SLC基因重组腺病毒的构建

Construction of Recombinant Adenovirus Vector Carrying Secondary Lymphoid Organ Chemokine

目的构建携带次级淋巴组织趋化因子(secondary lymphoidorgan chemokine,SLC)基因的重组腺病毒。方法采用PCR技术从含有SLC基因的质粒上扩增出SLC基因,将PCR产物酶切后连接至pENTR11载体上,再通过pENTR11与腺病毒骨架载体pAd/CMV/V5-DEST之间的同源重组作用将SLC基因片段重组至pAd/CMV/V5-DEST上,最后经293细胞的包装扩增后得到携带SLC基因的重组腺病毒。结果成功将SLC基因片段克隆至pAd/CMV/V5-DEST载体上,并经293细胞包装出病毒颗粒;经测定病毒滴度为2.6×108pfu/mL。结论构建的重组SLC腺病毒可将SLC基因导入肿瘤细胞或组织内,为进一步研究SLC基因的抗肿瘤作用提供了基础。

Objective To construct a recombinant adenovirus vector earring the SLC gene for further studies on gene therapy for carcinoma. Methods The SLC gene was amplified from the pORF5 vector with polymerase chain reaction （PCR） technique. The amplified gene was cloned into the pENTRll vector. With the pENTR11-SLC plasmid and the backbone plasmid pAd/CMV/VS-DEST, the homologous recombination reaction took place in vitro. The reaction mixture was transferred into TOP10 E. coli strains （without F＇ episome）. The recombination adenovirus plasmid was then generated. The recombinant adenoviruses were packaged and amplified in 293A cells. Results The SLC gene was successfully cloned into the pAd/CMV/V5-DEST plasmid and the recombinant adenoviruses carrying SLC gene were detected by PCR, with a viral titer of 2. 6 ×10^8 pfu/mL. Conclusion The constructed recombinant adenovirus can introduce SLC gene into tumor tissues, which provides a foundation for the study of antitumor efficacy of SLC.