PKC抑制剂SP及激活剂PMA影响DEV增殖研究

发布时间：2018-08-20 09:22

【摘要】：鸭肠炎病毒(Duck enteritis virus,DEV)又称鸭瘟病毒(Duck plague virus,DPV),是阻碍养鸭业健康发展的重要病原之一。实验室前期研究发现了DEV核衣壳蛋白(Nucleocapsid protein,NP)及其宿主细胞受体即蛋白激酶C抑制蛋白(Protein kinase C inhibitor,PKCI)。PKCI是宿主细胞内蛋白激酶C(Protein kinase C,PKC)的抑制蛋白质,PKC是宿主细胞磷酸化通路的关键分子,参与多种病毒的增殖过程。但宿主细胞磷酸化通路是否影响DEV侵染过程,尚无文献报道。为此,本研究利用PKC经典抑制剂(Staurosporine,SP)和专性激活剂(Phorbol-12-myristate-13-acetate,PMA)开展磷酸化通路在DEV感染中的作用研究,以期为阐明DEV侵染机理提供一些新的线索和思路。1.鸭源PKC基因荧光定量PCR检测方法的建立:根据GenBank上PKC基因设计和合成特异性引物,通过PCR扩增从鸭胚成纤维(DEF)细胞mRNA提取样本中获取目的基因片段,构建重组质粒GV pXT19-T-PKC,并以其为阳性标准品建立荧光定量PCR检测方法和标准曲线,然后进行重复性、特异性和敏感性验证,结果显示:荧光定量PCR标准曲线的线性关系为Y=-3.3340x+44.199,相关系数为0.9954,扩增效率为99.19%;熔解曲线仅出现单特异峰,对H5亚型AIV、H7亚型AIV、H9亚型AIV、NDV和DHV均未检测到荧光信号。这表明本试验建立的荧光定量PCR方法具有良好的稳定性、特异性和灵敏性。2.SP和PMA对DEV毒力的影响:取DEV贵州强毒株,接种DEF细胞,测定TCID50值;取培养成单层的DEF细胞,分别用SP和PMA(浓度均为100nmol/L)处理4h,分别加入系列稀释的DEV进行孵育,观察和测定TCID50值变化;分别以不同浓度的SP和PMA处理DEF细胞后,再加入0.01MOI的DEV进行孵育,观察和分析病毒TCID50值变化,结果:DEV贵州强毒株的TCID50值为3.16×10-9/0.1mL;经SP处理,随处理浓度的加大,病毒TCID50值随之下降,但浓度到100nmol/L时,病毒TCID50值回升;经PMA处理,在20至50nmol/L时病毒TCID50值随之上升,但到100至200nmol/L时,病毒TCID50值反而下降。这表明经SP和PMA处理,可影响DEV毒力即DEV的增殖。3.SP和PMA对PKC、PKCI和DEV-NP基因转录的影响:取培养成单层的DEF细胞,经SP和PMA处理后,再用DEV感染,然后收集细胞培养物,应用前期建立的荧光定量PCR方法检测分析PKC、PKCI和DEV-NP基因的转录水平,结果:SP处理组PKC、PKCI和DEV-NP基因的转录水平分别为2.62×109copies/μL、2.61×102copies/μL和6.65×100copies/μL;PMA处理组相应为3.92×108copies/μL、5.24×101copies/μL和2.55×108copies/μL;病毒对照组相应为8.11×106copies/μL、5.83×101copies/μL和2.74×102copies/μL。这表明,SP处理能降低DEV增殖水平,而PMA处理能提高DEV增殖水平。
[Abstract]:Duck enteritis virus (Duck enteritis virus), also known as duck plague virus (Duck plague virus), is one of the most important pathogens hindering the healthy development of duck industry. Previous laboratory studies have found that DEV nucleocapsid protein NP and its host cell receptor, the protein kinase C inhibitor (Protein kinase C, are the key molecules of the host cell phosphorylation pathway. Participate in the proliferation of many viruses. However, whether the phosphorylation pathway of host cells affects the process of DEV infection has not been reported. Therefore, the phosphorylation pathway of PKC classical inhibitor (SP) and specific activator (Phorbol-12-Eritrestate-13-acetatePMA) were used to study the role of phosphorylation pathway in DEV infection, in order to provide some new clues and ideas for elucidating the mechanism of DEV infection. Establishment of fluorescent quantitative PCR Detection method for Duck PKC Gene: according to the design and synthesis of specific primers of PKC gene on GenBank, the target gene fragment was obtained by PCR amplification from mRNA samples of (DEF) cells derived from duck embryo fibroblasts. The recombinant plasmid GV pXT19-T-PKC was constructed, and the fluorescent quantitative PCR detection method and standard curve were established by using GV pXT19-T-PKC as positive standard. Then the reproducibility, specificity and sensitivity were verified. The results showed that the linear relationship of the standard curve of fluorescence quantitative PCR was Y-3.3340x 44.199.The correlation coefficient was 0.9954, and the amplification efficiency was 99.19.The melting curve showed only a single specific peak, and no fluorescence signals were detected for the H5 subtype AIV-H7 subtype AIVH9 subtype AIVNV and DHV. The results showed that the fluorescent quantitative PCR method established in this study had good stability, specificity and sensitivity. 2.The effects of SP and PMA on the virulence of DEV were as follows: take DEV Guizhou virulent strain, inoculate DEF cells, measure TCID50 value, and culture into monolayer DEF cells. After treated with SP and PMA (concentration of 100nmol/L) for 4 h, a series of diluted DEV was added to incubate, observe and measure the change of TCID50 value, after treated with SP and PMA at different concentrations, DEF cells were incubated with 0.01MOI DEV. By observing and analyzing the change of virus TCID50 value, the results showed that the TCID50 value of Guizhou virulent strain was 3.16 脳 10 ~ (-9) / 0.1 mL. After SP treatment, the virus TCID50 value decreased with the increase of treatment concentration, but when the concentration reached 100nmol/L, the virus TCID50 value increased, and PMA treatment, At 20 to 50nmol/L, the virus TCID50 increased, but at 100 to 200nmol/L, the virus TCID50 decreased. This indicated that SP and PMA could affect the virulence of DEV, I. E. the proliferation of DEV and the effect of PMA on the transcription of PKCPKCI and DEV-NP genes: DEF cells were cultured into monolayer DEF cells, then infected with DEV after SP and PMA treatment, and then the cell cultures were collected. The transcriptional levels of PKCU PKCI and DEV-NP genes were detected by fluorescence quantitative PCR assay. The results showed that the transcriptional levels of PKCU PKCI and DEV-NP gene were 2.62 脳 109copies/ 渭 L 2.61 脳 102copies/ 渭 L and 6.65 脳 100copies/ 渭 L PMA-treated group, respectively, and were 3.92 脳 108copies/ 渭 L 5.24 脳 101copies/ 渭 L and 2.55 脳 108copies/ 渭 L, respectively, and 8.11 脳 106copies/ 渭 L 5.83 脳 101copies/ 渭 L and 2.74 脳 102copies/ 渭 L in the virus control group. The results showed that the transcriptional levels of PKCI gene and DEV-NP gene in the control group were 8.11 脳 106copies/ 渭 L 5.83 脳 101copies/ 渭 L and 2.74 脳 102copies/ 渭 L, respectively. This indicated that SP treatment could decrease the proliferation of DEV, while PMA treatment could increase the proliferation of DEV.
【学位授予单位】：贵州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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