The invention relates to a construction, amplification and purification method of a porcine CD40L / GMCSF / PCV2Cap recombinant adenovirus. The method comprises the steps of: S1. connecting CD40L, GM-CSF and Cap to a vector pUC57 in order, and naming the product as pUC-CD40L-Cap-GMCSF; S2. connecting CD40L, Cap and GM-CSF to pShuttle-CMV, converting DH5a, conducting
bacteria picking,
bacteria shaking and
plasmid extraction, and naming the product as PS-CD40L-Cap-GMCSF; S3. linearizing the constructed PS-CD40L-Cap-GMCSF, then conducting electric transformation on BJ5183 with the linearized PS-CD40L-Cap-GMCSF and skeleton
plasmid pAdEasy-1, and then carrying out
bacteria picking, bacteria shaking and
plasmid extraction, and performing single
enzyme digestion identification; S4. when the identification result is right, using a kit to extract plasmid, transfecting HEK293A
cell, when
cell lesion appears, collecting cells, performing
centrifugation, then resuspending the precipitate in autoclaving PBS; S5. carrying out repeated freezing and thawing, performing
centrifugation to extract the supernatant, thus obtaining recombinant adenovirus; 6. amplifying the obtained recombinant adenovirus; and 7. purifying the recombinant adenovirus. The method provided by the invention for the first time adds porcine
tumor necrosis factor related activation
protein gen and porcine
granulocyte-
macrophage colony stimulating factor into the adenovirus vector simultaneously to improve expression of the PCV2 Cap recombinant adenovirus
immunogenicity.