蜂毒肽分离纯化与体内外抗HSV-1病毒作用研究
发布时间：2018-12-16 23:56
【摘要】： 近年来由单纯疱疹病毒I型引起的疾病发病率不断上升且伴有病毒耐药株的出现,为此我们开展了蜂毒肽抗HSV-1作用的研究。研究结果如下: 利用QF-1型超级蜜蜂电子自动取毒器采集意蜂蜂毒,通过葡聚糖凝胶层析分离纯化,再进行SDS-PAGE电泳鉴定,证实其为电泳级纯度的蜂毒肽,供后续实验使用。利用非洲绿猴肾细胞(Vero细胞)培养体外模型,采用细胞病变法,阿昔洛韦(ACV)作对照,探讨蜂毒肽有无体外抗HSV-1活性。结果显示,通过MTT法测得蜂毒肽对Vero细胞的半数毒性浓度(CC50)为10-2.1 mg/100μL。当利用100μL 9CC50的蜂毒肽时能够使病毒抑制率达到66.51 %。病毒的半数感染量(TCID50)为10-5.2/100μL。用HSV-1病毒建立小鼠脑炎模型,以ACV作为阳性对照物,对蜂毒肽体内抗HSV-1病毒的活性进行评估。结果显示,HSV-1病毒对小鼠的半数致死量(LD50)为10-3 mg/kg;蜂毒肽可以使染毒小鼠存活率从20 %提高到50 %,具有明显抑制HSV-1病毒的作用,并具有延长小鼠存活时间的作用。 设计四组蜂毒肽抗HSV-1病毒作用靶点作为四个试验组,分别探讨蜂毒肽对HSV-1病毒的直接作用、病毒的吸附、合成及释放是否有影响。结果显示:(1)四个浓度梯度1CC50,3CC50,6CC50,9CC50的蜂毒肽对HSV-1病毒的吸附均有抑制作用,抑制率分别为31.25 %,32.92 %,32.92 %,32.08 %,其中蜂毒肽对HSV-1病毒的最高抑制率为32.92 %;(2)1CC50,3CC50,6CC50,9CC50四个浓度梯度的蜂毒肽对HSV-1病毒的合成均有抑制作用,抑制率分别为29.05 %,29.91 %,31.69 %,33.06 %,其中蜂毒肽对HSV-1病毒合成的最高抑制率为33.06 %;(3)1CC50,3CC50,6CC50,9CC50四个浓度梯度的蜂毒肽对HSV-1病毒的释放均有抑制作用,抑制率分别为29.31 %,18.37 %,13.28 %,10.70 %,其中蜂毒肽对HSV-1病毒释放的最高抑制率为29.31 %,可见蜂毒肽能够抑制HSV-1病毒的释放,但是随着蜂毒肽剂量的增加,对病毒释放的抑制率明显降低。(4)蜂毒肽对病毒直接作用的实验显示,蜂毒肽实验组与病毒对照组的细胞存活率基本相同,说明蜂毒肽没有直接杀死病毒的作用。
[Abstract]:In recent years, the incidence of herpes simplex virus type I disease has been increasing and accompanied by the emergence of virus resistant strains. Therefore, we have carried out a study on the antiviral effect of propolis peptide against HSV-1. The results are as follows: the bee venom of Italian bee was collected by QF-1 super bee electronic automatic venom extractor, purified by dextran gel chromatography, and identified by SDS-PAGE electrophoresis, which was proved to be a pure bee venom peptide of electrophoretic grade. For subsequent experiments. Vero cells were cultured in vitro, cytopathic method and acyclovir (ACV) were used to investigate the anti- HSV-1 activity of propolis peptide in vitro. The results showed that the median toxic concentration (CC50) of propolis peptide to Vero cells was 10-2.1 mg/100 渭 L by MTT assay. The inhibition rate of the virus was 66.51% when the propolis peptide of 100 渭 L 9CC50 was used. The TCID50 of the virus was 10-5.2 / 100 渭 L. HSV-1 virus was used to establish mouse encephalitis model and ACV was used as positive control to evaluate the antiviral activity of propolis peptide against HSV-1 virus. The results showed that the half lethal dose (LD50) of HSV-1 virus to mice was 10-3 mg/kg;. Propolis peptide can increase the survival rate of mice from 20% to 50%. It can inhibit the HSV-1 virus obviously and prolong the survival time of mice. Four groups of bee venom peptides against HSV-1 virus were designed as four experimental groups to investigate the direct effect of bee venom peptide on HSV-1 virus, the adsorption, synthesis and release of HSV-1 virus. The results showed that: (1) the adsorption of HSV-1 virus was inhibited by bee venom peptide with four concentration gradient of 1CC50, 3CC50, CC509CC50, and the inhibition rates were 31.25%, 32.92%, 32.92%, 32.08%, respectively, and the inhibitory rates were 31.25%, 32.92%, 32.92% and 32.08%, respectively. The highest inhibitory rate of propolis peptide on HSV-1 virus was 32.92%. (2) (1) the synthesis of HSV-1 virus was inhibited by four concentration gradient of propolis peptide, CC50, 6CC509CC50, and the inhibition rates were 29.05%, 29.91%, 31.69%, 33.06%, respectively, the inhibitory rates were 29.05%, 29.91%, 31.69% and 33.06%, respectively. The highest inhibitory rate of propolis peptide on HSV-1 virus synthesis was 33.06%. (3) (1) the release of HSV-1 virus was inhibited by the four concentration gradient of propolis in CC50, CC50 and 9CC50, and the inhibition rates were 29.31%, 18.37%, 13.28%, 10.70%, respectively, the inhibitory rates were 29.31%, 18.37%, 13.28% and 10.70%, respectively. The highest inhibitory rate of propolis on HSV-1 virus release was 29.31%, which showed that propolis could inhibit the release of HSV-1 virus, but with the increase of the dose of propolis, The inhibition rate of virus release was significantly decreased. (4) the direct effect of propolis peptide on virus showed that the cell survival rate of the experimental group was basically the same as that of the control group, which indicated that the provenin had no direct effect on killing the virus.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R752.11
