基于miR30的靶向DcR_3慢病毒干扰载体的构建及鉴定

Construction and identification of DcR3 targeted RNA interfering lentivirus vector based on a new miR30 like sequence

目的构建基于miR30的靶向DcR3慢病毒干扰载体。方法人工设计针对DcR3编码区的干扰序列,使其转录后RNA结构类似于miR30结构,茎杆序列替换成DcR3序列,送上海生物工程技术服务有限公司合成。以该序列为模板,分别利用携带EcoRⅠ和XhoⅠ酶切位点的引物行PCR扩增,通过酶切连接的方式将插入片段连入p201慢病毒载体骨架中。经酶切和测序鉴定后,用包装质粒pMD2G及包膜质粒psAX2共同转染293FT细胞进行病毒包装。病毒包装上清液加入培养的人食管鳞癌细胞系KYSE150细胞中,用FACS分选后进行RT-PCR和Western blot检测DcR3表达情况。结果构建了针对DcR3的3条慢病毒干扰载体,成功包装出病毒,并不同程度地抑制了DcR3蛋白的表达。结论基于miR-30的DcR3能有效抑制DcR3表达,为进一步研究DcR3功能打下了基础。

Objective To construct new lentivirus interfering vectors target on DcR3, to produce virus to inhibit the expression of DcR3 in KYSE150 esophageal squamous cell line. Methods A miR30 like oligo was designed to target the coding region of DcR3 gene. After PCR amplification, the product were digested with Ecor Ⅰ and Xho Ⅰ, and then inserted into the downstream of the EGFP coding sequence in p201 vector. The verified recombined vectors were co-transfect with pMD2g and psAX2 into 293FT packing cell line to produced the virus. The virus-containing cell culture supernatants were added into the KYSE150 cell culture. Finally the virus infected cells were enriched by FACS and the DcR3 expression level were evaluated by Real-time PCR and Western blotting. Results Three DcR3 targeted lentivirus interfering vector were generated. The virus were produced successfully and inhibited the expression level of DcR3 in different degree respectively. Conclusion The new miR30 likfe sequence based lentivirus interfering vector was successfully constructed,which can provide a basis for further study of DcR3 function.