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背景:重组腺病毒质粒的构建是重组腺病毒制备过程中的中枢环节,传统的细胞内同源重组法步骤多、成功率低、实验周期长。目的:构建含有大鼠白细胞介素10(rIL-10)基因的重组复制缺陷型腺病毒Ad.rIL-10,为真核表达及其动物模型的研究提供实验基础。设计、时间及地点:开放性实验,于2005-07/2006-04在中山大学附属第一医院完成。材料:SD大鼠1只。AdEasy系统由美国JohnHopkins肿瘤研究中心惠赠。ThermoscriptTMRTkit和Trizol为Invitrogen产品;HEK-293保存于中山大学动物实验中心细胞库;克隆大鼠白细胞介素10基因引物合成及测序由上海博亚生物技术有限公司完成。方法:采用反转录聚合酶链反应的方法从健康SD大鼠脾脏新鲜组织中提取的总RNA中克隆rIL-10基因,采用AdEasy-1腺病毒载体系统经E.ColiBJ5183细菌内同源重组及HEK-293包装而获得重组腺病毒Ad.rIL-10。主要观察指标:用Westernblot和RT-PCR的方法鉴定Ad.rIL-10并进行滴度测定。结果:自健康SD大鼠脾脏组织中克隆rIL-10基因,构建出重组腺病毒Ad.rIL-10。经PCR扩增、酶切鉴定、DNA测序最终确定插入序列为rIL-10,Westernblot和RT-PCR均检测出细胞内rIL-10基因及其蛋白表达。再通过扩增、纯化后获得滴度为1.0×1014pfu/mL的重组腺病毒。结论:采用AdEasy-1系统,经细菌内同源重组及HEK-293细胞包装、扩增,纯化后获得足够数量和质量的病毒,方法简便易行,结果稳定可靠。
BACKGROUND: The construction of recombinant adenovirus plasmid is the central part of the preparation of recombinant adenovirus. The traditional method of intracellular homologous recombination has many steps with low success rate and long experimental period. OBJECTIVE: To construct recombinant replication-deficient adenovirus Ad.rIL-10 containing rat interleukin 10 (rIL-10) gene and provide experimental basis for the study of eukaryotic expression and animal model. DESIGN, TIME AND SETTING: The open experiment was performed at the First Affiliated Hospital of Sun Yat-sen University from July 2005 to April 2006. Material: 1 SD rat. AdEasy system by the United States John Hopkins Cancer Research Center. ThermoscriptTMRTkit and Trizol are Invitrogen products; HEK-293 is stored in the cell bank of Sun Yat-Sen Animal Experiment Center; and the synthesis and sequencing of cloned rat interleukin-10 gene primers are completed by Shanghai Bea Biotech Co., Ltd. Methods: The rIL-10 gene was cloned by RT-PCR from the total RNA extracted from fresh spleen tissue of healthy SD rats. The recombinant plasmid was transfected into E. coli BJ5183 by homologous recombination in E. coli BJ5183 using AdEasy-1 adenovirus vector system. HEK-293 to obtain the recombinant adenovirus Ad.rIL-10. MAIN OUTCOME MEASURES: Ad.rIL-10 was identified by Western blot and RT-PCR and titer was determined. Results: The rIL-10 gene was cloned from the spleen of healthy SD rats and the recombinant adenovirus Ad.rIL-10 was constructed. The recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing. The sequence of rIL-10 was confirmed by DNA sequencing. The expression of rIL-10 gene and its protein was detected by Western blot and RT-PCR. The recombinant adenovirus with the titer of 1.0 × 1014 pfu / mL was obtained by amplification and purification. Conclusion: The AdEasy-1 system was successfully constructed with homologous recombination and HEK-293 cell packaging, amplification and purification to obtain a sufficient quantity and quality of virus. The method is simple and easy to perform, and the method is stable and reliable.