载有hTERT基因逆转录病毒感染日本血吸虫童虫的研究

发布时间：2018-01-05 23:35

本文关键词：载有hTERT基因逆转录病毒感染日本血吸虫童虫的研究　出处：《中南大学》2010年博士论文　论文类型：学位论文

【摘要】： 迄今为止,应用于血吸虫病防治实践的技术仍存在许多不足,尤在控制日本血吸虫病流行与传播方面尚无理想方法。30年前有学者提出,建立稳定生长和连续传代培养的血吸虫细胞系,可为寻求新的防治技术提供基础和条件,但通过数十年的细胞培养技术探索,一直未能实现建立可连续传代生长的血吸虫细胞系目标。本研究受哺乳动物体细胞经转导外源永生化基因后成功建立永生化细胞系的启示,开展了采用逆转录病毒载体对日本血吸虫童虫细胞进行外源基因转导的生物学理论探索,制备了载有永生化基因(hTERT)的双嗜性逆转录病毒和泛嗜性逆转录病毒,并观察了这2种逆转录病毒载体转导外源基因到日本血吸虫童虫细胞或虫体后所发生的整合、转录和表达以及对Sj细胞增殖的影响。本研究目的在于为血吸虫细胞永生化研究提供理想的转导外源基因的载体,验证外源hTERT基因能否在血吸虫体内整合、表达及其表达部位,同时探索hTERT基因表达诱导细胞增殖的可行性。 [目的]探讨用双嗜性逆转录病毒载体将外源基因导入日本血吸虫(Sj)细胞的生物学理论与实验依据。[方法]从GenBank中收集褐家鼠双嗜性逆转录病毒受体(rRam-1)的氨基酸序列,应用Blastp工具对其进行序列相似性搜索,并用Cluster W2工具对相似性较高的氨基酸序列进行同源性分析；采用RPS-blast与InterproScan在线工具对rRam-1受体及其同源的氨基酸序列进行保守区域分析；进一步应用多个在线分析工具对与rRam-1同源的Sj蛋白进行蛋白二级结构、疏水性、跨膜性、信号肽、亚细胞定位以及翻译后修饰点等进行分析与预测；在生物信息学预测的基础上采用含短片段外源基因的双嗜性逆转录病毒载体感染Sj-12d童虫细胞培养物,并应用PCR与RT-PCR方法检测外源基因在Sj细胞中的整合与表达。[结果]rRam-1氨基酸残基序列相似性搜索及同源性分析显示,其氨基酸序列与多种脊椎动物的钠离子依赖性的磷酸盐运载体家族的氨基酸序列有很高的同源性,一致性均在59%以上,其中,与中国仓鼠Ram-1受体(cRam-1)和人类Ram-1 (h Ram-1)受体的氨基酸序列一致性均为93%；此外,与多种无脊椎动物磷酸盐运载体家族的氨基酸序列也有较高的同源性,氨基酸一致性在42%以上；Sj中存在2种与rRam-1受体有较高同源性的蛋白SJCHGC09605和SjCHGC05362,它们与rRam-1受体之间的氨基酸序列一致性分别为54%和61%,相似性分别为74%和72%。2种血吸虫蛋白与人类、褐家鼠及中国仓鼠的Ram-1受体蛋白处于平行的进化分枝上。保守性分析显示,2种Sj蛋白与人类、褐家鼠及中国仓鼠的Ram-1受体存在相同的PH04 Superfamily保守结构域,均为磷酸盐运载体超家族成员；二级结构显示,SJCHGC09605和SJCHGC05362蛋白中,α螺旋分别占68.97%和39.22%,跨膜区预测显示分别有7个和5个可能的区域,与疏水性预测结果完全一致；亚细胞定位及翻译后修饰位点分析显示,2种蛋白均不含信号肽序列和亚细胞定位信号,也不含糖基化、磷酸化和脂酰化等翻译后修饰位点。利用携带外源E77.43基因的双嗜性逆转录病毒感染Sj童虫细胞后,经PCR与RT-PCR检测到目的基因存在与表达,扩增的目的片段大小为330 bp,与理论值相符。[结论]双嗜性逆转录病毒rRam-1受体与日本血吸虫细胞膜上起离子转运通道或受体蛋白作用的SjCHGC09605和SjCHGC05362两种跨膜蛋白成分存在较高同源性；用载有E77.43基因的双嗜性逆转录病毒感染Sj童虫细胞获得成功,推测SjCHGC09605和SjCHGC05362两种与rRam-1受体同源的蛋白可能是Sj感染过程中起作用的分子。该结果为下一步用双嗜性逆转录病毒载体转导永生化基因至Sj细胞提供了生物学理论与实验依据。 [目的]建立含永生化基因(hTERT)的稳定产逆转录病毒细胞株,观察hTERT基因转导Sj童虫细胞后的整合和表达情况以及对细胞增殖作用的影响。