广西医学
廣西醫學
엄서의학
GUANGXI MEDICAL JOURNAL
2014年
1期
10-12
,共3页
分泌性白细胞蛋白酶抑制剂%果蝇表达系统%稳定表达
分泌性白細胞蛋白酶抑製劑%果蠅錶達繫統%穩定錶達
분비성백세포단백매억제제%과승표체계통%은정표체
Secretary leukocyte protease inhibitor%Drosophila expression system%Stable expression
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。
目的：在果蠅S2細胞中錶達人分泌性白細胞蛋白酶抑製劑（ SLPI ）併對其生物學性質進行初步測定。方法以A549細胞RNA為模闆，利用反轉錄PCR方法擴增得到SLPI蛋白的編碼序列，併將其剋隆到錶達載體pMT/V5-His A中，構建pMT/V5-SLPI 錶達質粒，通過與抗性篩選質粒pCoHygro 共轉染S2細胞，經潮黴素B篩選3～4週，得到能夠穩定錶達SLPI蛋白的多剋隆細胞繫。經CuSO 4誘導錶達後，用RT-PCR及Western blot分析SLPI基因的轉錄和錶達。結果穫得瞭抗性穩定的多剋隆S2細胞株S2/SLPI，CuSO4誘導後，RT-PCR證明SLPI在S2/SLPI細胞中能高效轉錄，Western blot 在細胞培養上清中檢測到錶達的重組SLPI蛋白。結論在果蠅S2細胞中成功錶達瞭人SLPI，SLPI的成功錶達為進一步開展SLPI促進免疫應答及其在抗病毒感染等方麵的作用研究提供瞭重要基礎。
목적：재과승S2세포중표체인분비성백세포단백매억제제（ SLPI ）병대기생물학성질진행초보측정。방법이A549세포RNA위모판，이용반전록PCR방법확증득도SLPI단백적편마서렬，병장기극륭도표체재체pMT/V5-His A중，구건pMT/V5-SLPI 표체질립，통과여항성사선질립pCoHygro 공전염S2세포，경조매소B사선3～4주，득도능구은정표체SLPI단백적다극륭세포계。경CuSO 4유도표체후，용RT-PCR급Western blot분석SLPI기인적전록화표체。결과획득료항성은정적다극륭S2세포주S2/SLPI，CuSO4유도후，RT-PCR증명SLPI재S2/SLPI세포중능고효전록，Western blot 재세포배양상청중검측도표체적중조SLPI단백。결론재과승S2세포중성공표체료인SLPI，SLPI적성공표체위진일보개전SLPI촉진면역응답급기재항병독감염등방면적작용연구제공료중요기출。
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 .
