Construction and Identification of Recombinant replication-defective Adenovirus Vector Containing interleukin-10 in a Rat

BACKGROUND: Construction of recombinant adenovirus plasmid plays a central role in preparation of recombinant adenovirus. However, conventionally intracellular homologous recombination method is limited by complex procedures, low successful ratio, and long experimental cycle. OBJECTIVE: To construct the recombinant replication-defective adenovirus vector containing interleukin-10 (Ad.rIL-10) in a SD rat, and to provide experimental evidences for eukaryotic expression and animal model studying. DESIGN, TIME AND SETTING: Opening study was conducted in the First Affiliated Hospital of Sun Yat-sen University from July 2005 to April 2006. MATERIALS: A SD rat, AdEasy system (USA), ThermoscriptTMRT kit & Trizol (USA), and HEK-293 (Guangzhou, China) were used in this study. The primer of rIL-10 gene of clone rat was synthesized and sequenced in Shanghai, China. METHODS: rIL-10 gene was cloned from total RNA of healthy SD rat spleen tissue with RT-PCR, and then recombinant adenovirus plasmid named pAd.rIL-10 was obtained by homologous recombination within E.ColiBJ5183 carried with AdEasy-1 system. Last, the recombinant adenovirus was packaged, proliferated by HEK-293 cells and purified. MAIN OUTCOME MEASURES: The Ad.rIL-10 was identified using Western blot and RT-PCR methods. RESULTS: rIL-10 gene was successfully cloned from fresh spleen tissue of a SD rat and incorporated into recombinant adenovirus plasmid pAd in order to obtain the Ad.rIL-10. Western blot and RT-PCR showed the rIL-10 gene and protein expressions in the cells. Finally, the rIL-10 recombinant adenovirus was obtained with the titers of 1.0×10^14 pfu/mL after amplification and purification. CONCLUSION: AdEasy-1 system characterizing by simple operation and reliable results is commonly used to obtain enough quantity and quality adenovirus after homologous recombination, and HEK-293-induced package, amplification, and purification.