[方法]将从美国引进的pBABE-puro-hTERT质粒经核酸内切酶酶切、PCR扩增和测序鉴定确认；倍比稀释测定PA317细胞和NIH3T3细胞对嘌呤霉素的最高耐受浓度；用脂质体将质粒转染至PA317细胞内,经嘌呤霉素筛选获得抗性克隆并扩大培养,并通过PCR、测序、免疫荧光、Western-blot及透射电镜对抗性细胞株进行鉴定,并以NIH3T3细胞测定收集的逆转录病毒液滴度。常规制备Sj-12d童虫细胞,并在体外培养中用BrudU-ELISA法检测细胞增殖情况,PCR法检测兔线粒体特异性基因确定无宿主来源细胞污染,倍比稀释法测定Sj细胞对嘌呤霉素的最高耐受浓度；用浓缩的双嗜性逆转录病毒感染Sj-12d童虫细胞并以嘌呤霉素连续筛选培养获得抗性Sj细胞克隆,扩大培养后用PCR、RT-PCR、Western-blot检测外源hTERT基因和puror基因在细胞内的整合与表达；用3H-TdR掺入法检测嘌呤霉素抗性Sj-12d细胞的增殖能力,利用细胞计数法绘制其生长曲线,并应用TRAP-ELISA法测定其端粒酶活性。[结果]pBABE-puro-hTERT质粒经酶切、PCR和测序鉴定为目的质粒；PA317细胞和NIH3T3细胞对嘌呤霉素的最高耐受浓度为6μg/ml和3μg/ml；嘌呤霉素抗性PA317克隆扩大培养物经PCR、测序、免疫荧光以及Western-blot检测到外源hTERT基因和puror基因的整合、转录及蛋白质表达；透射电镜检测到抗性PA317细胞的培养上清及胞浆内有逆转录病毒颗粒的存在,经浓缩后测得其滴度为2×105cfu/ml。Sj-12d童虫细胞培养3d后即可见部分细胞分裂相,10-14d后可见较多分裂相,BrdU-ELISA也显示培养14d后有明显的DNA合成与增殖,其对嘌呤霉素最高耐受浓度为0.5μg/ml;双嗜性逆转录病毒感染Sj-12d细胞后经嘌呤霉素连续筛选21d可见抗性克隆形成,对扩大培养后的抗性细胞做PCR、RT-PCR和Western blot,检测到外源hTERT基因和puror基因在抗性Sj-12d细胞内的整合、转录及蛋白表达,但整合的拷贝数少,转录水平低下；3H-TdR掺入法检测显示抗性Sj-12d细胞与常规培养的Sj-12d细胞均有一定增殖能力,但二者间差异无显著性(P0.05)；TRAP-ELISA实验未能从抗性Sj-12d细胞内检测到端粒酶活性；在培养4周内的抗性Sj-12d细胞生长相对较快,此后生长逐渐减慢,死亡细胞和退变细胞的数目逐渐增多,最后全部死亡。[结论]用pBABE-puro-hTERT逆转录病毒质粒转染PA317细胞后,成功建立含hTERT基因稳定产双嗜性逆转录病毒颗粒的细胞株；该病毒感染Sj-12d童虫细胞后可检测到外源hTERT基因和puror基因的整合、转录及蛋白表达,但整合拷贝数少,转录水平低下,未能激活Sj-12d细胞的端粒酶活性和改善细胞的增殖能力。 [目的]为提高逆转录病毒对血吸虫的感染能力,探讨应用pVSV-G质粒和pBABE-puro-hTERT质粒共转染包装细胞制备泛嗜性逆转录病毒的可行性,并观察该病毒感染Sj童虫后外源基因在虫体内的整合、转录、表达及具体的表达部位情况。[方法]将pVSV-G质粒和pBABE-puro-hTERT质粒共转染GP2-293包装细胞,转染后48h收集细胞培养上清,用浓缩的上清液与Polybrene混合液感染NIH3T3细胞系,经嘌呤霉素连续筛选12d后获得抗性克隆,计数抗性克隆数目并计算病毒滴度；挑取抗性NIH3T3克隆扩大培养,以PCR检测外源hTERT基因和puror基因在细胞内的整合,采用免疫细胞化学染色法检测hTERT基因在细胞内的表达情况；将泛嗜性逆转录病毒加入到体外培养的Sj-12d童虫,感染24h后更换培养基,将虫体连续培养6d；采用PCR和Southern杂交检测外源hTERT基因在虫体内的整合,同时应用RT-PCR、Western blot及免疫组织化学染色检测外源hTERT基因在虫体内的转录、表达及定位情况。