本文選題：新城疫病毒 + 血凝素-神經(jīng)氨酸酶�。� 參考：《河北北方學(xué)院》2016年碩士論文

【摘要】：隨著醫(yī)療水平的不斷提高,癌癥的治療受到越來越多的關(guān)注,溶瘤病毒為腫瘤的治療及治愈提供了一個(gè)新的視角。新城疫病毒(Newcastle disease virus,NDV)是溶瘤病毒中的一員。研究發(fā)現(xiàn),NDV在人類腫瘤細(xì)胞中的復(fù)制效率是正常細(xì)胞中的10000倍[1],其對(duì)腫瘤細(xì)胞的選擇性殺傷作用使其成為潛在的腫瘤生物治療因子。NDV為不分節(jié)段的單負(fù)鏈RNA病毒,屬于副黏病毒科副黏病毒亞科的腮腺炎病屬。主要受其感染的是禽類,可引起禽類呼吸道炎癥,感染人類的概率極小,表現(xiàn)為輕微的結(jié)膜炎或溫和的呼吸道癥狀,且為自限性疾病。已有研究結(jié)果提示,NDV溶瘤譜廣且不良反應(yīng)輕微,不同的NDV株系其抗腫瘤譜不同其抗腫瘤效果也不盡相同。在NDV外殼上的HN蛋白不僅可以識(shí)別唾液酸受體,而且還可以水解唾液酸受體,即它是一種同時(shí)具備兩種功能的病毒刺突蛋白,在病毒吸附和感染細(xì)胞時(shí)發(fā)揮著重要作用。近年來隨著RNA病毒基因工程的反向遺傳學(xué)技術(shù)的不斷完善,使得人們可以在c DNA水平上對(duì)病毒基因組進(jìn)行改造和探索,我們實(shí)驗(yàn)室保存著一株抗腫瘤新城疫病毒NDV-HBNU/LSRC/F3,是具有強(qiáng)毒株裂解位點(diǎn)的NDV弱毒株。其抗腫瘤效果已被初步探索和證實(shí),且也已經(jīng)測(cè)定了其全基因組序列。研究F3的HN蛋白的結(jié)構(gòu)和功能可以促進(jìn)更好的認(rèn)識(shí)和解讀全病毒抗腫瘤等功能,同時(shí)繼續(xù)探索F3誘導(dǎo)人食管癌ECA109細(xì)胞凋亡的信號(hào)通路。對(duì)更好掌握F3抗腫瘤機(jī)制,及更好的改造和發(fā)揮其抗腫瘤特性有著重要意義。HN基因及其編碼蛋白Gen Bank的登錄號(hào)分別為KC246549.1和AGL94562.1,首先利用蛋白質(zhì)分析專家系統(tǒng)(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等生物信息學(xué)在線分析程序,結(jié)合PSORT II Prediction、rasmol等生物信息學(xué)軟件,分析、預(yù)測(cè)HN蛋白的主要特性及T/B細(xì)胞抗原表位等。提取F3的總RNA。據(jù)HN基因全長(zhǎng)編碼序列(登錄號(hào)為KC246549.1)的開放閱讀框架和載體C-flag pc DNA3的多克隆位點(diǎn),設(shè)計(jì)帶酶切位點(diǎn)的HN引物,兩步法RT-PCR行目的片段的擴(kuò)增,并將擴(kuò)增產(chǎn)物克隆至真核表達(dá)載體C-flag pc DNA3的多克隆位點(diǎn)上,構(gòu)建C-flag pc DNA3-F3/HN真核表達(dá)系統(tǒng),重組質(zhì)粒經(jīng)細(xì)菌PCR、重組質(zhì)粒雙酶切、測(cè)序鑒定,將測(cè)序正確的重組質(zhì)粒C-flag pc DNA3-F3/HN轉(zhuǎn)染人食管癌ECA109細(xì)胞,轉(zhuǎn)染后通過間接免疫熒光法和RT-PCR鑒定轉(zhuǎn)染后細(xì)胞內(nèi)HN蛋白和基因的表達(dá),流式細(xì)胞儀測(cè)被重組質(zhì)粒轉(zhuǎn)染后腫瘤細(xì)胞的凋亡率。課題組前期研究已經(jīng)證實(shí)NDV F3在體外誘導(dǎo)食管癌細(xì)胞凋亡時(shí)有caspase-8的激活,提示死亡受體途徑可能在其誘導(dǎo)食管癌細(xì)胞凋亡中發(fā)揮著重要作用,因此我們?cè)贔3感染ECA109細(xì)胞后的36h內(nèi)每隔4h收集細(xì)胞總蛋白一次,運(yùn)用Western-blot法檢測(cè)ECA109細(xì)胞被感染后細(xì)胞內(nèi)的cleaved Caspase-3、cleaved PARP、FASL、FAS、FADD等蛋白的動(dòng)態(tài)表達(dá)變化情況,來研究NDV F3體外誘導(dǎo)腫瘤細(xì)胞凋亡的信號(hào)通路中死亡受體途徑變化情況,同時(shí)我們還運(yùn)用全Caspase抑制劑Z-VAD-FMK觀察抑制劑對(duì)NDVF3在體外誘導(dǎo)食管癌細(xì)胞凋亡的影響,以此觀察Caspase依賴性凋亡途徑在NDVF3體外誘導(dǎo)食管癌細(xì)胞凋亡中的作用。