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目的:构建pTARGETTM-AANAT真核表达载体并转化L6成肌细胞,检测目的基因的表达;制备pTARGETTMAANAT转基因细胞微囊,初步探讨AANAT转基因细胞微囊的生物活性。方法:RT-PCR技术扩增大鼠AANATcDNA,酶切连接pTARGETTM载体,脂质体法转染L6细胞并检测pTARGETTM-AANAT的基因表达产物;将高表达活性的L6细胞进行APA微囊化处理,倒置显微镜观察微囊形态,Western-blot检测囊内细胞AANAT蛋白表达活性。结果:酶切、DNA测序及凝胶电泳检测结果证实,pTARGETTM-AANAT真核表达载体构建成功。转染后的L6细胞表达AANATmRNA,胞浆中能检测到AANAT蛋白的表达。转染细胞微囊体外存活良好,微囊外形完整无粘连,大小较均一,囊内细胞清晰可见,体外培养两周后破囊检测证实囊内细胞具有AANAT蛋白表达活性。结论:pTARGETTM-AANAT真核表达载体在L6细胞中成功表达,转染细胞微囊体外存活良好,囊内细胞具备AANAT蛋白表达活性。
OBJECTIVE: To construct the eukaryotic expression vector pTARGETTM-AANAT and transform it into L6 myoblasts to detect the expression of the target gene. The pTARGETTMAANAT transgenic microencapsulated cells were prepared and the biological activity of AANAT transgenic microencapsulated cells was investigated. METHODS: The rat AANAT cDNA was amplified by RT-PCR and ligated into pTARGETTM vector. The L6 cells were transfected into L6 cells by lipofectamine and the gene expression products of pTARGETTM-AANAT were detected. The highly expressed L6 cells were treated with APA microencapsulation, The morphology of the microcapsules was observed by inverted microscope. The expression of AANAT protein in cystic cells was detected by Western-blot. Results: The results of restriction enzyme digestion, DNA sequencing and gel electrophoresis confirmed that pTARGETTM-AANAT eukaryotic expression vector was successfully constructed. After transfected L6 cells expressed AANAT mRNA, AANAT protein was detected in cytoplasm. The transfected cells showed excellent viability in vitro and intact microcapsules with no adhesions. The size of the cells was uniform, and the cells in the cysts were clearly visible. After two weeks of the in vitro culture, the ruptured cysts showed that the cells in the cysts had AANAT protein expression activity. CONCLUSION: The pTARGETTM-AANAT eukaryotic expression vector was successfully expressed in L6 cells. The transfected cells showed good survival in vitro and AANAT protein expression in cystic cells.