[结果]经计数后,泛嗜性逆转录病毒颗粒滴度为3.2×108,抗性NIH3T3细胞经PCR扩增出外源hTERT基因和puror基因特异性145bp和204bp目的条带,免疫细胞化学染色法检测到hTERT基因在细胞内发生了蛋白质表达,表达部位以细胞核内为主；泛嗜性逆转录病毒感染后的虫体经PCR和RT-PCR扩增出了外源hTERT基因和puror基因特异性145bp和204bp目的产物,其中,hTERT基因的转录水平较高,而puror基因的转录水平则较低,Southern杂交也显示出虫体内有外源hTERT基因的多个拷贝整合；Western blot实验显示病毒感染后的虫体内有外源hTERT基因的表达,其表达部位经免疫组织化学染色确定为在虫体的口吸盘、腹吸盘和后部体壁皮层下的表达量最多。[结论]用pVSV-G质粒和pBABE-puro-hTERT质粒共转染包装细胞后成功制备携带外源hTERT基因泛嗜性逆转录病毒,并证明该病毒感染Sj童虫活虫体后可在吸盘和后部体壁皮层下产生外源hTERT基因的多拷贝整合、转录与蛋白质表达。
[Abstract]:So far, there are still many problems in the technologies used for schistosomiasis control, especially in controlling the spread of schistosomiasis japonica is the ideal method of.30 years ago, some scholars put forward the establishment of schistosome cell lines with stable growth and continuous passage, can provide the basis and conditions for prevention of new technology, but through decades of cells culture exploration, has failed to achieve a continuous passage of growth of schistosome cells target. This study by mammalian somatic cell transduction by exogenous immortalized gene successfully established immortalized cell lines of enlightenment, to carry out exploration of the biological theory of exogenous gene transduction on schistosomulum cells by retroviral vector, preparation an immortalized gene (hTERT) of the amphotropic retrovirus and retroviral, and observe the 2 kinds of reverse transcriptase disease Virus vector exogenous gene to integrate the schistosomulum cells or body of transcription and expression and effects on proliferation of Sj cells. The purpose of this study is to provide an ideal carrier exogenous gene for the study of schistosome cell immortalization, verify whether exogenous hTERT gene in schistosome integration, expression and expression at the same time, the expression of hTERT gene to explore the feasibility of inducing cell proliferation.
