CAJ | 학술논문

目的：应用 AdEasyTM 腺病毒载体系统构建含人 NME1基因的重组腺病毒,检测 NME1基因在肺癌细胞的表达。方法从人肝脏标本克隆带有 KpnⅠ,XhoⅠ双酶切位点的 NME1cDNA,利用细菌体内同源重组法将 NME1正向连接至腺病毒载体上,测序鉴定正确后,于 QBI-293A 细胞中包装扩增,TCID 50法检测重组腺病毒滴度,重组腺病毒转染至肺癌细胞 GLC-82,检测 NME1在细胞内表达。结果肝脏组织克隆出470bp 条带,测序与 NME1编码序列相符,测得重组腺病毒滴度为1010 TCID 50/ mL ,免疫实验显示 NME1在 GLC-82细胞成功表达。结论成功构建含人 NME1基因的重组腺病毒并转染肺癌细胞,检测到 NME1在肺癌细胞中正常表达。
목적：응용 AdEasyTM 선병독재체계통구건함인 NME1기인적중조선병독,검측 NME1기인재폐암세포적표체。방법종인간장표본극륭대유 KpnⅠ,XhoⅠ쌍매절위점적 NME1cDNA,이용세균체내동원중조법장 NME1정향련접지선병독재체상,측서감정정학후,우 QBI-293A 세포중포장확증,TCID 50법검측중조선병독적도,중조선병독전염지폐암세포 GLC-82,검측 NME1재세포내표체。결과간장조직극륭출470bp 조대,측서여 NME1편마서렬상부,측득중조선병독적도위1010 TCID 50/ mL ,면역실험현시 NME1재 GLC-82세포성공표체。결론성공구건함인 NME1기인적중조선병독병전염폐암세포,검측도 NME1재폐암세포중정상표체。
Objective To construct the recombinant containing human NME1 gene by AdEasyTM adenovirus vector, and to examine the expression of the target gene in lung cancer cells. Methods The NME1 cDNA with double restriction-enzyme rec-ognition sites (Kpn I, XhoI) was cloned from human liver tissue, and then being connected in correct direction to adenovirus vec-tor by homologous recombination in bacteria. After identifying correct by sequencing, the recombinant adenovirus were packaged into QBI-293A cells and then amplified. The recombinant adenovirus were transfected into the lung cancer cells (GLC-82) after detecting the virus titer by TCID 50 methods, and expression of the HME1 gene was detected by a series of immunological assays. Results The 470bp band was detected in the products after cloning from liver tissue. The recombinant adenovirus were confirmed to be accordance with NME1 gene coding region sequence by sequencing. The recombinant adenovirus titer were 3. 7 × 10 10 TCID 50 / mL. A series of immunological experiments confirmed that NME1 gene had expressed successfully in GLC-82 cells. Con-clusion The recombinant adenovirus containing NME1 gene were successfully constructed and transfected to the lung cancer cell as well as expressed normally.