Expression of Two Reporter Genes in Clonal Cell Line RIRI-PaDe-2-C6 Developed from <i>Papilio demoleus</i> Linnaeus (Lepidoptera: Papilionidae)

LIU Zhi-gang; DING Wei-feng; SUN Na; ZHANG Xin; LI Xian; XIE Shi-cong; FENG Ying

doi:10.13275/j.cnki.lykxyj.2018.04.005

Volume 31 Issue 4

Jul. 2019

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Expression of Two Reporter Genes in Clonal Cell Line RIRI-PaDe-2-C6 Developed from Papilio demoleus Linnaeus (Lepidoptera: Papilionidae)

Research Institute of Resource Insects, Chinese Academy of Forestry, Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Kunming 650224, Yunnan, China

Corresponding author: FENG Ying, rirify@139.com
Received Date: 2018-01-27

Abstract

Objective In the previous research, a monoclonal cell line RIRI-PaDe-2-C6 was established from Papilio demoleus cell line RIRI-PaDe-2 and it was found that the two cell lines could be infected by wild-type Autographa californica multiple nucleopolyhedrosis virus (AcMNPV). Especially, RIRI-PaDe-2-C6 was susceptible to AcMNPV and exhibited a higher production of AcMNPV polyhedral per infected cell averagely compared to the parent RIRI-PaDe-2 cells. The aim of this study is to further understand the characteristics of RIRI-PaDe-2-C6 in expressing exogenous genes. Method The Bac-to-Bac baculovirus expression system was used to establish recombinant baculovirus carrying β-galactosidase gene and secreted alkaline phosphatase (SEAP) gene. The RIRI-PaDe-2-C6 was infected with recombinant virus. The expression levels of the two recombinant proteins were detected at 24, 48, 72, 96, 120, 144, and 168 hours after infection and compared with RIRI-PaDe-2 cells. The methods of cells morphology analysis, growth analysis and chromosome analysis were used to obtain the biological characteristics of RIRI-PaDe-2-C6. Result RIRI-PaDe-2-C6 and RIRI-PaDe-2 could be infected by recombinant baculovirus. The expression of β-galactosidase (β-Gal) was significantly higher than that of RIRI-PaDe-2 (P < 0.05), but no significant difference in expression level of secreted alkaline phosphatase (SEAP) was observed between RIRI-PaDe-2-C6 and RIRI-PaDe-2 (P>0.05). All cells were spindle-shaped in RIRI-PaDe-2-C6 which was more homogeneous than RIRI-PaDe-2. The cell population doubling time of RIRI-PaDe-2-C6 was 94.94 hours which was longer than those of RIRI-PaDe-2 (67.42 hous). The averages chromosome numbers of RIRI-PaDe-2-C6 was 52.26±30.48 which was significant different from that of RIRI-PaDe-2 (73.19±24.27). Conclusion The differences in expressing exogenous genes and biological characteristics are significant between clone cell line RIRI-PaDe-2-C6 and its parent cell line RIRI-PaDe-2.
- Papilio demoleus,
- single cell cloning,
- insect cell line,
- β-galactosidase,
- secreted alkaline phosphatase,
- recombinant baculovirus

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昆虫细胞-杆状病毒表达系统(Insect Cell-Baculovirus Expression Victor System，IC-BEVS)是近年来发展迅速的真核细胞表达系统，具有完备的翻译后加工修饰能力、生物安全性高、对外源基因容量大的特点，能够高效表达外源基因^[1-2]。在基因工程、蛋白工程、工程疫苗、药物研究和开发、生物农药等领域具有重要的应用价值^[3-4]。据统计，已有上千种外源基因在IC-BEVS中得到了成功表达，且有95%的外源重组蛋白能够被正确的转译、加工以及修饰成具有生物活性的产物^[5]。IC-BEVS常用的宿主细胞来自鳞翅目和双翅目昆虫细胞系，特别是来源鳞翅目蛾类的昆虫细胞系，如草地夜蛾(Spodoptera frugiperda)细胞系Sf21^[6]及其克隆细胞株Sf9^[7]，粉纹夜蛾(Trichoplusia ni)细胞系Tn5^[8]及其高产克隆株High Five^[9]，被广泛用于IC-BEVS^[10]；但目前用于外源蛋白表达的昆虫细胞株数量有限，且随着这些细胞系传代次数的增加，细胞开始出现一些退化，如外源蛋白产量减少，细胞增殖速率降低等^[11-12]。因此，建立和筛选具有高效表达外源基因潜力的新细胞系具有重要的意义。

