人颗粒溶素活性肽和小鼠白细胞介素-12基因真核穿梭共表达质粒的构建与真核表达

Eukaryotic expression and identification of recombinant shuttle plasmid for co-expression of human granulysin active peptide and murine IL-12

目的构建含分枝杆菌复制子Orim,可分泌表达人颗粒溶素相对分子质量为9000的活性肽与小鼠白细胞介素-12(mIL-12)的真核穿梭共表达质粒,并检测其蛋白表达。方法以含有颗粒溶素cDNA序列的质粒为模板,通过PCR扩增获得相对分子质量为9000的颗粒溶素活性肽基因片段后,定向插入真核表达载体pBudCE4.1中,获得重组表达质粒pBudCE4.1-S9K,经双酶切及插入片段测序对质粒进行鉴定。同时,将mIL-12亚克隆入pBudCE4.1另一多克隆位点,构建真核共表达质粒pBudCE41-S9K/mlL-12。然后,将分支杆菌复制子Orim亚克隆入真核共表达质粒pBudCE4.1-S9K/mlL-12,形成穿梭共表达质粒。采用RT-PCR、免疫细胞化学法,检测颗粒溶素活性肽在RAW264.7细胞的瞬时表达与分泌。ELISA检测mIL-12的表达。结果酶切、PCR及测序鉴定证实,颗粒溶素活性肽基因插入片段正确;RT-PCR和免疫细胞化学检测表明其可以在细胞中瞬时表达;ELISA检测细胞培养上清有mIL-12表达。结论成功构建穿梭共表达质粒pBM9,并能体外表达。

Objective To construct an eukaryotic co-expression plasmid pBM9 carrying human granulysin active peptide and murine IL-12 and determine its expression. Methods The primer pairs including granulysin leader peptide DNA sequence were designed to amplify granulysin from the plasmid pZM03 carrying granulysin gene by polymerase chain reaction （PCR）. PCR product was directly cloned into an eukaryotic co-expression plasmid pBudCE4.1 to construct plasmid pBudCE4.1-S9K, pBudCE4.1-S9K plasmid was identified by DNA sequencing. Murine IL-12 gene was subcloned into pBudCE4. 1-S9K to construct eukaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12. Mycobacteria replicon Orim from plasmid pZM03 was subeloned into Not Ⅰ site of pBudCE4.1-S9K/mIL-12 to construct eukaryotic co-expression shuttle plasmid pBM9. pBM9 was tansfected into RAW264.7 cells. RT-PCR was used to detect the mRNA expressions of granulysin and IL-12. The protein expression of them were observed by immunocytochemical method and ELISA respectively. Results RT-PCR identified the expressions of granulysin and mIL-12 in transfected cells and culture supernatant. Immunocytochemical method and ELISA verified the protein expressions. Conclusion The recombinant shuttle plasmid pBM9 is successfully constructed and expressed in vitro, which laid a foundation of gene therapy for tumors with granulysin and mIL-12.