CN108265067A - The cloning process of novel duck reovirus L group genes - Google Patents

Abstract

The invention provides a method for cloning the L group gene of novel duck reovirus, comprising the steps of: 1) extracting the total RNA of the allantoic fluid of novel duck reovirus with Trizol reagent; 2) using PrimeScript ^TM One Step RT‑ PCR Kit kit clones the total RNA extracted in step 1), wherein the specific primers used include any pair of L1 primer pair, L2 primer pair and L3 primer pair, and the L1 primer pair includes L1-F primer and L1-R primers, the pair of L2 primers includes L2-F primers and L2-R primers, and the pair of L3 primers includes L3-F primers and L3-R primers. The beneficial effects of the present invention are: establishing a one-step RT-PCR system for amplifying the full-length gene sequence of group L of NDRV, avoiding the need for cloning, connecting, and multiple sequencing of gene fragments after conventional PCR amplification. The full-length amplification also lays the foundation for the research on the protein encoded by the NDRV L group.

Description

Cloning Method of L Group Gene of Novel Duck Reovirus

technical field

The invention relates to the technical field of biotechnology, in particular to a method for cloning a novel duck reovirus L group gene.

Background technique

"New duck reovirus (NDRV)" is a new major epidemic that broke out in Fujian, Zhejiang and other provinces since 2005. The incidence rate is 5% to 32.5%, and the case fatality rate varies greatly, ranging from 4% to 20%. The younger the duck, the higher the morbidity and mortality. The disease mainly causes different degrees of spot/plaque hemorrhage and necrosis of the liver, spleen enlargement and necrosis, heart and bursa hemorrhage, etc.

NDRV is a segmented ds RNA orthoreovirus, which contains 10 RNA segments and encodes 14 proteins; the total genome includes 3 large gene segments (L1, L2, L3), and 3 medium gene segments ( M1, M2, M3) and 4 small gene segments (S1, S2, S3, S4). At present, there are many studies on the S group genes, but few studies on the L group gene sequences. Studies have shown that the gene sequence fragments of the NDRVL group are 3959bp, 3830bp, and 3907bp in length, encoding λA protein (1294 amino acids), λB protein (1260 amino acids), and λC protein (1286 amino acids); λA is one of the main structures of the inner shell, and λB Protein is a minor component of the inner capsid, and λC protein is a pentameric protein that can penetrate the core of the virus and the outer capsid, and has multiple functions.

Conventional PCR reactions are only suitable for amplifying gene fragments within 3kb, and are not suitable for the amplification of long fragments. For long fragment gene sequences, primers are generally designed in sections to amplify the target sequence, which can be used for subsequent cloning, ligation, and multiplexing of target gene fragments. Sequencing and so on increase the complicated work.

Contents of the invention

The present invention aims to provide 3 pairs of full-length specific primer sequences for amplifying NDRV L group genes, which effectively reduces the complicated work of subsequent target gene fragment cloning, connection, and multiple sequencing.

The technical scheme that the present invention takes is:

A kind of cloning method of novel duck reovirus L group gene, comprises steps:

1) extract the total RNA of novel duck reovirus allantoic fluid;

2) The total RNA extracted in step 1) was cloned using PrimeScript ^™ One Step RT-PCR Kit kit, wherein the specific primers used included any pair of L1 primer pair, L2 primer pair and L3 primer pair, said L1 primer pair includes L1-F primer and L1-R primer, described L2 primer pair includes L2-F primer and L2-R primer, described L3 primer pair includes L3-F primer and L3-R primer, the sequence of each primer They are shown in Table 1 respectively.

Table 1 L group gene-specific amplification primers of NDRV

The above-mentioned kit reaction system includes:

The operation program is: 50°C for 30 minutes; denaturation at 94°C for 3 minutes; 35 cycles of 98°C for 10 s and 68°C for 4 minutes; extension at 72°C for 10 minutes.

The beneficial effects of the present invention are: establishing a one-step RT-PCR system for amplifying the L-group full-length gene sequence of NDRV, avoiding the need for cloning, linking, and multiple sequencing of gene fragments after conventional PCR amplification. The full-length amplification also lays the foundation for the study of the protein encoded by the NDRV L group.

Detailed ways

The present invention will be further described in detail through specific embodiments below. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, the third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Embodiment 1: the cloning method of novel duck reovirus L group gene

1) Extract the total RNA of the new duck reovirus allantoic fluid with Trizol reagent;

2) The total RNA extracted in step 1) was cloned using PrimeScript ^™ One Step RT-PCR Kit kit, wherein the specific primers used included any pair of L1 primer pair, L2 primer pair and L3 primer pair, said The L1 primer pair includes L1-F primer and L1-R primer, the L2 primer pair includes L2-F primer and L2-R primer, and the L3 primer pair includes L3-F primer and L3-R primer.

Kit reaction system 25μl, including:

The operation program is: 50°C for 30 minutes; denaturation at 94°C for 3 minutes; 35 cycles of 98°C for 10 s and 68°C for 4 minutes; extension at 72°C for 10 minutes.

Embodiment 2: Identification of novel duck reovirus L group gene

(1) The reaction system in Example 1 was expanded to 50 μl, and the purified target gene fragment was obtained through a gel recovery kit.

10 μl of reaction system using pMD18-T vector ligation kit: 4.5 μl of purified product, 5 μl of 2×LigaseBuffer (Ligation Solution), 0.5 μl of pMD18-T vector. Operate on an ice box, add the above various reaction reagents into a sterilized PCR tube, mix well, react at 16°C for 4 hours, and continue to stand at 4°C overnight.

(2) Transfer the above liquid into 100 μl DH5α competent cells, mix gently, place in ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, then quickly put it back on ice for 10 minutes, add 500 μl fresh LB liquid medium, 37°C 230r/ After shaking for 60 minutes, pipette 50 μl of bacterial solution from the tip of the pipette to the bottom of the EP tube and spread evenly on the self-made blue-white LB agar plate, and incubate the plate upside down at 37°C for 14 hours.

(3) Pick suspicious white single colonies, inoculate them in LB broth containing ampicillin, and culture them with shaking at 37°C for 10 hours. During the 5th to 10th hours of shaking culture, you can use the PCR reaction system of Premix Ex Taq ^TM hot start enzyme to carry out bacterial liquid PCR identification.

PCR reaction system of Premix Ex Taq ^TM hot start enzyme: enzyme 12.5 μl; F primer and R primer 1 μl each; RNaseFree Water 8.5 μl.

SEQUENCE LISTING

<110> Foshan Institute of Science and Technology

<120> Cloning method of novel duck reovirus L group gene

<130> 2018

<160> 6

<170> PatentIn version 3.5

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Claims (1)

1. a kind of cloning method of novel duck reovirus L group gene, is characterized in that, comprises steps: 1) extract the total RNA of novel duck reovirus allantoic fluid; 2) The total RNA extracted in step 1) was cloned using PrimeScript ^™ One Step RT-PCR Kit kit, wherein the specific primers used included any pair of L1 primer pair, L2 primer pair and L3 primer pair, said L1 primer pair includes L1-F primer and L1-R primer, described L2 primer pair includes L2-F primer and L2-R primer, described L3 primer pair includes L3-F primer and L3-R primer, the sequence of each primer They are respectively shown in SEQ ID №1-6.