結(jié)果顯示,擴(kuò)增的HN基因約為1716bp,經(jīng)序列分析該蛋白由571個(gè)氨基酸組成,分子式為C2766H4316N752O852S24,分子質(zhì)量單位為62.5k Da,等電點(diǎn)理論值為6.24,HN蛋白富含無規(guī)則卷曲(Cc)和α-螺旋(Hh)的跨膜蛋白其主要定位在線粒體和胞質(zhì)中,共有12個(gè)親水性較高區(qū)域(參數(shù)得分≥1.9)、17個(gè)表面可及性較高區(qū)域(參數(shù)得分≥1.9)、9個(gè)柔韌性較高區(qū)域(參數(shù)得分≥2.0),36個(gè)翻譯后修飾位點(diǎn),17個(gè)潛在的B細(xì)胞表位,12個(gè)CTL表位和輔助性T細(xì)胞聯(lián)合表位。將其克隆到C-flag pc DNA3載體的多克隆位點(diǎn)上后,測(cè)序結(jié)果經(jīng)比對(duì)與Genbank上報(bào)告的HBNU/LSRC/F3株HN基因序列比對(duì)測(cè)序結(jié)果完全一致,將測(cè)序正確的重組質(zhì)粒轉(zhuǎn)染ECA109細(xì)胞,36h后轉(zhuǎn)染重組質(zhì)粒組的ECA109細(xì)胞胞質(zhì)內(nèi)可見黃綠色熒光,RT-PCR擴(kuò)增到了HN的m RNA,而對(duì)照組則無,證明構(gòu)建的重組質(zhì)粒能在體外真核細(xì)胞內(nèi)表達(dá)目的蛋白。經(jīng)初步探索,重組質(zhì)粒C-flag pc DNA3-F3/HN具有一定的抗腫瘤特性,轉(zhuǎn)染48h時(shí)重組質(zhì)粒組的ECA109細(xì)胞的凋亡率高于空質(zhì)粒對(duì)照組。F3感染后ECA109細(xì)胞中檢測(cè)到Fas及Fasl蛋白的表達(dá),證實(shí)了F3誘導(dǎo)ECA109細(xì)胞凋亡通路FAS-FASL-FADD的存在,FADD蛋白的表達(dá)在感染后4h時(shí)達(dá)最多,4h后有遞減趨勢(shì),而活化的caspase-3、PARP的表達(dá)則隨感染時(shí)間的延長(zhǎng)有遞增趨勢(shì),加上課題組前期研究證實(shí)的相同濃度的F3誘導(dǎo)ECA109細(xì)胞凋亡時(shí),caspase-8和caspase-9被活化激活,證實(shí)F3抗腫瘤途徑中內(nèi)源性凋亡通路和外源性凋亡通路共同發(fā)揮誘導(dǎo)腫瘤細(xì)胞凋亡作用;又因顯微鏡下觀察到在感染F3 24h后全caspase抑制劑Z-VAD-FMK不能抑制F3誘導(dǎo)的ECA109細(xì)胞病變,提示F3抗腫瘤機(jī)制通過半胱氨酸蛋白酶(cysteinyl aspartate specific proteinase,caspase)依賴性途徑,同時(shí)可能也通過半胱氨酸蛋白酶非依賴性途徑。本課題主要完成內(nèi)容如下:第一,獲得F3株HN蛋白的主要特性,為后期研究HN的結(jié)構(gòu)與功能做好了理論基礎(chǔ);第二,實(shí)現(xiàn)了F3株HN蛋白的真核表達(dá)并初步探索其抗腫瘤特性;第三,進(jìn)一步證實(shí)F3誘導(dǎo)細(xì)胞凋亡通過內(nèi)源性及外源性caspase依賴性途徑,24h后細(xì)胞又開始繼續(xù)發(fā)生細(xì)胞病變現(xiàn)象,提示F3株誘導(dǎo)ECA109細(xì)胞凋亡可能有半胱氨酸蛋白酶依賴性途徑,同時(shí)可能也有半胱氨酸蛋白酶非依賴性的凋亡通路。為進(jìn)一步研究新城疫病毒抗腫瘤的作用機(jī)制,從而更好的改造和發(fā)揮其抗腫瘤特性奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96

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