3种检测牛白细胞源抗菌肽抗病毒活性方法的建立
|
摘要
为了建立牛白细胞源抗菌肽抗病毒活性的检测方法,采用半数组织细胞感染量(TCID_(50))测定、荧光试验、MTT比色3种方法,用Vero ccl-81作为细胞模型,以猪水疱性口炎病毒(VSV)作为模式病毒,对牛白细胞源抗菌肽抗病毒活性进行评价。依据3种方法浓度水平测定的结果和由此结果推算出的抗病毒指标值建立回归模型,并对3种方法的稳定性进行试验。结果表明:抗菌肽浓度在0～62.5μg/mL时对Vero ccl-81细胞安全,在此浓度区间检测方法有效。在3种检测方法中,TCID_(50)法最准确,荧光试验特异直观迅速,MTT比色法准确又直观。TCID_(50)方法回归模型方程为:y=0.285x+1.890(R~2=0.928),荧光试验回归模型方程为:y=2.138x+6.136(R~2=0.903),CPE法回归模型方程为:y=10.229x+3.859(R~2=0.979),3种方法稳定性均较好。说明牛白细胞源抗菌肽基于TCID_(50)法、荧光试验、MTT比色法3种方法联合检测模型可以定量化地准确计算其抗病毒活性。
In order to establish a method for detecting the antiviral activity of bovine leukocyte-derived antimicrobial peptides, this experiment used the 50% tissue cell infection(TCID_(50)) method, the fluorescence test, and the MTT colorimetric method to evaluate the antiviral activity of bovine leukocyte-derived antimicrobial peptides. In the experiment, Vero ccl-81 was used as a cell model, and the porcine vesicular stomatitis virus(VSV) was used as a model virus. A regression model was established based on the results of the three methods and the antiviral index values derived from the results, and the stabilities of the three methods were tested. The results showed that bovine leukocyte-derived antimicrobial peptides at concentrations ranging from 0 to 62.5 μg/mL were safe for Vero ccl-81 cells, and the detection methods were effective in this concentration range. Among the three detection methods, the TCID_(50) method was the most accurate, and the fluorescence test was specific and intuitive, and the MTT colorimetry was both accurate and intuitive. The regression model equations of the TCID_(50) method, fluorescence and CPE regression were y=0.285x+1.890(R~2=0.928), y=2.138x+6.136(R~2=0.903), and y=10.229x+3.859(R~2=0.979), respectively; and the stabilities of the three methods were all fine. These results suggested that the antiviral activities of bovine leukocyte-derived antibacterial peptides could be quantitatively and accurately calculated using the TCID_(50) method, the fluorescence test and the MTT colorimetric method.
In order to establish a method for detecting the antiviral activity of bovine leukocyte-derived antimicrobial peptides, this experiment used the 50% tissue cell infection(TCID_(50)) method, the fluorescence test, and the MTT colorimetric method to evaluate the antiviral activity of bovine leukocyte-derived antimicrobial peptides. In the experiment, Vero ccl-81 was used as a cell model, and the porcine vesicular stomatitis virus(VSV) was used as a model virus. A regression model was established based on the results of the three methods and the antiviral index values derived from the results, and the stabilities of the three methods were tested. The results showed that bovine leukocyte-derived antimicrobial peptides at concentrations ranging from 0 to 62.5 μg/mL were safe for Vero ccl-81 cells, and the detection methods were effective in this concentration range. Among the three detection methods, the TCID_(50) method was the most accurate, and the fluorescence test was specific and intuitive, and the MTT colorimetry was both accurate and intuitive. The regression model equations of the TCID_(50) method, fluorescence and CPE regression were y=0.285x+1.890(R~2=0.928), y=2.138x+6.136(R~2=0.903), and y=10.229x+3.859(R~2=0.979), respectively; and the stabilities of the three methods were all fine. These results suggested that the antiviral activities of bovine leukocyte-derived antibacterial peptides could be quantitatively and accurately calculated using the TCID_(50) method, the fluorescence test and the MTT colorimetric method.
