【作者】 郑桂玲；

【导师】 薛明； 李国勋；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 自1962年成功建立传代昆虫细胞系以来,昆虫细胞系已广泛地应用于生物学、农业和医学等领域,其中鳞翅目昆虫细胞系应用最为广泛,特别是随着杆状病毒表达载体系统(Baculovirus expression victor system,BEVS)的发明,使昆虫细胞在重组蛋白生产、工程疫苗和蛋白组学等领域得到了越来越多的应用,被认为是有巨大开发潜力的新型生物反应器。因此,建立新型昆虫细胞系、筛选用于病毒和重组蛋白生产等方面更加理想的细胞系,将具有重要的理论意义和实践价值。本文以重要农业害虫-棉铃虫(Helicoverpa armigera)和粘虫(Mythimna separata)为材料建立新细胞系,并对其生物学特性进行研究,丰富了有限的昆虫细胞系资源,为离体条件下进行昆虫生理生化、杆状病毒感染和细胞凋亡机制的研究提供实验材料;同时对本实验室建立的高产悬浮性粉纹夜蛾(Trichoplusia ni)胚胎细胞系QB-Tn9-4s进行克隆,从中筛选高蛋白表达的克隆株,进行无血清培养驯化,并阐明其生物学特性,为昆虫细胞这一新型生物反应器的深入研究和开发奠定基础。1.由棉铃虫胚胎组织建立了两株细胞系,分别命名为QB-Ha-E-1和QB-Ha-E-5,在含10%胎牛血清的TNM-FH培养基中已传代60余代。两株细胞系均以圆形和短梭形细胞为主;DAF和RAPD鉴定结果表明两株细胞系均来源于棉铃虫胚胎;染色体均为典型的鳞翅目昆虫细胞染色体特征;QB-Ha-E-1和QB-Ha-E-5的第30代细胞群体倍增时间分别为63.7 h和66.9 h。两株细胞系均能被棉铃虫核型多角体病毒(HaSNPV)感染,4 d的感染率分别为86.6%和56.5%;对甘蓝夜蛾(Mamestra brassicae)核型多角体病毒(MbNPV)7 d的感染率均为15%左右;但对苜蓿银纹夜蛾(Autographa californica)核型多角体病毒(AcMNPV)侵染的反应不同,DAPI染色和基因组DNA电泳结果表明,AcMNPV可诱导QB-Ha-E-5细胞发生凋亡,极少数细胞内可形成病毒多角体,但不能诱导QB-Ha-E-1细胞发生凋亡,其感染率为55.3%,两株细胞系均可被1.25μg/mL的放线菌素D诱导发生凋亡。2.由粘虫胚胎组织建立了四株细胞系,分别命名为QB-Ms-E-1、QB-Ms-E-2、QB-Ms-E-3和QB-Ms-E-4,在含10%胎牛血清的TNM-FH培养基中已传代50余代。四株细胞系均以圆形和短梭形细胞为主;DAF和RAPD鉴定结果表明四株细胞系均来源于粘虫胚胎;染色体均呈典型的鳞翅目昆虫细胞染色体特征;QB-Ms-E-1、QB-Ms-E-2、QB-Ms-E-3和QB-Ms-E-4的群体倍增时间分别为44.9 h、46.6 h、46.5 h和47.1 h。四株细胞系均能被同源的粘虫核型多角体病毒(MsNPV)感染,7 d的感染率分别为56.6%、53.6%、49.3%和62.5%;对MbNPV 10 d的感染率均在5%以下;但对苜蓿银纹夜蛾核型多角体病毒(AcMNPV)都很敏感,4 d的感染率均在90%以上。3.对粉纹夜蛾胚胎细胞系QB-Tn9-4s进行克隆获得了9个克隆株,分别命名为克隆株QB-Tn-Ⅰ、QB-Tn-Ⅱ、QB-Tn-Ⅲ、QB-Tn-Ⅳ、QB-Tn-Ⅴ、QB-Tn-Ⅵ、QB-Tn-Ⅶ、QB-Tn-Ⅷ和QB-Tn-Ⅸ。对各克隆株基因组DNA进行RAPD鉴定,结果表明各克隆株与原始细胞系QB-Tn9-4s具有相同的DNA扩增谱带。病毒侵染试验表明,各克隆株对AcMNPV均很敏感,感染率都在87%以上,平均每个细胞病毒多角体产量在70～87个之间,其中克隆株QB-Tn-Ⅰ的多角体产量最高(86.7±1.5 OBs/cell),高于对照BTI-Tn5B1-4和原始细胞QB-Tn9-4s的多角体产量,是Sf-9多角体产量的2.8倍。比较各细胞第6 d重组蛋白β-半乳糖苷酶(β-gal)的表达量,克隆株QB-Tn-Ⅴ的表达量最高(3.06×10~4 IU/mL),其次为克隆株QB-Tn-Ⅰ(2.70×10~4 IU/mL),均高于原始细胞QB-Tn9-4s和对照细胞BTI-Tn5B1-4及Sf-9的表达量。比较各细胞克隆株第7 d分泌型碱性磷酸酶(SEAP)的表达量,克隆株QB-Tn-Ⅴ的表达量最高(2.96 IU/mL),其次为QB-Tn-Ⅰ(2.41 IU/mL),均显著高于原始细胞QB-Tn9-4s和对照细胞BTI-Tn5B1-4及Sf-9的表达量。4.将原始细胞系QB-Tn9-4s及其克隆株QB-Tn-Ⅰ和QB-Tn-Ⅴ分别在无血清培养基Sf-900Ⅲ及EX-CELL 420中进行驯化培养,在Sf-900Ⅲ中驯化均获得成功,而在EX-CELL 420中只有克隆株QB-Tn-Ⅴ最终驯化成功。无血清驯化后各细胞形态和大小都发生了一定的改变。生长曲线测定结果表明,各细胞在Sf-900Ⅲ中的生长速度变快,群体倍增时间缩短,细胞最大密度均有提高,但是克隆株QB-Tn-Ⅴ在EX-CELL 420中生长速度变慢,倍增时间延长,细胞最大密度有所下降。QB-Tn9-4s及克隆株QB-Tn-Ⅰ和克隆株QB-Tn-Ⅴ在有血清培养基TNM-FH中的群体倍增时间分别为27.4 h、28.0 h和27.0 h,在Sf-900Ⅲ中的倍增时间分别为24.1 h、26.1 h和25.4 h,克隆株QB-Tn-Ⅴ在EX-CELL 420中的群体倍增时间为28.8 h。QB-Tn9-4s及其克隆株QB-Tn-Ⅰ和QB-Tn-Ⅴ在Sf-900Ⅲ中细胞最大密度分别是TNM-FH中的1.8、1.7和1.7倍,而在EX-CELL 420中的细胞最大密度是TNM-FH中的0.9倍。AcMNPV感染各细胞的结果表明,克隆株QB-Tn-Ⅰ在Sf-900Ⅲ中的病毒感染率为80.3%,平均每个细胞病毒多角体(OBs)产量为33.9个,其他细胞的感染率均在90%以上,平均每个细胞多角体产量在81～87个之间,与对照细胞BTI-Tn5B1-4的产量差异不显著。测定了各细胞在有血清培养基和无血清培养基中的重组蛋白表达水平,结果表明,克隆株QB-Tn-Ⅴ在Sf-900Ⅲ中第6 dβ-半乳糖苷酶的表达量最高(3.27×10~4 IU/mL);对比各细胞有血清和无血清培养下第6 dβ-半乳糖苷酶的表达量可以看出,各细胞在无血清培养基Sf-900Ⅲ中的表达量均高于在有血清培养基TNM-FH中的表达量,但克隆株QB-Tn-Ⅴ在无血清培养基EX-CELL 420中的表达量低于其在Sf-900Ⅲ中和在有血清培养基TNM-FH中的表达量。BTI-Tn5B1-4在Sf-900Ⅲ中第7 d分泌型碱性磷酸酶的表达量(4.43 IU/mL)最高,其次为QB-Tn-Ⅴ(4.30 IU/mL)在Sf-900Ⅲ中的表达量。对比各细胞在有血清和无血清培养下第7 d的分泌型碱性磷酸酶表达量可以看出,各细胞在无血清培养基Sf-900Ⅲ中的表达量均显著高于有血清培养基TNM-FH中的表达量,但克隆株QB-Tn-Ⅴ在无血清培养基EX-CELL 420中的表达量低于其在Sf-900Ⅲ中和有血清培养基TNM-FH中的表达量。更多还原

【Abstract】 The development of insect cell lines have been played a significant role in research and application of virology, medicine, agriculture, and biology since the first insect cell line was established in 1962. Lepidopteran insect cell lines are used broadly in insect virology research and recombinant protein expression. With the advent of the baculovirus-insect cell expression system a new exciting application for insect cells in biotechnology was realized. Insect cell lines have become more important as a new bioreactor for the production of virus biopesticides and recombinant proteins. In this study establishment of two new cell lines from two important agricultural pest insects, Helicoverpa armigera and Mythimna separata, identification of the biological characterization, screening of cell clones with higher production of virus and recombinant protein from Trichoplusia ni cell line QB-Tn9-4s were performed. These rerults can provide new resources in studies of apoptosis, biochemical mechanisms and virology, and the basis for potential application and development of insect cell lines.1. Two new cell lines, QB-Ha-E-1 and QB-Ha-E-5, were established from the embryonic tissue of Helicoverpa armigera (Lepidoptera: Noctuidae). The cell lines have been subcultured over 60 passages in TNM-FH medium supplemented with 10% FBS. Each cell line consists of two major morphological types: round cells and spindle-shaped cells. The analyses of DNA amplification fingerprinting (DAF) and random amplified polymorphic DNA (RAPD) showed similar DNA profiles between the two cell lines and the embryonic tissue of Helicoverpa armigera. Two cell lines had a typical lepidopteran chromosomes pattern. The cell doubling times of QB-Ha-E-1 and QB-Ha-E-5 were 63.7 h and 66.9 h at the 30th passage, respectively, and they can be infected by Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) with infection rates of 86.6% and 56.5%, respectively, at 4 d