前期研究中，项目组建立了4个来源于鳞翅目凤蝶科达摩凤蝶(Papilio demoleus)新孵幼虫的细胞系(RIRI-PaDe-1、RIRI-PaDe-2、RIRI-PaDe-3、RIRI-PaDe-4)^[13]。观察发现，这4个细胞系均为贴壁细胞，其细胞组成、形态大小都存在不同程度的差异。使用野生型苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrosis virus, AcMNPV)侵染4个细胞系发现均能表现出病理学特征，且不同个体对AcMNPV的敏感性不同，如有的细胞受病毒侵染后能够产生大量包涵体(occlusion bodies, OBs)，但有的细胞个体却没有明显病变特征。笔者使用半固体培养法结合显微操作系统对这4个细胞系进行了单细胞克隆操作，共获得61个单细胞克隆株，使用AcMNPV侵染这些克隆株并统计细胞产生OBs数量，发现其中1个克隆株RIRI-PaDe-2-C6平均每个细胞产生的OBs数量较原细胞系RIRI-PaDe-2有显著增高。AcMNPV具有较广的宿主范围，常作为外源基因的载体在蛾类昆虫细胞中表达外源蛋白，为研究以AcMNPV为载体的重组病毒能否在达摩凤蝶细胞系中的表达，在本研究中，笔者利用Bac-to-Bac表达系统分别构建了携带β-半乳糖苷酶(β-galactosidase, β-Gal)和分泌型碱性磷酸酶(secreted alkaline phosphatase, SEAP)基因的2个重组杆状病毒(AcMNPV-Gal和AcMNPV-SEAP)，这2种基因是常用的报告基因，其检测手段较为成熟。用这2种重组病毒侵染RIRI-PaDe-2细胞系和克隆株RIRI-PaDe-2-C6，检测宿主细胞对这2个报告基因的表达水平；同时对RIRI-PaDe-2-C6的细胞形态、倍增时间、染色体数目等基础生物学特性进行研究，为后续利用提供依据。

1. 材料与方法

1.1. 供试细胞

达摩凤蝶细胞系RIRI-PaDe-2及其单细胞克隆株RIRI-PaDe-2-C6由中国林业科学研究院资源昆虫研究所建立并保存。培养基为Zhang等^[14]根据经典Grace昆虫培养基培养经过改良获得的Grace⁺培养基^[14-15]，培养时添加20%的HyClone胎牛血清(货号SV30087.02)以促进细胞生长。培养条件为27℃避光恒温培养。

1.2. 供试病毒

外源基因分别来自表 1所列的2个商业质粒产品，通过PCR扩增获得目的片段并添加酶切位点。使用赛默飞世尔科技有限公司(Thermo Fisher Scientific Inc.)的Bac-to-Bac杆状病毒表达系统(货号10359-016)构建重组杆状病毒AcMNPV-Gal和AcMNPV-SEAP，并取P2代病毒贮备液用于试验。

质粒Plasmid	引物和酶切位点Primers and restriction sites
pSV-β-Galactosidase Control Vector (Promega, Cat. E1081, GenBank: X65335)	5’-GGCGAATTCGTCGTTTTACAACGTCGTGA-3’ 5’-GACAAGCTTATTTTTGACACCAGACCA-3’	(EcoRI) (HindⅢ)
pSEAP2-Control Vector (Clontech, Cat. 631717, GenBank: U89938)	5’-ATGCTGCTGCTGCTGCTGCT-3’ 5’-GGATCCTGTCTGCTCGAAGCGGCCGG-3’	(BamHⅠ)