引文
[1] Yeung A T,Gellatly S L,Hancock R E.Multifunctional cationic host defence peptides and their clinical applications[J].Cell Mol Life Sci,2011,68(13):2161-2176.
[2] Yeaman M R,Yount N Y.Unifying themes in host defence effector polypeptides[J].Nat Rev Microbiol,2007,5(9):727-740.
[3] Prado Montes de Oca E.Antimicrobial peptide elicitors:new hope for the post-antibiotic era[J].Innate Immunity,2013,19(3):227-241.
[4] 尚田田,王青,杭柏林,等.牛血液白细胞源抗菌肽的纯化及活性鉴定[J].广东农业科学,2012,39(11):130-132.
[5] 段文斗.牛白细胞提取物的抗菌活性及其对小鼠免疫功能的影响[D].郑州:河南农业大学,2007.
[6] 谷素美.牛血白细胞抗菌肽的粗提取及防腐保藏功效检测[D].郑州:河南农业大学,2012.
[7] He M,Zhang H,Li Y,et al.Cathelicidin-Derived Antimicrobial Peptides Inhibit Zika Virus Through Direct Inactivation and Interferon Pathway[J].Front Immunol,2018,9:722.
[8] 王剑,侯林,陈亚乔,等.金银花多糖的提取纯化及抗病毒活性研究[J].中国医院药学杂志,2018,38(8):810-812.
[9] Wang H,Liu R,Cui J,et al.Characterization and utility of phages bearing peptides with affinity to porcine reproductive and respiratory syndrome virus nsp7 protein[J].J Virol Methods,2015,222:231-241.
[10] 余春林,夏波,胡陈明,等.沐川乌骨黑鸡生长曲线拟合与分析[J].黑龙江畜牧兽医,2018(10):195-198.
[11] Feng T,Sun T,Li G,et al.DEAD-Box Helicase DDX25 Is a Negative regulator of type I interferon pathway and facilitates RNA virus infection[J].Front Cell Infect Microbiol,2017,7:356-365.
[2] Yeaman M R,Yount N Y.Unifying themes in host defence effector polypeptides[J].Nat Rev Microbiol,2007,5(9):727-740.
[3] Prado Montes de Oca E.Antimicrobial peptide elicitors:new hope for the post-antibiotic era[J].Innate Immunity,2013,19(3):227-241.
[4] 尚田田,王青,杭柏林,等.牛血液白细胞源抗菌肽的纯化及活性鉴定[J].广东农业科学,2012,39(11):130-132.
[5] 段文斗.牛白细胞提取物的抗菌活性及其对小鼠免疫功能的影响[D].郑州:河南农业大学,2007.
[6] 谷素美.牛血白细胞抗菌肽的粗提取及防腐保藏功效检测[D].郑州:河南农业大学,2012.
[7] He M,Zhang H,Li Y,et al.Cathelicidin-Derived Antimicrobial Peptides Inhibit Zika Virus Through Direct Inactivation and Interferon Pathway[J].Front Immunol,2018,9:722.
[8] 王剑,侯林,陈亚乔,等.金银花多糖的提取纯化及抗病毒活性研究[J].中国医院药学杂志,2018,38(8):810-812.
[9] Wang H,Liu R,Cui J,et al.Characterization and utility of phages bearing peptides with affinity to porcine reproductive and respiratory syndrome virus nsp7 protein[J].J Virol Methods,2015,222:231-241.
[10] 余春林,夏波,胡陈明,等.沐川乌骨黑鸡生长曲线拟合与分析[J].黑龙江畜牧兽医,2018(10):195-198.
[11] Feng T,Sun T,Li G,et al.DEAD-Box Helicase DDX25 Is a Negative regulator of type I interferon pathway and facilitates RNA virus infection[J].Front Cell Infect Microbiol,2017,7:356-365.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |