新城疫病毒F3-HN的生物信息学分析、抗瘤功能初探及Caspase抑制剂对F3诱导ECA109细胞凋亡的影响

发布时间：2018-06-17 08:59

本文选题：新城疫病毒 + 血凝素-神经氨酸酶　；参考：《河北北方学院》2016年硕士论文

【摘要】：随着医疗水平的不断提高,癌症的治疗受到越来越多的关注,溶瘤病毒为肿瘤的治疗及治愈提供了一个新的视角。新城疫病毒(Newcastle disease virus,NDV)是溶瘤病毒中的一员。研究发现,NDV在人类肿瘤细胞中的复制效率是正常细胞中的10000倍[1],其对肿瘤细胞的选择性杀伤作用使其成为潜在的肿瘤生物治疗因子。NDV为不分节段的单负链RNA病毒,属于副黏病毒科副黏病毒亚科的腮腺炎病属。主要受其感染的是禽类,可引起禽类呼吸道炎症,感染人类的概率极小,表现为轻微的结膜炎或温和的呼吸道症状,且为自限性疾病。已有研究结果提示,NDV溶瘤谱广且不良反应轻微,不同的NDV株系其抗肿瘤谱不同其抗肿瘤效果也不尽相同。在NDV外壳上的HN蛋白不仅可以识别唾液酸受体,而且还可以水解唾液酸受体,即它是一种同时具备两种功能的病毒刺突蛋白,在病毒吸附和感染细胞时发挥着重要作用。近年来随着RNA病毒基因工程的反向遗传学技术的不断完善,使得人们可以在c DNA水平上对病毒基因组进行改造和探索,我们实验室保存着一株抗肿瘤新城疫病毒NDV-HBNU/LSRC/F3,是具有强毒株裂解位点的NDV弱毒株。其抗肿瘤效果已被初步探索和证实,且也已经测定了其全基因组序列。研究F3的HN蛋白的结构和功能可以促进更好的认识和解读全病毒抗肿瘤等功能,同时继续探索F3诱导人食管癌ECA109细胞凋亡的信号通路。对更好掌握F3抗肿瘤机制,及更好的改造和发挥其抗肿瘤特性有着重要意义。HN基因及其编码蛋白Gen Bank的登录号分别为KC246549.1和AGL94562.1,首先利用蛋白质分析专家系统(Ex PASy)提供的Prot Param、protscale、TMpred、Coil、Motify Scan及SWISS-MODEL等,NCBI上的CONSERVE DOMON及SMART、Signal P4.0、Bepipred1.0及Net CTL等和Bcepred、ABCpred、SOPMA、SYFPEITHI等生物信息学在线分析程序,结合PSORT II Prediction、rasmol等生物信息学软件,分析、预测HN蛋白的主要特性及T/B细胞抗原表位等。提取F3的总RNA。据HN基因全长编码序列(登录号为KC246549.1)的开放阅读框架和载体C-flag pc DNA3的多克隆位点,设计带酶切位点的HN引物,两步法RT-PCR行目的片段的扩增,并将扩增产物克隆至真核表达载体C-flag pc DNA3的多克隆位点上,构建C-flag pc DNA3-F3/HN真核表达系统,重组质粒经细菌PCR、重组质粒双酶切、测序鉴定,将测序正确的重组质粒C-flag pc DNA3-F3/HN转染人食管癌ECA109细胞,转染后通过间接免疫荧光法和RT-PCR鉴定转染后细胞内HN蛋白和基因的表达,流式细胞仪测被重组质粒转染后肿瘤细胞的凋亡率。课题组前期研究已经证实NDV F3在体外诱导食管癌细胞凋亡时有caspase-8的激活,提示死亡受体途径可能在其诱导食管癌细胞凋亡中发挥着重要作用,因此我们在F3感染ECA109细胞后的36h内每隔4h收集细胞总蛋白一次,运用Western-blot法检测ECA109细胞被感染后细胞内的cleaved Caspase-3、cleaved PARP、FASL、FAS、FADD等蛋白的动态表达变化情况,来研究NDV F3体外诱导肿瘤细胞凋亡的信号通路中死亡受体途径变化情况,同时我们还运用全Caspase抑制剂Z-VAD-FMK观察抑制剂对NDVF3在体外诱导食管癌细胞凋亡的影响,以此观察Caspase依赖性凋亡途径在NDVF3体外诱导食管癌细胞凋亡中的作用。结果显示,扩增的HN基因约为1716bp,经序列分析该蛋白由571个氨基酸组成,分子式为C2766H4316N752O852S24,分子质量单位为62.5k Da,等电点理论值为6.24,HN蛋白富含无规则卷曲(Cc)和α-螺旋(Hh)的跨膜蛋白其主要定位在线粒体和胞质中,共有12个亲水性较高区域(参数得分≥1.9)、17个表面可及性较高区域(参数得分≥1.9)、9个柔韧性较高区域(参数得分≥2.0),36个翻译后修饰位点,17个潜在的B细胞表位,12个CTL表位和辅助性T细胞联合表位。将其克隆到C-flag pc DNA3载体的多克隆位点上后,测序结果经比对与Genbank上报告的HBNU/LSRC/F3株HN基因序列比对测序结果完全一致,将测序正确的重组质粒转染ECA109细胞,36h后转染重组质粒组的ECA109细胞胞质内可见黄绿色荧光,RT-PCR扩增到了HN的m RNA,而对照组则无,证明构建的重组质粒能在体外真核细胞内表达目的蛋白。经初步探索,重组质粒C-flag pc DNA3-F3/HN具有一定的抗肿瘤特性,转染48h时重组质粒组的ECA109细胞的凋亡率高于空质粒对照组。F3感染后ECA109细胞中检测到Fas及Fasl蛋白的表达,证实了F3诱导ECA109细胞凋亡通路FAS-FASL-FADD的存在,FADD蛋白的表达在感染后4h时达最多,4h后有递减趋势,而活化的caspase-3、PARP的表达则随感染时间的延长有递增趋势,加上课题组前期研究证实的相同浓度的F3诱导ECA109细胞凋亡时,caspase-8和caspase-9被活化激活,证实F3抗肿瘤途径中内源性凋亡通路和外源性凋亡通路共同发挥诱导肿瘤细胞凋亡作用;又因显微镜下观察到在感染F3 24h后全caspase抑制剂Z-VAD-FMK不能抑制F3诱导的ECA109细胞病变,提示F3抗肿瘤机制通过半胱氨酸蛋白酶(cysteinyl aspartate specific proteinase,caspase)依赖性途径,同时可能也通过半胱氨酸蛋白酶非依赖性途径。本课题主要完成内容如下:第一,获得F3株HN蛋白的主要特性,为后期研究HN的结构与功能做好了理论基础;第二,实现了F3株HN蛋白的真核表达并初步探索其抗肿瘤特性;第三,进一步证实F3诱导细胞凋亡通过内源性及外源性caspase依赖性途径,24h后细胞又开始继续发生细胞病变现象,提示F3株诱导ECA109细胞凋亡可能有半胱氨酸蛋白酶依赖性途径,同时可能也有半胱氨酸蛋白酶非依赖性的凋亡通路。为进一步研究新城疫病毒抗肿瘤的作用机制,从而更好的改造和发挥其抗肿瘤特性奠定了基础。
[Abstract]:With the continuous improvement of medical level, the treatment of cancer is being paid more and more attention. The Newcastle disease virus (NDV) is a member of the oncolytic virus. The study found that the replication efficiency of NDV in human tumor cells is 10000 of the normal cells. [1], its selective killing effect on tumor cells makes it a potential tumor biotherapy factor.NDV as an unsegmented single negative chain RNA virus, belonging to parotitis of paramyxovirus subfamily paramyxovirus subfamily. It is mainly infected by poultry and can cause respiratory inflammation in poultry. Conjunctivitis or mild respiratory symptoms and self limiting disease. The results of the study have suggested that NDV has a wide spectrum and a slight adverse reaction. Different NDV strains have different antitumor effects. The HN protein on the NDV shell can not only identify the sialic acid receptor, but also hydrolyze the sialic acid receptor. It is a viral spike protein with two functions at the same time. It plays an important role when the virus adsorb and infect cells. In recent years, with the continuous improvement of the reverse genetics technology of RNA virus gene engineering, people can modify and explore the virus genome at the level of C DNA. Our laboratory preserves a strain of resistance to the virus genome. The tumor Newcastle disease virus (NDV) NDV-HBNU/LSRC/F3, a NDV strain with the lysate site of the strong strain, has been preliminarily explored and confirmed, and its whole genome sequence has been determined. The structure and function of the HN protein of F3 can promote better understanding and interpretation of the function of the whole virus anti tumor, and continue to explore the F3 lure. The signal pathway of ECA109 cell apoptosis in human esophageal carcinoma. It is of great significance to better master the anti-tumor mechanism of F3 and to better transform and develop its anti-tumor properties. The login number of.HN gene and its encoded protein Gen Bank is KC246549.1 and AGL94562.1 respectively. First of all, Prot Param by the protein segregation expert system (Ex PASy), protsc Ale, TMpred, Coil, Motify Scan and SWISS-MODEL, CONSERVE DOMON and SMART, Signal P4.0, etc. The total RNA. of F3 was extracted from the open reading frame of the HN gene and the polyclonal loci of the carrier C-flag PC DNA3, designed the HN primers with the enzyme cut site, the amplification of the RT-PCR line of the two step method, and cloned the amplified products to the polyclonal loci of the eukaryotic expression vector C-flag PC DNA3. The eukaryotic expression system of C-flag PC DNA3-F3/HN was constructed. The recombinant plasmid was identified by bacterial PCR, double enzyme digestion and sequencing. The recombinant plasmid C-flag PC DNA3-F3/HN was transfected into human esophageal cancer ECA109 cells. The expression of HN protein and gene in the cells after transfection was identified by indirect immunofluorescence and RT-PCR, and the flow cytometry was used to determine the expression of the protein and gene in the transfected cells. The apoptosis rate of the tumor cells after the transfection of recombinant plasmids. Previous studies have confirmed that NDV F3 has caspase-8 activation in inducing apoptosis of esophageal cancer cells in vitro, suggesting that the death receptor pathway may play an important role in inducing apoptosis of esophageal cancer cells. Therefore, we collect every 4H within 36h after F3 infection of ECA109 cells. Western-blot assay was used to detect the changes in the dynamic expression of cleaved Caspase-3, cleaved PARP, FASL, FAS, FADD and other proteins in the cells infected by ECA109 cells after infection, to study the change of the death receptor pathway in the signal pathway of NDV F3 inducing tumor cell apoptosis in vitro, and we also used full Caspase inhibition. The effect of inhibitor on the apoptosis of esophageal cancer cells induced by NDVF3 in vitro was observed by Z-VAD-FMK in order to observe the role of Caspase dependent apoptosis pathway in the