postinfection (p.i.). Approximately 15% of both cell lines were infected by Mamestra brassicae nucleopolyhedrovirus (MbNPV) at 7 d p.i.. Two cell lines had differential responses to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. The results from the fluorescent staining with DAPI and electrophoresis of genomic DNA indicated that QB-Ha-E-5 cell line had typical apoptotic response following AcMNPV infection, but QB-Ha-E-1 cell line with AcMNPV infection rate of 55.3% appeared anti- apoptosis. Actinomycin D could induce apoptosis of the both cell lines at the concentrations of 1.25μg/mL.2. Four cell lines, QB-Ms-E-1, QB-Ms-E-2, QB-Ms-E-3, and QB-Ms-E-4,were established from the embryonic tissue of Mythimna separata (Lepidoptera: Noctuidae). The cell lines have been subcultured over 50 passages in TNM-FH medium supplemented with 10% fetal bovine serum (FBS). Four cell lines consist of two major morphological types: round cells and spindle-shaped cells. The DAF and RAPD analyses indicated similar DNA profiles between the four cell lines and the embryonic tissue of Mythimna separata. The four cell lines showed a typical chromosomes pattern of lepidopteran insect. The cell population doubling times of QB-Ms-E-1, QB-Ms-E-2, QB-Ms-E-3, and QB-Ms-E-4 were 44.9 h, 46.6 h, 46.5 h, and 47.1 h, respectively, and they can be infected by Mythimna separata nucleopolyhedrovirus (MsNPV) with infection rates of 56.6%, 53.6%, 49.3%, and 62.5%, respectively, at 7 d p.i.. Less than 5% of four cell lines were infected by Mamestra brassicae nucleopolyhedrovirus (MbNPV) at 10 d p.i.. Four cell lines were highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with infection rates of over 90% at 4 d p.i..3. Nine cell clones were successfully derived from the established Trichoplusia ni cell line QB-Tn9-4s and designated QB-Tn-Ⅰ, QB-Tn-Ⅱ, QB-Tn-Ⅲ, QB-Tn-Ⅳ, QB-Tn-Ⅴ, QB-Tn-Ⅵ, QB-Tn-Ⅶ, QB-Tn-Ⅷ, and QB-Tn-Ⅸ, respectively. RAPD analysis of the genomic DNA of these clones confirmed their genetic identity as their parent cell line QB-Tn9-4s. Each clone was highly susceptible to AcMNPV virus with infection rates of over 87% cells with production of around 70-87 OBs per cell. The clone QB-Tn-Ⅰproduced 86.7±1.5 OBs per cell, which was higher than BTI-Tn-5B1-4, QB-Tn9-4s and Sf-9. The expression ofβ-galactosidase (β-gal) was determined and compared at 6 d p.i., the clone QB-Tn-Ⅴshowed the highest expression level (3.06×104 IU/mL), closely followed by QB-Tn-Ⅰ(2.70×104 IU/mL), both cells were higher than BTI-Tn-5B1-4, QB-Tn9-4s