人颗粒溶素活性肽与小鼠白细胞介素-12共表达质粒的构建与真核表达

Construction of Recombinant Plasmid for Co-expression of Human Granulysin Active Peptide and Murine Interleukin-12 in Eukaryotic Cells and Identification of Expressed Product

目的构建能分泌表达相对分子质量为9000的人颗粒溶素活性肽与小鼠白细胞介素-12(mIL-12)的真核共表达质粒,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序列测定对质粒进行鉴定。同时,将小鼠IL-12亚克隆入pBudCE4.1另一多克隆位点,构建真核共表达质粒pBudCE4.1-S9K/mIL-12。采用RT-PCR、免疫细胞化学与Dot-ELISA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;Dot-ELISA检测表明颗粒溶素可分泌到细胞外。ELISA检测细胞培养上清有mIL-12表达。结论已成功构建真核表达质粒pBudCE4.1-S9K/mIL-12,并能体外表达,为下一步利用颗粒溶素与mIL-12基因治疗肿瘤奠定了基础。

Objective To construct the recombinant plasmid for co-expression of human granulysin active peptide（Mr 9 000） and routine interleukin-12（mIL-12） and identify the expressed product. Methods Amplify the gene encoding human granulysin active peptide by PCR using the plasmid containing the eDNA sequence of granulysin as template and insert into eukaryotic expression vector pBudCE4. 1. Identify the constructed recombinant plasmid pBudCE4 1-SgK by restriction analysis and DNA sequencing,to which miL- l2 gene was subcloned. Transfect RAW204. 7 cells with the constructed co-expression vector pBudCE4. 1-SgK/mIL-12 and identify the expressed granulysin active peptide by RT-PCR ,immunocytochemical method and Dot-ELISA, and the expressed mIL-12 by ELISA. Results The results of restriction analysis, PCR and DNA sequencing proved that human granulysin active peptide gene was correctly insealod into plasmid pBudCE4. 1. RT-PCR and immunocytochemical method showed transient expression of human granulysin active peptide in RAW264. 7 cells. However, Dot-ELISA showed extracellular secretion of expressed human granulysin active peptide. ELISA proved ea：pression of mIL-12 in culture supernatant of RAW264. 7 cells. Conclusion The recombinant plasmid pBudCE4. 1-SgK/mIL-12 was successfully constructed and expressed in vitro,which laid a foundation of gene therapy of tumors with granulysin and mIL-12.