Recombinant H7N9 subtype avian influenza virus, inactivated marked vaccine and preparation method thereof

A method for preparing a recombinant H7N9 subtype avian influenza virus, the method comprising the steps of:

• (1) extracting the total RNA of the H7N9 subtype avian influenza JD/17 strain, and reverse transcribing the total RNA to obtain the cDNA of the H7N9 subtype avian influenza JD/17;
• (2) obtaining HA-1 and an HA-2 gene fragments via amplifying at least a portion of the HA-1 gene of the cDNA of the H7N9 subtype avian influenza JD/17 with the primer pair KS-H7-1 and JDH7H3-1-R, and at least a portion of the HA-2 gene of the cDNA of the H7N9 subtype avian influenza JD/17 with the primer pair JDH7H3-2-F and KS-H7-2;
• wherein the nucleotide sequence of the KS-H7-1 primer is the nucleotide sequence of SEQ ID No. 3;
• wherein the nucleotide sequence of the JDH7H3-1-R primer is the nucleotide sequence of SEQ ID No. 4;
• wherein the nucleotide sequence of the JDH7H3-2-F primer is the nucleotide sequence of SEQ ID No. 5;
• wherein the nucleotide sequence of the KS-H7-2 primer is the nucleotide sequence of SEQ ID No. 6;
• (3) using the HA-1 and HA-2 gene fragments obtained in step (2) as templates, performing overlapping PCR amplification to obtain sequence-replaced HA gene fragments;
• and
• (4) obtaining a recombinant H7N9 subtype avian influenza virus strain via ligating the sequence-replaced HA gene fragment obtained in step 3 to a Blunt3 vector to obtain an intermediate transition plasmid, digesting the intermediate transition plasmid with a BsmBI enzyme to obtain a digested target product, cloning the digested target product into a pHW2000 vector to obtain a HA gene transfection vector plasmid, and transfecting the HA gene transfection vector plasmid with an expression plasmid constructed from the other 7 genes of the JD/17 strain in pHW2000 vectors.