摘要
目的利用细菌内同源重组法构建携带凋亡素VP3基因的重组腺病毒,观察其与姜黄素单独或联合对结肠癌SW480细胞凋亡及周期分布的影响。方法设计VP3 cDNA扩增引物,从PET15b-VP3质粒中扩增VP3 DNA序列,与pShuttle-IRES-hrGFP连接构建pShuttle-VP3-hrGFP穿梭质粒,PCR和EcoRⅤ酶切电泳鉴定;线性化穿梭质粒pShuttle-VP3-hrGFP,转化含pAdeasy-1的超感受态BJ5183大肠杆菌,构建pAd-VP3重组腺病毒质粒并经PCR、电泳及测序鉴定,线性化腺病毒质粒经脂质体转染AD293细胞进行pAd-VP3重组腺病毒的包装和扩增,CsCI密度梯度离心法进行病毒浓缩和纯化并将其单独或联合姜黄素作用人结肠癌SW480细胞,观察其对细胞凋亡及周期分布的影响。结果pShuttle-VP3-hrGFP穿梭质粒构建鉴定成功,pAd-VP3重组腺病毒质粒经酶切获得一大于23 kb的大片段和4.5kb的片段,PCR反应扩增出了402 bp的片段,重组质粒测序证实VP3-hrGFP编码区成功地克隆入了腺病毒pAd中,且其序列与GeneBank中VP3 CDNA序列完全一致。成功包装出携带VP3基因的腺病毒,滴度为1.738×1012opu·ml-1,获得的重组腺病毒及姜黄素单独或合用均可阻滞细胞周期于G0/G1期,诱导细胞凋亡(P<0.05,P<0.01),二药联用效果更为显著(P<0.01)。结论细菌内同源重组法可快速、高效地制备携带凋亡素VP3基因的重组腺病毒,并能与姜黄素协同通过阻滞细胞周期于G0/G1期诱导结肠癌SW480细胞凋亡。
Objective To construct recombinant adenovirus containing Apoptin VP3 gene by using the method of homologous recombination in bacteria, and observe its inducing apoptosis effect on human colonic carcinoma cells alone or combination with Curcumin in vitro. Methods The DNA sequence of VP3 was amplified from PETISb - VP3 and ligated into pShuttle - IRES - hrGFP The recombinant plasmid was named after pShuttle - VP3 - hrGFP and was identified with PCR and EcoR V digestion; pShuttle - VP3 - hrGFP was linealized and transformed into uhracompletent B J5183 containing pAdeasy - 1, then recombinant ad- enovirus pad - vp3 was constructed by homologous recombination in bacteria. The recombinant adenoviral plasmid pAd - VP3 was identified by PCR ,digestion and DNA sequencing, subsequently linealized recombinant advenovirus vector pAd - VP3 was trans- fected into AD293 cells by liposome to generate recombinant adenovirus particles which were purified and concentrated by CsCI density gradient centrifugation. Adenovirus particles ,Curcumin alone or combination were used to infect the human colonic carci- noma cells SW480, and apoptosis rate and cell phase were observed. Results Reeombined adenovlrus plasmid pSbuttle - VP3 - hrGFP was generated successfully. Linealized pShuttle- VP3 -hrGFP transformed into uhracompletent colibacillus BJ5183, There were two bands 4.5kb and larger than 23kb when pAd - VP3 was digested with Pad. A 402bp VP3 cDNA fragment was amplified by PCR. The target gene VP3 - hrGFP was successfully cloned into Adenovirus , The sequence of VP3 segment was identical with that published in GenBank. we have successfully constructed recombinant advenovirus pad - VP3. Adenovirus encoding Apoptin VP3 gene was generated with titers of about 1. 738×10^12opu·L-j ,pAd-VP3 Adenovirus and Curcumin all could arrest cell cycle in G0/G1 phase. The effect above of Curcumin combination with pAd - VP3 was better than that of pAd - VP3 , Curcumin alone（ P〈0.01 ）. Conclusion Homologous recombination in bacteria can efficien
出处
《时珍国医国药》
CAS
CSCD
北大核心
2014年第8期1865-1867,共3页
Lishizhen Medicine and Materia Medica Research
基金
安徽省自然科学基金(No.1208085MH134)
湖北省卫生厅科研基金(No.QJX2008-42)
关键词
姜黄素
同源重组
凋亡素
腺病毒
人结肠癌细胞
Curcumin
Homologous recombination
Apoptin VP3
Adenovirus
Colonic carcinoma
