靶向EBV阳性鼻咽癌小环质粒的构建及表达

Construction and expression of minicircle targeted to EBV positive nasopharyngeal carcinoma

目的构建表达人IFNγ的靶向小环质粒及其对照质粒,并观察其在EBV阳性鼻咽癌细胞中的靶向表达。方法采用分子克隆的方法构建中间靶向质粒p SP72-ori P-IFNγ-poly A(p SP72-ori P-IFNγ)和对照质粒p SP72-CMV-IFNγ-poly A(p SP72-CMV-IFNγ)。对其分别做双酶切,定向克隆于母环质粒p2ФC31中,构建成p2ФC31-ori P–IFNγ和p2ФC31-CMV-IFNγ质粒。转化TOP10细菌后,通过介导分子内重组,降解原核骨架,获取只含目的基因表达盒的靶向小环质粒mc-ori P-IFNγ和对照质粒mc-CMV-IFNγ。将小环质粒分别转染EBV阴性细胞和EBV阳性细胞,采用ELISA方法定量检测小环质粒介导目的基因的靶向表达。结果构建了靶向性母环质粒p2ФC31-ori P–IFNγ及其对照质粒p2ФC31-CMVIFNγ,并诱导纯化出相应的靶向性小环质粒mc-ori P-IFNγ及其对照质粒mc-CMV-IFNγ,通过转染真核细胞,检测出靶向性小环质粒能够介导IFNγ在EBV阳性鼻咽癌细胞中特异表达。结论成功构建了靶向性小环质粒mc-ori P-IFNγ,并证明其能够特异性地介导目的基因在EBV阳性鼻咽癌细胞中表达,而在EBV阴性鼻咽癌细胞及正常细胞中不表达。

Objective To construct the targeted minicircle vector which expresses IFNγ, then detect its selective expression in EBV positive nasopharyngeal carcinoma（NPC）. Methods Targeted intermediate plasmid p SP72-ori P-IFNγ and control plasmid p SP72-CMV-IFNγ were constructed by molecular cloning methods. Parent plasmids p2ФC31-ori P-IFNγ and p2ФC31-CMV-IFNγ were constructed by inserting the Sal I-ori P-IFNγ-poly A-Spe I fragment and Sal I-CMV-IFNγ-poly A-Apa I fragment from intermediate plasmids into the empty parent plasmid p2ФC31, respectively. Then parent plasmids were transformed into E. coli strain Top 10, and mediated intramolecular recombination. Targeted minicircle-ori P-IFNγ and control minicircle-CMV-IFNγ were produced using plasmid purification kits. To evaluate transgene expression from mc-ori P-IFNγ or mc-CMV-IFNγ in EBVnegative and-positive cells, the concentration of IFNγ in the culture supernatant of transfected cell lines was measured with an anti- human IFNγ ELISA kit. Results Targeted parent plasmid p2ФC31-ori P-IFNγ and control plasmid p2ФC31-CMV-IFNγ were successfully constructed. Then the corresponding minicircles were purified. All of these plasmids contained an IFNγ expression cassette driven by an ori P promoter or CMV promoter were transfected into EBV-negative or EBV-positive cells. These results showed that the expression level of IFNγ was obviously lower when driven by ori P promoter than by CMV promoter in EBV-negative cells. However, there was no significant difference in IFNγ expression levels when driven by the ori P or CMV promoter in EBV-positive C666-1 cells. Conclusion Targeted minicircle plasmid mc-ori P-IFNγ was successfully constructed. It can mediate selective expression of targeted gene in EBV-positive NPC cell line, but not in EBV-negative cell lines.