[of amphotropic retroviral vector exogenous gene of Schistosoma japonicum (Sj)] to the biological basis of theoretical and experimental methods. Cell collection of Rattus norvegicus amphotropic retrovirus receptor from GenBank (rRam-1) amino acid sequence, the application of Blastp tools for sequence similarity search, and the amino acid sequence similarity high homology analysis with Cluster W2 tools; conservative region analysis of the amino acid sequence of rRam-1 receptor and its homologous with RPS-blast online tools and InterproScan; the further application of multiple online analysis tools for protein secondary structure of two homologous with rRam-1 Sj protein, hydrophobicity, transmembrane, signal peptide, subcellular localization and post translation modification sites for analysis and prediction; in bioinformatics prediction based on the exogenous gene fragment containing short amphotropic retrovirus disease Sj-12d schistosomula cell culture virus vector infection, and]rRam-1. The amino acid sequence similarity search and homology analysis showed that the expression of the integration and application of PCR and RT-PCR for the detection of exogenous gene in Sj cells, the sodium ion of its amino acid sequence with a variety of vertebrate dependent phosphate transport carrier with amino acid sequence family high homology, consistency in more than 59%, and Chinese hamster Ram-1 receptor (cRam-1) and human Ram-1 (H Ram-1) amino acid sequence identity of the receptor was 93%; in addition, the homology of amino acid sequence with a variety of invertebrate transport phosphate carrier family also has a higher consistency in 42% amino acids. There are 2 or more; Sj and rRam-1 receptor homology protein SJCHGC09605 and SjCHGC05362 amino acid sequence consistency between them and rRam-1 receptor were 54% and 61%. It is 74% and 72%.2 respectively for schistosome proteins and human Chinese, Rattus norvegicus and hamster Ram-1 receptor protein in parallel evolutionary branches. Conservative analysis showed that 2 Sj protein and human, Rattus norvegicus and Ram-1 receptor PH04 China hamster Superfamily conserved domain of the same carrier are phosphate superfamily; two level structure, SJCHGC09605 and SJCHGC05362 protein, alpha helix accounted for 68.97% and 39.22%, transmembrane prediction showed there were 7 and 5 possible areas, was exactly consistent with hydrophobicity prediction; subcellular localization and posttranslational modification site analysis showed that all the 2 proteins without signal peptide and subcellular localization sequence signal, without glycosylation, phosphorylation and lipid acylation post-translational modification sites. By carrying exogenous E77.43 gene amphotropic retrovirus infected Sj cells of schistosomula, and by PCR The presence of RT-PCR detected and expression of target gene, the amplified fragments were 330 BP, consistent with the ion channels or the role of the SjCHGC09605 receptor protein and SjCHGC05362 two transmembrane protein has high homology up. Conclusion] amphotropic retrovirus receptor rRam-1 of Schistosoma japonicum and on the cell membrane and the theoretical value; for carrying E77.43 gene amphotropic retrovirus infection Sj schistosomula cells successfully, suggesting that SjCHGC09605 and SjCHGC05362 two and rRam-1 receptor homologous protein may play a role in the molecular process of Sj infection. The results provide a theoretical and experimental basis for the biological amphotropic retrovirus vector transduction immortalized gene into Sj cells under a step.
[Objective] to establish immortalized gene (hTERT) of the stable production of retroviral plasmid pBABE-puro-hTERT cell lines, to observe hTERT gene transduction of Sj insect cells after children integration and expression and effect on cell proliferation. Methods will be introduced from America by enzyme digestion, PCR amplification and sequencing confirmed times; dilution determination of PA317 cells and NIH3T3 cells of the highest puromycin resistant concentration; plasmid by liposome was transfected into PA317 cells by puromycin resistant clones screened and expanded in culture, and by PCR, sequencing, immunofluorescence and transmission electron microscopy, Western-blot resistant cells were identified by NIH3T3 the cell titer of retrovirus was determined. The routine preparation of Sj-12d cells of schistosomula, and in vitro cell proliferation was assessed by BrudU-ELISA method, PCR method for detection of rabbit mitochondrial specific base Because of no pollution from host cells, dilution of Sj cells was determined by the highest puromycin resistant concentration; with concentrated amphotropic retrovirus infected Sj-12d cells of schistosomula and puromycin resistance continuous screening culture Sj cell clones after culture expansion by PCR, RT-PCR, integration and expression of exogenous detection hTERT gene and puror gene in Western-blot cells; detection of puromycin resistant Sj-12d cells by 3H-TdR incorporation assay proliferation and draw the growth