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PCR (polymerase chain reaction) amplification primers of influenza A virus gene and rapid cloning method of influenza A virus gene
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An influenza virus and amplification primer technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc. Superior, high success rate results
Active Publication Date: 2015-03-25
YANGZHOU UNIV
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[0003] The purpose of the present invention is to provide primers for PCR amplification of type A influenza virus genes and a method for rapid cloning of type A influenza virus genes, thereby greatly simplifying the gene cloning process in traditional influenza virus reverse genetic technology and solving the problem of traditional influenza virus reverse genetics. The Cloning Process of Influenza Virus Genes in Genetic Technology is Complicated and Inefficient
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[0047] Design primers for influenza virus gene fragments and primers for influenza virus reverse genetic linearization vectors, the design process is as follows:
[0048] a) Each upstream primer used to amplify the influenza virus gene has 15 conserved bases and 2-3 specific bases for each gene segment;
[0049] b) Each downstream primer used to amplify the influenza virus gene has 15 conserved bases and 5-8 specific bases for each gene segment;
[0050] c) The upstream primer used to amplify the influenza virus reverse genetic linearization vector has 15 bases that are reverse complementary to the downstream primer used to amplify the influenza virus gene;
[0051] d) The downstream primer used to amplify the influenza virus reverse genetic linearization vector has 15 bases that are reverse complementary to the upstream primer used to amplify the influenza virus gene.
[0052] The sequences of the designed primers are show...
Embodiment 2
[0066] Rapid Cloning of H9N2 Influenza Virus Genes:
[0067] The design process of the primers of the influenza virus gene fragments, the primers of the influenza virus reverse genetic linearization vector, and the designed primers are the same as in Example 1.
[0068] The steps of H9N2 influenza virus gene cloning are as follows:
[0069] (1) Type A influenza virus gene and influenza virus reverse genetic linearization vector PCR amplification.
[0070] Using the cDNA of H9N2 influenza virus and the reverse genetic vector of influenza virus pDP2002 as templates, the primers in Table 1 were used to amplify influenza virus gene fragments PB2, PB1, PA, HA, NP, NA, MP, NS and Linearized influenza virus reverse genetic vectors, such as figure 1 Step 1 in.
[0071] Among them, the PCR amplification reaction system is: 40 μl water, 5 μl 10 times buffer, 1 μl 10 mmol / L dNTP, 1 μl 10 μmol / L upstream primer, 1 μl 10 μmol / L downstream primer, 1 μl influenza virus cDNA or 1 μl 10ng / μ...
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Abstract
The invention relates to a cloning method of an influenza virusgene, in particular to PCR (polymerasechain reaction) amplification primers of an influenza Avirusgene and a rapid cloning method of the influenza Avirusgene. According to the PCR amplification primers of the influenza A virus gene and the rapid cloning method of the influenza A virus gene, the PCR amplification primers of eight gene segments of the influenza A virus and primers of linearized influenza virus reverse genetic vectors are provided and the rapid cloning method of the influenza A virus gene is further provided, the influenza virus gene segments and the linearized influenza virus reverse genetic vectors are amplified by the primers through PCR, and then the influenza virus gene segments and the linearized influenza virus reverse genetic vectors are recombined and cloned in vitro by recombinase and transformed to escherichia coli for cloning. According to the invention, the gene cloning process of a traditional influenza virus reverse genetic technology is greatly simplified, and the problems of complex influenza virus gene cloning process and low efficiency of the traditional influenza virus reverse genetic technology are solved.
Description
Technical field [0001] The invention involves a cloning method of influenza virus gene, which specifically involves type A influenza virus genes of PCR amplification primers and type A influenza virus genes. Background technique [0002] The establishment of the reverse genetic technology of influenza virus has made important contributions to understanding the diversity of influenza virus, clarifying the relationship between genotype and phenotype, the development of influenza virus vaccine, and the effective prevention and control of influenza virus.However, in the traditional influenza virus reverse genetic technology, the cloning of the influenza virus gene depends on the enzymecutting of the restricted internal cutase and the connection of the connection enzyme.Practice has shown that the use of 8 gene fragments of an influenza virus in a traditional method to the reverse genetic rescue carrier of the influenza virus is a very time -consuming and labor -consuming process. Es...
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