Construction and identification of recombinant replication- defective adenovirus vector containing interleukin-10 in a rat / 中国组织工程研究

ABSTRACT

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.dL-10) in a SD rat, and to provide experimental evidences for eukaryotic expression and animat model studying.DESIGN, TIME AND SEI-FING: 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), ThermoscdptTMRT kit & Tdzol (USA), and HEK-293 (Guangzhou, China) were used in this study. The primer of rlL-10 gene of clone rat was synthesized and sequenced in Shanghai, China.METHODS: rlL-10 gene was cloned from total RNA of healthy SD rat spleen tissue with RT-PCR, and then recombinant adenovirus plasmid named pAd.rlL-10 was obtained by homologous recombination within E.ColiBJ5183 carded with AdEasy-1 system. Last, the recombinant adenovirus was packaged, proliferated by HEK-293 cells and purified.MAIN OUTCOME MEASURES: The Ad.rlL-10 was identified using Western blot and RT-PCR methods.RESULTS: rlL-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.rlL-10. Western blot and RT-PCR showed the rlL-10 gene and protein expressions in the cells. Finally, the rlL-10 recombinant adenovirus was obtained with the titers of 1.0x1014 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.