H9N2 subtype avian influenza virus rescue method based on seamless cloning technology

Abstract

The invention belongs to the field of biology, and discloses an H9N2 subtype avian influenza virus rescue method based on a seamless cloning technology. The H9N2 subtype avian influenza virus rescue method comprises the following steps that step 1, RNA of an H9N2 subtype avian influenza virus is extracted, and cDNA of the virus is obtained according to the RNA; step 2, amplifying is carried out by using eight pairs of primers by taking the cDNA as a template to obtain PCR products of eight fragments of the H9N2 subtype avian influenza virus; step 3, enzyme digestion is carried out on the PHW2000 plasmid to obtain a linearized fragment; step 4, homologous recombination is correspondingly carried out on the PCR products of the eight fragments of the H9N2 subtype avian influenza virus and the linearized PHW2000 vector to obtain eight recombinant plasmids; and step 5, 293T and MDCK mixed culture cells are co-transfected with the eight recombinant plasmids to obtain a recombinant H9N2 virus. The sequences of the eight pairs of primers are correspondingly shown as SEQ ID NO: 2 to SEQ ID NO: 17 in a sequence table. The H9N2 subtype avian influenza virus rescue method based on the seamless cloning technology has the advantages of simple operation, accurate recombination and high rescue efficiency.

Description

technical field [0001] The invention relates to the field of biology, in particular to a rescue method for H9N2 subtype avian influenza virus based on seamless cloning technology. Background technique [0002] Avian influenza virus (AIV) belongs to the Orthomyxoviridae family, and is a type A influenza virus in the genus Influenza virus. Its genome is single-stranded negative-sense RNA and has a wide range of hosts. Influenza A viruses can be divided into 18 subtypes based on hemagglutinin HA and 11 subtypes based on neuraminidase NA. Among the many subtypes, although the H9N2 subtype avian influenza virus is a low-to-medium pathogenic strain, its spread and damage are the most extensive. First of all, the virus can infect poultry through direct infection, co-infection and immunosuppression, causing serious losses to the poultry farming industry; second, the virus can not only infect mammals, but also can be the new highly pathogenic influenza virus H5N1, H7N9 etc. provide...

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Problems solved by technology

[0007] (2) The design of PCR primers needs to introduce enzyme cutting sites on the carrier, and the PCR products are then directional cloned into the target carrier after enzyme digestion, gel recovery, and ligation, which is time-consuming and laborious;

[0008] (3) It is necessary to find a suitable enzyme cutting site. If there is no suitable enzyme cutting site in the vector or genome, mutations need to be carried out, which reduces the fidelity of the gene and the probability of successful virus rescue, and it is necessary to purchase various Endonuclease;

[0009] (4) Positive rate is low

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