蜂毒肽分離純化與體內(nèi)外抗HSV-1病毒作用研究
發(fā)布時(shí)間：2018-12-16 23:56
【摘要】： 近年來由單純皰疹病毒I型引起的疾病發(fā)病率不斷上升且伴有病毒耐藥株的出現(xiàn),為此我們開展了蜂毒肽抗HSV-1作用的研究。研究結(jié)果如下: 利用QF-1型超級蜜蜂電子自動(dòng)取毒器采集意蜂蜂毒,通過葡聚糖凝膠層析分離純化,再進(jìn)行SDS-PAGE電泳鑒定,證實(shí)其為電泳級純度的蜂毒肽,供后續(xù)實(shí)驗(yàn)使用。利用非洲綠猴腎細(xì)胞(Vero細(xì)胞)培養(yǎng)體外模型,采用細(xì)胞病變法,阿昔洛韋(ACV)作對照,探討蜂毒肽有無體外抗HSV-1活性。結(jié)果顯示,通過MTT法測得蜂毒肽對Vero細(xì)胞的半數(shù)毒性濃度(CC50)為10-2.1 mg/100μL。當(dāng)利用100μL 9CC50的蜂毒肽時(shí)能夠使病毒抑制率達(dá)到66.51 %。病毒的半數(shù)感染量(TCID50)為10-5.2/100μL。用HSV-1病毒建立小鼠腦炎模型,以ACV作為陽性對照物,對蜂毒肽體內(nèi)抗HSV-1病毒的活性進(jìn)行評估。結(jié)果顯示,HSV-1病毒對小鼠的半數(shù)致死量(LD50)為10-3 mg/kg;蜂毒肽可以使染毒小鼠存活率從20 %提高到50 %,具有明顯抑制HSV-1病毒的作用,并具有延長小鼠存活時(shí)間的作用。 設(shè)計(jì)四組蜂毒肽抗HSV-1病毒作用靶點(diǎn)作為四個(gè)試驗(yàn)組,分別探討蜂毒肽對HSV-1病毒的直接作用、病毒的吸附、合成及釋放是否有影響。結(jié)果顯示:(1)四個(gè)濃度梯度1CC50,3CC50,6CC50,9CC50的蜂毒肽對HSV-1病毒的吸附均有抑制作用,抑制率分別為31.25 %,32.92 %,32.92 %,32.08 %,其中蜂毒肽對HSV-1病毒的最高抑制率為32.92 %;(2)1CC50,3CC50,6CC50,9CC50四個(gè)濃度梯度的蜂毒肽對HSV-1病毒的合成均有抑制作用,抑制率分別為29.05 %,29.91 %,31.69 %,33.06 %,其中蜂毒肽對HSV-1病毒合成的最高抑制率為33.06 %;(3)1CC50,3CC50,6CC50,9CC50四個(gè)濃度梯度的蜂毒肽對HSV-1病毒的釋放均有抑制作用,抑制率分別為29.31 %,18.37 %,13.28 %,10.70 %,其中蜂毒肽對HSV-1病毒釋放的最高抑制率為29.31 %,可見蜂毒肽能夠抑制HSV-1病毒的釋放,但是隨著蜂毒肽劑量的增加,對病毒釋放的抑制率明顯降低。(4)蜂毒肽對病毒直接作用的實(shí)驗(yàn)顯示,蜂毒肽實(shí)驗(yàn)組與病毒對照組的細(xì)胞存活率基本相同,說明蜂毒肽沒有直接殺死病毒的作用。
[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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R752.11
本文編號：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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R752.11
【引證文獻(xiàn)】
相關(guān)期刊論文 前2條
1 曹锫沛;張朝鳳;許翔鴻;張勉;;天然產(chǎn)物中毒性多肽的研究進(jìn)展[J];藥學(xué)與臨床研究;2012年03期
2 楊小浪;董江濤;陳文彬;繆曉青;;蜂毒肽抗腫瘤研究新進(jìn)展[J];中國蜂業(yè);2013年21期
，本文編號：2383274
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