人分泌性白细胞蛋白酶抑制剂在果蝇细胞中的稳定表达及鉴定

Stable Expression and Characterization of Human Secretary Leukocyte Protease Inhibitor in Drosophila Cells

目的：在果蝇S2细胞中表达人分泌性白细胞蛋白酶抑制剂（ SLPI ）并对其生物学性质进行初步测定。方法以A549细胞RNA为模板，利用反转录PCR方法扩增得到SLPI蛋白的编码序列，并将其克隆到表达载体pMT/V5-His A中，构建pMT/V5-SLPI 表达质粒，通过与抗性筛选质粒pCoHygro 共转染S2细胞，经潮霉素B筛选3～4周，得到能够稳定表达SLPI蛋白的多克隆细胞系。经CuSO 4诱导表达后，用RT-PCR及Western blot分析SLPI基因的转录和表达。结果获得了抗性稳定的多克隆S2细胞株S2/SLPI，CuSO4诱导后，RT-PCR证明SLPI在S2/SLPI细胞中能高效转录，Western blot 在细胞培养上清中检测到表达的重组SLPI蛋白。结论在果蝇S2细胞中成功表达了人SLPI，SLPI的成功表达为进一步开展SLPI促进免疫应答及其在抗病毒感染等方面的作用研究提供了重要基础。

Objective To explore the expression and characterization of human secretary leukocyte protease inhibitor（SLPI） in Drosophila S2 cell lines.Methods Using total RNA extracted from A549 cells,SLPI-encoding cDNA was synthesized by RT-PCR and inserted into vector pMT/V5-His A.The resultant recombinant plasmid of pMT/V5-SLPI was contransfected into S 2 cells with a selection vector of pCoHygro containing hygromycin-resistant gene.The stable cell lines were established following repeated screening by hygromycin-B for 3-4 weeks.The recombinant SLPI was induced by CuSO 4 and detected by RT-PCR and Western blot .Results We obtained stable S2 cell lines, designated S2-SLPI.The effective expression of SLPI induced by CuSO 4 in S2-SLPI cells was confirmed by RT-PCR, and the recombinant SLPI in culture supernatant was detected by Western blot .Conclusion The stable expression of SLPI in S2 cell lines provides an important foundation for further reseach on the influence of SLPI on promoting the immune response and anti-virus infection .