apoptosis of esophageal cancer cells induced by NDVF3 in vitro. The results showed that the amplified HN gene was about 1716bp, and the protein was composed of 571 amino acids by sequence analysis, and the molecular formula was C2766H4316N752O852S2 4, the molecular mass unit is 62.5k Da, the isoelectric point theory value is 6.24, the HN protein is rich in the irregular curl (Cc) and the alpha helix (Hh), which mainly locates in the mitochondria and cytoplasm, and there are 12 hydrophilic regions (parameter score > 1.9), 17 surface accessibility regions (parameter score > 1.9), 9 flexible regions. The number of points more than 2), 36 posttranslational modifier sites, 17 potential B cell epitopes, 12 CTL epitopes and auxiliary T cells were cloned on the polyclonal site of the C-flag PC DNA3 vector, and the sequencing results were identical to the sequencing results compared to the HN gene sequence of the HBNU/LSRC/F3 strain on the Genbank, and the sequencing was correct. The recombinant plasmid transfected to ECA109 cells, after 36h transfected to the recombinant plasmid group, the cytoplasm of ECA109 cells showed yellowish green fluorescence, RT-PCR amplified to HN m RNA, while the control group did not, proved that the constructed recombinant plasmid could express the target protein in eukaryotic cells in vitro. After preliminary exploration, the recombinant plasmid C-flag PC DNA3-F3/HN had some antitumor. When transfected with 48h, the apoptosis rate of ECA109 cells in the recombinant plasmid group was higher than that in the empty plasmid control group, and the expression of Fas and Fasl protein was detected in ECA109 cells after.F3 infection. It proved that F3 induced FAS-FASL-FADD in the apoptosis pathway of ECA109 cells. The expression of FADD protein was the most in 4h after infection. The expression of PARP increased with the prolongation of the time of infection. In addition, when the same concentration of F3 induced apoptosis in ECA109 cells, the Caspase-8 and caspase-9 were activated. It proved that the endogenous apoptosis pathway and exogenous apoptosis pathway in the anti-tumor pathway of F3 could induce the apoptosis of the tumor cells. It was observed under microscopically that all caspase inhibitor Z-VAD-FMK after infection of F3 24h did not inhibit the F3 induced ECA109 cell lesion, suggesting that the anti tumor mechanism of F3 is dependent on cysteine protease (cysteinyl aspartate specific proteinase, caspase), and may also pass through the non dependent pathway of cysteine protease. The main contents are as follows: first, the main characteristics of F3 strain HN protein were obtained, and the theoretical basis for the study of the structure and function of HN was done. Second, the eukaryotic expression of HN protein of F3 strain was realized and its anti-tumor properties were preliminarily explored. Third, further confirmed that F3 induced apoptosis through endogenous and exogenous caspase dependent pathway and 24h fine. Cell lesions begin to occur, suggesting that F3 strain may induce ECA109 cell apoptosis with cysteine protease dependent pathway, and may also have non dependent apoptosis pathway of cysteine protease, which can further study the anti-tumor mechanism of Newcastle disease virus and improve its antitumor properties. The foundation is laid.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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