Construction And Identification Of A VCIP IRES-Mediated Recombinant Adenovirus Vector Co-expressing HLYZ And BoIFN-γ

Internal ribosome entry site (IRES) sequence is a bicistronic sequence which can recruit ribosomes to expressing proteins independently, and it plays an important role in the process of building a multi-gene co-expression vector. Lysozyme(LYZ) is an alkaline enzyme which can hydrolyse the mucopolysaccharide of cell wall, having a very broad antibacterial spectrum. So it is capable of dissolving the Gram-positive bacterias and some Gram-negative bacterias, and it even has a certain inactivation effect on viruses. Bovine gamma-interferon (BoIFN-y) is a small amount of glycoproteins which can anti virus、affect cell growth and differentiation、anti tumors and regulate immune function. It is secreted under the action of suitable stimulation and inducers. So the combination of BoIFN-y and LYZ has a very high value in the prevention and treatment of dairy cow mastitis. As a novel reporter gene and positioning marker, the GFP (Green Fluorescent Protein, GFP), firstly discovered in the jellyfish, is a protein composed of238amino acids, and it has a unique light-emitting mechanism. Because of its high efficiency of exogenous gene expression, transient expression, security and ease of obtaining high viral titer vectors, the Adenovirus vector (Ad vector) has been widely used in genetic engineering.In order to obtaining efficient adenoviral vector co-expressing the human’s lysozyme (hLYZ) and BoIFN-y, the present experiment cloned human’s vascular collagen inducible protein (hVCIP) IRES sequence, named for the hIRES and mouse’s vascular collagen inducible protein (mVCIP) IRES sequence, named for the hIRES from the genomes of human and mouse; cloned the hLYZ gene from the pCDNAK-LYZ vector which contained hLYZ; cloned GFP gene from pEGFP-N1vector which contained the GFP gene. After sequencing correctly, they were inserted into the corresponding sites of the adenovirus vector Pshuttle-cmv, obtaining two recombinant adenovirus vectors Pshuttle-hLYZ-hIRES-GFP and Pshuttle-hLYZ-mIRES-GFP. Using liposome to transfected the PK15, AAV-293and bovine kidney cells. By fluorescence microscopy observing and FACS detection, the results showed that the two IRESs were able to express the downstream gene. And the expression level of reporter gene GFP has a large difference in different type of cells. Replacing the GFP gene with the BoIFN-y gene, we obtained the recombinant Adenovirus vector Pshuttle-hLYZ-mIRES-BoIFN-y. We Packaged it into adnovirus in AAV-293. By RT-PCR, the result showed that the adnovirus co-expressing hLYZ and BoIFN-y had been successfully obtained.