人IL-8正义和反义真核表达载体的构建与表达

Construction and expression of human IL-8 sense or antisense eukaryotic expression vectors

目的:构建人IL-8正义和反义真核表达载体。方法:RT-PCR扩增正义和反义IL-8基因片段后,将其克隆入真核表达载体pcDNA3.1(+),通过菌落PCR、双酶切及测序进行鉴定后,采用脂质体法分别瞬时转染至人卵巢癌细胞系A2780和SKOV3细胞中,并用RT-PCR和ELISA法检测细胞转染后IL-8的表达水平。结果:经验证,重组人IL-8正义/反义真核表达载体构建正确。pcDNA3.1(+)-ssIL-8转染A2780细胞后,其IL-8 mRNA及蛋白表达水平均显著增加;而pcDNA3.1(+)-asIL-8转染SKOV3细胞后,其IL-8分泌水平降低。结论:成功构建了人IL-8正义/反义真核表达质粒pcDNA3.1(+)-ssIL-8/pcDNA3.1(+)-asIL-8,为后续研究IL-8在卵巢癌及其他肿瘤中的作用打下基础。

AIM：To construct the sense or antisense IL-8 eukaryotic expression vectors. METHODS：Sense or antisense IL-8 full length gene were amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1（＋）. After the identification by PCR,restriction endonuclease digestion and the nucleotide sequencing,the recombinant vectors were transfected into human ovarian carcinoma A2780 and SKOV3 cell lines transiently by lipofectamine mediation. The expression of IL-8 gene and protein were detected by RT-PCR and ELISA. RESULTS： The sense or antisense IL-8 eukaryotic expression vectors were constructed and verified. The expression of IL-8 gene and protein in A2780 cells transfected with pcDNA3, 1 （ ＋ ）-sslL-8 were increased, whereas the expression of IL-8 protein in SKOV3 cells transfected with pcDNA3. 1 （ ＋ ）-aslL-8 was decreased. CONCLUSION： The eukaryotic expression vectors pcDNA3.1 （ ＋ ）-sslL-8 or pcDNA3, 1 （ ＋ ）-aslL-8 have been constructed successfully, which lays a base for further study on roles of IL-8 in ovarian cancer and other tumors.