curve by using cell counting method, determination of the activity of]pBABE-puro-hTERT. The plasmid was verified by enzyme digestion of telomerase and the application of TRAP-ELISA method, PCR and DNA sequencing plasmids and PA317 cells; NIH3T3 cells of the highest puromycin resistant concentration was 6 g/ml and 3 g/ml; puromycin resistant clones were cultured by PA317 PCR, immunofluorescence and Western-blot sequencing. Detection of exogenous hTERT gene and puror gene expression, transcription and protein; the culture supernatant and cytoplasm by electron microscopy to resistant PA317 cells with retroviral particles exist, after concentration measured the titer was 2 * 105cfu/ml.Sj-12d schistosomula cultured 3D cells after visible cell division phase, can be seen after 10-14d more splitting phase, BrdU-ELISA showed that after 14d culture, DNA synthesis and proliferation, the highest puromycin tolerance concentration was 0.5 g/ml; amphotropic retrovirus infected Sj-12d cells by puromycin screening for 21d visible clone formation, expansion of resistant cells cultured in PCR and RT-PCR. Western blot, detection of exogenous hTERT gene and puror gene in resistant Sj-12d cells, the expression of mRNA and protein, but the integration of the copy number of small, low level of transcription; 3H-TdR incorporation assay. In resistant Sj-12d cells and normal cultured Sj-12d cells have certain ability of proliferation, but the difference between the two groups had no significant difference (P0.05); TRAP-ELISA experiment failed to detect from the resistant Sj-12d cells to telomerase activity in the culture; within 4 weeks of the resistance of Sj-12d cell growth is relatively fast, then the growth gradually slowed down, the number of cell death and cell degeneration increased gradually, finally died. Conclusion pBABE-puro-hTERT with retroviral vector transfected PA317 cells was successfully established with hTERT gene stably producing amphotropic retrovirus particles in cell lines; the virus infection Sj-12d schistosomula cells can be detected after exogenous hTERT gene and puror gene expression, transcription and protein, but copy number of integrated, low level of transcription, failed to activate Sj-12d cells telomerase activity and improve the proliferation of the cells.
[Objective] to improve the ability of retroviral infection of Schistosoma japonicum and explore the application of pVSV-G plasmid and pBABE-puro-hTERT plasmid were transfected into the packaging cell preparation feasibility of retroviral infection, and observe the transcription of Sj gene in schistosomula after integration, parasite, expression and specific expression site. Methods] the pVSV-G plasmid and pBABE-puro-hTERT plasmid were transfected into GP2-293 packaging cells, transfected 48h supernatants were collected to infect NIH3T3 cell lines with the supernatant and Polybrene mixed solution concentration, by puromycin resistant clones obtained after continuous screening of 12D, the number of counting resistant clones and calculate the virus titer were NIH3T3; resistant clones were cultured to detect exogenous PCR hTERT gene and puror gene in the cells of the integration, expression of staining detection of hTERT gene in cells by immunocytochemistry; will Retroviral entry into cultured Sj-12d schistosomula, 24h infection after replacement of the medium will worm continuous culture of 6D; PCR and Southern hybridization detection of exogenous hTERT gene in the integration of the parasite by simultaneous application of RT-PCR, Western blot and immunohistochemical detection of exogenous hTERT gene transcription in the parasite., the expression and localization. The results by counting after retroviral particles titer was 3.2 * 108, the resistance of NIH3T3 cells was amplified by PCR hTERT gene and puror gene specific 145bp and 204bp band, immunocytochemical staining method to detect the hTERT gene in the cell occurs in protein expression, the expression in the nucleus of the main parts of the body; retroviral infection after PCR and RT-PCR were amplified with exogenous hTERT gene and puror gene specific 145bp and 204bp products, including hTERT based Because of the high transcription level and puror gene transcription level is low, Southern hybridization also showed the parasite multiple copies of exogenous hTERT gene; Western blot experiment showed the expression of exogenous hTERT gene in infected larvae, the expression by immunohistochemical staining to determine the oral sucker in insects the expression of ventral sucker and posterior wall. Most subcortical conclusion with pVSV-G plasmid and pBABE-puro-hTERT plasmid were transfected into the packaging cell prepared successfully carrying exogenous hTERT gene retroviral, and prove that the virus infection Sj schistosomula live parasites can produce hTERT gene multi copy integration in sucker and the rear wall cortex, and protein expression of transcription.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

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