[Construction and identification of recombinant adenovirus vector expressing IkappaBalpha-IRES2-shCD40L]

To construct adenovirus expressing vector secretory human CD40L extracellular domain (shCD40L) and IkappaBalpha.

The gene of the shCD40L and the secreting signal peptide was amplified with PCR respectively. Then the CD40L and signal peptide was colligated to get shCD40L segment in vitro. The gene which had cuted from PODB7I kappaBalpha and IRES2 were amplified with PCR respectively. After colligated successfully, the gene shCD40L, IRES2, IkappaBalpha were inserted into pGEMT-easy to amplify. The gene shCD40L was linked with EGFP and the gene IkappaBalpha was linked with IRES2. The two pieces of recombinant gene were inseeted into pshuttle-cmv vector. The pshuttle-cmv-IkappaBalpha-EGFP plasmid and pshuttle-cmv-shCD40L-IRES2 plasmid were transdcut into AdEasy adenovirus vector. System and acquired the recombinant plasmid pAdvIkappaBalpha-IRES2-shCD40L. The pAdvIkappaBalpha-IRES2-shCD40L plasmid harboring was constructed by homologous recombination in E.coil AdEasy-1-BJ5183. Then recombinant vector was propagated in 293 cells and obtain the recombination replication-deficient adenovirus AdvIkappaBalpha-IRES2-shCD40L. PCR method was used in identification of recombinant adenovirus vector harboring IkappaBalpha-IRES2-shCD40L gene. Expressing products in supernatant adenovirus was identified by PCR method. The safety was evaluated by morphology of PK15 and 293 cells after adenovirus infection.

The recombinant IkappaBalpha-IRES2-shCD40L adenovirus was generated by homologous and identified by PCR methods. The adenovirus titre reached 6.561 x 10(12) pfu/L. The adenovirus didn't replicate in PK15 cells.

The IkappaBalpha-IRES2-shCD40L adenovirus is prepared successfully and its biotic safety is confirmed.