本文编号：2383274
[Abstract]:In recent years, the incidence of herpes simplex virus type I disease has been increasing and accompanied by the emergence of virus resistant strains. Therefore, we have carried out a study on the antiviral effect of propolis peptide against HSV-1. The results are as follows: the bee venom of Italian bee was collected by QF-1 super bee electronic automatic venom extractor, purified by dextran gel chromatography, and identified by SDS-PAGE electrophoresis, which was proved to be a pure bee venom peptide of electrophoretic grade. For subsequent experiments. Vero cells were cultured in vitro, cytopathic method and acyclovir (ACV) were used to investigate the anti- HSV-1 activity of propolis peptide in vitro. The results showed that the median toxic concentration (CC50) of propolis peptide to Vero cells was 10-2.1 mg/100 渭 L by MTT assay. The inhibition rate of the virus was 66.51% when the propolis peptide of 100 渭 L 9CC50 was used. The TCID50 of the virus was 10-5.2 / 100 渭 L. HSV-1 virus was used to establish mouse encephalitis model and ACV was used as positive control to evaluate the antiviral activity of propolis peptide against HSV-1 virus. The results showed that the half lethal dose (LD50) of HSV-1 virus to mice was 10-3 mg/kg;. Propolis peptide can increase the survival rate of mice from 20% to 50%. It can inhibit the HSV-1 virus obviously and prolong the survival time of mice. Four groups of bee venom peptides against HSV-1 virus were designed as four experimental groups to investigate the direct effect of bee venom peptide on HSV-1 virus, the adsorption, synthesis and release of HSV-1 virus. The results showed that: (1) the adsorption of HSV-1 virus was inhibited by bee venom peptide with four concentration gradient of 1CC50, 3CC50, CC509CC50, and the inhibition rates were 31.25%, 32.92%, 32.92%, 32.08%, respectively, and the inhibitory rates were 31.25%, 32.92%, 32.92% and 32.08%, respectively. The highest inhibitory rate of propolis peptide on HSV-1 virus was 32.92%. (2) (1) the synthesis of HSV-1 virus was inhibited by four concentration gradient of propolis peptide, CC50, 6CC509CC50, and the inhibition rates were 29.05%, 29.91%, 31.69%, 33.06%, respectively, the inhibitory rates were 29.05%, 29.91%, 31.69% and 33.06%, respectively. The highest inhibitory rate of propolis peptide on HSV-1 virus synthesis was 33.06%. (3) (1) the release of HSV-1 virus was inhibited by the four concentration gradient of propolis in CC50, CC50 and 9CC50, and the inhibition rates were 29.31%, 18.37%, 13.28%, 10.70%, respectively, the inhibitory rates were 29.31%, 18.37%, 13.28% and 10.70%, respectively. The highest inhibitory rate of propolis on HSV-1 virus release was 29.31%, which showed that propolis could inhibit the release of HSV-1 virus, but with the increase of the dose of propolis, The inhibition rate of virus release was significantly decreased. (4) the direct effect of propolis peptide on virus showed that the cell survival rate of the experimental group was basically the same as that of the control group, which indicated that the provenin had no direct effect on killing the virus.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R752.11
【引证文献】
相关期刊论文 前2条
1 曹锫沛;张朝凤;许翔鸿;张勉;;天然产物中毒性多肽的研究进展[J];药学与临床研究;2012年03期
2 杨小浪;董江涛;陈文彬;缪晓青;;蜂毒肽抗肿瘤研究新进展[J];中国蜂业;2013年21期
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