细菌诱导对昆虫细胞系NIH-SaPe-4生长与抗菌蛋白活性的影响

离体昆虫细胞系在昆虫免疫、抗菌肽及蛋白研究和药物开发方面具有较好的应用前景。该文对双翅目麻蝇科麻蝇成虫卵巢胚细胞系NIH-SaPe-4在藤黄微球菌诱导和非诱导条件下,细胞密度和活力的变化、诱导对细胞生长的影响、抗菌活性及其活性随时间的变化关系等进行了研究,并对所得抗菌蛋白进行了初步分离纯化和稳定性评估。结果表明,诱导使得细胞密度增长减缓,活力变弱。诱导和非诱导组细胞均可产生对3种革兰氏阳性菌具有抑菌活性的抗菌蛋白,其中对藤黄微球菌的抑菌活性最明显;诱导组细胞抗菌蛋白活性出现时间、稳定期抑菌活性均大于非诱导组,诱导菌消失一段时间后抗菌活性恢复到同等水平。抗菌蛋白具有酸碱稳定性和热稳定性。2组抗菌蛋白粗提液经凝胶、反相分离纯化后均得到一种60 kDa左右的抗菌蛋白,诱导组电泳后条带亮度大于非诱导组。该研究为昆虫细胞抗菌蛋白性质、分离纯化等研究奠定了科学基础。

Influence of Bacterial Induction on Cell Growth and Antimicrobial Peptide Activity in Insect Cell Lines NIH-SaPe-4

Insect cell lines can be applied in the studies of antimicrobial peptides, protein （Amps） and drug development with multiple advantages. In the present study we evaluated the changes of cell density and cellular vitality in the insect cell lines NIH-SaPe-4, which originated from ovarian germ cell of adult flesh fly （Diptera： Sarcophagidae）, after inducing by Micrococcus luteus. We also assayed the activity of antimicrobial protein and the changes of activity over time. Finally, the antimicrobial protein was isolated and purified and the stability of the protein was assessed. Our result demonstrated that the inducing by bacteria caused the insect cell growth slowed and impaired the cellular vitality. Both the induced group and control group could produce antimicrobial proteins with inhibitory activity against 3 types of Gram-positive bacteria. The protein showed the strongest antibacterial activity against Micrococcus luteus. The antimicrobial activity appeared earlier in the induced cells group, and the inhibitory activity of antimicrobial protein in induced cells group was higher than in the control group. Furthermore, the antibacterial activity in the induced group returned to the same level as in the control group after the disappearance of inducing bacteria. A kind of 60 kDa antimicrobial protein was obtained, after electrophoresis and reversedphase purification, from the crude extracts of both induced and control groups. The electrophoresis band intensity from induced group was greater than which from the control group. The obtained antimicrobial protein showed pH stability and thermal stability. Our result could provide basic information for future studies which focus on purification and characterization of antimicrobial peptides and proteins from insect cell.