in the field of whole genome cloning of fish bacillus-like virus, can solve problems such as no reports, and achieve the effect of convenient subsequent purification and sequencing, and simple and practical operation.
Inactive Publication Date: 2019-05-10
SHAOXING UNIVERSITY
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Problems solved by technology
At present, there are no reports on the genome cloning methods of Pelyophyllum-like virus and Chinook salmon virus
Method used
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Embodiment 1
[0011] Example 1: This example describes the design and synthesis of primers for cloning the entire genome of the Pelyobacterium-like virus provided by the present invention.
[0012] Design and optimize the primers: design based on the genome sequence of P. japonica-like virus. The above primer design principles are as follows: ①The distance between the upstream and downstream positions is not greater than 4kb; ②The primer itself does not form a secondary structure; ③The primer Tm value is moderate, G+C The content is between 40% and 60%; ④The primer itself cannot have 4 consecutive complementary bases. The primer sequences are shown in the table below:
Embodiment 2
[0013] Example 2: This example describes the RNA extraction strategy of the Pelyobacterium-like virus provided by the present invention.
[0014] Take 200 μL virus sample and place it in a 1.5ml centrifuge tube, add 200 μL tissue lysate, and lyse at room temperature for 30 minutes;
[0015]
[0016] Genomic RNA Extraction Kit (Axygen Company) provided the operating steps to extract viral RNA; finally dissolved in 40 μL sterile water and stored at low temperature for future use.
Embodiment 3
[0017] Example 3: This example describes the reaction conditions for the reverse transcription of viral cDNA provided by the present invention.
[0018] (1) Use PrimeScript TM 1st StrandcDNA Synthesis Kit (Takara Company) prepared the following reaction system:
[0019]
[0020]
[0021] For different fragment clones, add corresponding reverse transcription primers P1-R, P2-R, P3-R, P4-R, P5-R, P6-R, P7-R, P8-R, P9-R, P10 -R, P11-R, P12-R, P13-R, and P14-R.
[0022] (2) After keeping warm in a water bath at 65°C for 5 minutes, put it on ice to cool rapidly.
[0023] (3) Prepare the following reaction system:
[0024]
[0025] (4) Carry out reverse transcription reaction according to the following conditions: 30°C for 10min., 42°C for 60min., 95°C for 5min.
[0026] (5) After the above reaction is completed, place it on ice and perform PCR immediately or store it in a -80°C refrigerator for later use.
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Abstract
The invention relates to the technical field of biology and discloses a whole genome cloning method of pelteobagrus fulvidraco bacillus-shaped virus. The method comprises the following steps of: designing 14 specific primers for reverse transcription and 14 pairs of specific primers for PCR, wherein the primer sequences are shown as SEQ ID No.1-SEQ ID No.14; extracting pelteobagrus fulvidraco bacillus-shaped virus RNA; taking the extracted viral RNA as a template, respectively adding the extracted viral RNA into a reverse transcription reaction system and a PCR reaction system by using the primers, cloning the whole genome of the pelteobagrus fulvidraco bacillus-shaped virus by sections under reaction conditions, wherein 13 fragments of the amplified product are 2000bp in size, one fragment is 1000bp in size, a total of 14 fragments contain the entire genome sequence of the pelteobagrus fulvidraco bacillus-shaped virus. The invention provides a cloning method of the whole genome of thepelteobagrus fulvidraco bacillus-shaped virus. The method not only can be used for rapid cloning of the whole genome of the pelteobagrus fulvidraco bacillus-shaped virus, but also can be used for researching molecular biology pathogenic mechanism and molecular epidemiology investigation of the pelteobagrus fulvidraco bacillus-shaped virus, and is convenient to use and has good effect.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for cloning the entire genome of a pelturobacterium-like virus. Background technique [0002] Yellow catfish is a kind of bony fish belonging to the family Cyprinidae. Due to its excellent meat quality, yellow catfish has become one of the important freshwater aquaculture species in China, Japan, South Korea, East Asia and South Asia and other countries and regions. Yellow catfish bacilliform virus (YCBV) is a virus infecting yellow catfish isolated from yellow catfish by the present inventors, and there is no related report about this virus so far. Because the electron microscopic observation showed that the virion was Cheng Bacillus-like, it was named as Pelyobacterium-like virus. Shown through the gene sequence analysis of the Bacillus coliformis-like virus cloned by the present inventors, the Bacillus codiflora-like virus belongs to Obaniviridae (there is no Chinese trans...
Claims
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