NME1重组腺病毒构建及表达鉴定

Construction and Expression Identification of Recombinant Adenovirus Expressing Human NME1 Gene by AdeasyTM Adenovirus Vector System

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,TCID50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1010TCID50/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。

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 recognition sites （Kpn I, XhoI） was cloned from human liver tissue, and then being connected in correct direction to adenovirus vector 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. Conclusion The recombinant adenovirus containing NME1 gene were successfully constructed and transfected to the lung cancer cell as well as expressed normally.