【摘要】 目的在原核表达家蝇抗菌肽Attacin基因的研究基础上,为进一步提高抗菌肽的活性,利用重组杆状病毒Bacmid-pFastBac^TM-Attacin表达系统转染家蝇胚胎细胞系MDEC-07114,对其活性进行探索研究。方法构建pFastBac^TM HTA质粒,将重组质粒转化Escherichia coli（E.coli）DH10细胞,通过蓝白斑筛选,挑取阳性克隆。构建抗菌肽Attacin的家蝇胚胎细胞真核表达系统。构建Bacmid-pFastBac^TM-Attacin表达系统并转染家蝇胚胎细胞,提取培养上清和细胞裂解物粗蛋白,纯化,SDS-PAGE和Western-Blot对蛋白表达情况进行检测、鉴定。结果重组杆状病毒Bacmid DNA成功转染家蝇胚胎细胞,细胞出现分离、停止生长、直径变大等现象;培养上清和细胞裂解物蛋白经SDS-PAGE检测,在22 KD出现了1条特异性条带;通过Western-Blot实验检测目的蛋白在家蝇胚胎细胞中成功表达;通过细菌实验测得其产物有较好的抑菌活性。结论与E.coli BL21（DE3）原核表达系统相比,家蝇胚胎细胞系灵敏度较好,但仍有较大提升空间。更多还原

【Abstract】 Objective To express antimicrobial peptide in housefly embryos cells by using the recombinant baculovirus Bacmid-p Fast BacTM-Attacin,and then further investigate the biologic activity of Attacin. Methods The recombinant plasmid p Fast BacTMHTA was constructed,and then transformed into competent cell of Escherichia coli( E. coli) DH10 Bac. Blue & white selection was performed for choosing positive clone. Subsequently,the housefly embryonic cells( MDEC-07114) were transfected with the prepared recombinant baculovirus Bacmid-p Fast BacTM-Attacin. The crude proteins were extracted from the culture supernatant and cells,and then purified with the nickel column. The protein expression was detected by SDS-PAGE and Western Blot. Results The housefly embryonic cells could be successfully transfected by recombinant baculovirus. And the transfected cells showed series of abnormal phenomena such as separation,growth arrest,bigger diameter,etc. The crude proteins extracted from the culture supernatant and cells were analyzed by SDS-PAGE. In the results,a specific band with the molecular weight of 22 KD was observed,which was identified as Attacin through Western Blot. The peptide was certified to have a good antibacterial activity. Conclusion Compared with the prokaryotic expression of antimicrobial peptide in E. coli BL21,the eukaryotic expression in MDEC-07114 cell is more sensitive,but there is still room for improvement.更多还原