and Sf-9 cells. The expression of secreted alkaline phosphatase (SEAP) at 7 d p.i. clone QB-Tn-Ⅴwas highest (2.96 IU/mL) among the cell lines, followed by QB-Tn-Ⅰ(2.41 IU/mL), both cells were higher than BTI-Tn-5B1-4, QB-Tn9-4s and Sf-9 cells.4. The cell line QB-Tn9-4s and its clones QB-Tn-Ⅰand QB-Tn-Ⅴwere successfully adapted to a serum-free medium, Sf-900Ш, but one clone QB-Tn-Ⅴwas successfully adapted to a serum-free medium, EX-CELL 420, only. Their morphology and size in serum-free medium differed greatly from serum-containing medium, the results of growth kinetics showed that their cell population doubling times in Sf-900Шwere shorter than the cultures in serum-containing medium TNM-FH, the densities of all cells in Sf-900Шwere higher also than the cultures in serum-containing medium. But the clone QB-Tn-Ⅴgrew slower in EX-CELL 420 medium, its cell population doubling time was longer, and the density was lower than in serum-containing medium. The cell population doubling times of QB-Tn9-4s and its clones QB-Tn-Ⅰand QB-Tn-Ⅴin serum-containing medium were 27.4 h, 28.0 h, and 27.0 h, respectively, and in Sf-900ШSFM were 24.1 h, 26.1 h, 25.4 h, respectively, the doubling times of QB-Tn-Ⅴin EX-CELL 420 medium was 28.8 h. The maximum densities of QB-Tn9-4s and its clones QB-Tn-Ⅰand QB-Tn-Ⅴin Sf-900ШSFM were higher than in serum-containing medium (TNM-FH) by factors of 1.8, 1.8, and 1.7-fold, respectively.The virus infection rate of QB-Tn-Ⅰwas 80.3% with production of 33.9 OBs per cell in Sf-900ШSFM, the remainder of the cell lines were highly susceptible to AcMNPV with an infection rates of over 90% cells with production of around 81-87 OBs per cell, no significant difference were observed between in both Sf-900ШSFM and serum-containing medium TNM-FH.The expression ofβ-galactosidase (β-gal) of QB-Tn9-4s, QB-Tn-Ⅰ, QB-Tn-Ⅴand BTI-Tn5B1-4 in Sf-900ШSFM medium was higher than in serum-containing cultures, the clone QB-Tn-Ⅴexpressed the highest level (3.27×104 IU/mL) among these cell lines in Sf-900ШSFM medium at 6 d p.i.. The expression of secreted alkaline phosphatase (SEAP) of QB-Tn9-4s, QB-Tn-Ⅰ, QB-Tn-Ⅴand BTI-Tn5B1-4 in Sf-900ШSFM medium was higher than in serum-containing cultures also, BTI-Tn5B1-4 showed the highest expression level (4.43 IU/mL) in Sf-900ШSFM medium at 7 d p.i., closely followed by QB-Tn-Ⅴ(4.30 IU/mL) in Sf-900ШSFM medium. Whereas the expression levels ofβ-gal and SEAP of QB-Tn-Ⅴin EX-CELL 420 medium were lower than in Sf-900ШSFM and serum-containing medium TNM-FH.更多还原