Construction And Expression In Vitro Of Recombinant Adenoviral Vector Expressing CRT/MAGE-A3

ObjectiveCRT has been reported to be an endothelial cell inhibitor and exerts antitumor effects via antiangiogenesis,and it has been shown to associated with peptides delivered into the ER by transporters associated with antigen processing ad with MHC classâ… -β₂ microglobulin molecules to aid in antigen presenting;MAGE-A3 is a member of tumor antigen gene family-MAGE,which is expressed in a wide varity of tumors but not in normal cells,with the exception of the male germ cells and placenta. It has been used as target antigen in cancer gene therapy for its recognizing by CTL.In this study,we aimed to construct CRT/MAGE-A3 recombinant adenovirus vector, which holds a potency of antigen-specific cancer immunotherapy and antiangiogenesis, for further investigation in vivo and in vitro.Methods1.The CRT DNA fragment was isolated from pcDNA3-CRT and cloned into Nheâ… +Pmeâ… sites of pShuttle-GFP-CMV(NheI and Xbaâ… are isocaudarner).Screen the pShuttle-(â–³GFP)-CRT by anti-kanamycin and identify it by incision enzyme Hindâ…¢;2.MAGE-A3 was amplified with Tonch Down RT-PCR using human lung cancer tissue total RNA as a template(and a set of primers,MAGEA3-F-Bglâ…¡-GGA AGA TCT CCA CCA TGC CTC TTG AGC A,MAGEA3-R-Xhoâ… -AAC CTC TCG AGC TCA GAC TCA CTC TTC).The amplified product was then cloned into the Bglâ…¡/Xhoâ… sites of pShuttle-(â–³GFP)-CRT.Sreen the pShuttle-CRT/MAGE-A3 by anti-kanamycin and identify it by incision enzyme Bglâ…¡/Xhoâ… ;3.The CRT/MAGE-A3 DNA fragment was isolated from pShuttle-CRT/MAGE-A3 and cloned intoâ… -Ceuâ… +â… -Sceâ… sites of ethanol precipitated pAdxsi.Screen the Ad-CRT/MAGE-A3 by anti-ampmycin and identify it by incision enzyme Xhoâ… ;4.Linearizated the succeeded recombinant Ad vector with incision enzyme Pacâ… , and transfected it into 293LP cells for further amplication,purification and titer measure;5.Western blot assay was used to determine the protein expression of CRT and MAGE-A3 in 293LP.Resultsl.Agarose gel electrophoresis confirmed that pShuttle-(AGFP) -CRT was successfully constructed;2.Agarose gel electrophoresis confirmed that pShuttle-CRT / MAGE-A3 was successfully constructed;3.Agarose gel electrophoresis confirmed that Ad-CRT/MAGE-A3 was successfully constructed;4.Titer measure of purified amplified Ad-CRT/MAGE-A3 was 2×10⁷PFU/μl;5.We characterized the expression of CRT and MAGE-A3 in 293LP cells transfected with Ad-CRT/MAGE-A3.ConclusionsWe successfully constructed the recombinant adenoviral vector Ad-CRT/MAGE-A3 and identified the expression of CRT and MAGE-A3 in 293LP.