Table 1. Two reported gene sources

1.3. 重组蛋白表达水平检测

1.3.1. 接种病毒

取指数生长期的供试细胞，密度稀释至1×10⁵个·mL^-1，接种于24孔细胞培养板(Corning，货号3524)，每孔1 mL细胞悬液。每个细胞系接种4孔，其中，3孔作为平行对照，1孔作为阴性对照。使用Clontech Laboratories公司的BacPAK杆状病毒快速滴度检测试剂盒(货号631406)测定病毒滴度，稀释病毒液，使每个细胞感染的病毒数达到5(MOI=5)。培养板置于摇床(杭州米欧仪器有限公司，型号GS-20)避光轻摇2 h，使细胞充分吸收病毒；之后小心将每孔中培养基全部吸出，注意避免吸起细胞。然后在每孔中补加2 mL不含病毒的新鲜培养基，置于27℃恒温避光培养。

1.3.2. 重组蛋白酶活性检测

接种重组病毒后24 h进行第1次检测，之后每隔24 h检测1次，共检测7次。取接种重组病毒的24孔细胞培养板置于摇板机(DragonLab，型号MX-M)上轻摇15 min，使细胞分泌的重组蛋白均匀分散到细胞液中。每孔取30 μL细胞上清液作为待测样品，并向原孔补加30 μL新鲜培养基，使每孔培养液总体积保持不变。使用β-半乳糖苷酶检测试剂盒((Promega，货号E2000)测定样品中β-Gal的酶活性。使用Phospha-Light试剂盒(Applied Biosystems，货号T1017)检测样品中SEAP的酶活性。检测使用的多功能酶标仪型号是赛默飞世尔科技有限公司的Varioskan Flash。

1.3.3. 酶活性计算

每个细胞系3个平行对照与1个空白对照的酶活性记为S1、S2、S3以及C，计算S1-C、S2-C以及S3-C的平均值(Mean±Std.)作为重组蛋白在待测细胞中的表达量。使用Excel(Ver. 1710)以病毒侵染时间为横坐标，重组蛋白酶活性为纵坐标绘制直方图。使用SPSS(Ver. 19)对供试细胞系表达的酶活性进行t检验，确定它们对重组蛋白的表达水平是否存在显著差异(P＜0.05)。

1.4. 细胞生物学特性研究

1.4.1. 细胞形态学观察

使用Olympus Ⅸ71倒置生物显微镜进行观察并拍照，使用Adobe Photoshop CS6 Extended中的“计数工具”分别对每张照片中不同形态的细胞(分为圆形、梭形及多边形)进行计数统计，计算各细胞系不同形态细胞所占比例的平均值和标准差。使用Olympus CellSens图像分析软件对细胞系中圆形细胞的直径进行测量(梭形细胞计算长轴)，每个细胞类型统计的样本数(n)不少于100个细胞，并计算其平均值和标准差。

1.4.2. 细胞生长曲线绘制及倍增时间计算

取指数生长期的供试细胞，密度稀释至2×10⁵个·mL^-1，接种于96孔细胞培养板(Corning，货号3997)，每孔100 μL细胞悬液。每个细胞系接种4孔，作为平行对照，共接种7板，置于27℃恒温避光培养。每隔24 h取1板进行细胞活力检测。使用普洛麦格生物产品有限公司的Cell Titer 96 Aqueous单溶液细胞增殖检测试剂盒(货号G3582)进行细胞活力检测，连续检测7天。使用Excel(Ver. 1710)以检测时间作为横坐标，活细胞吸光度值作为纵坐标绘制细胞密度随培养时间变化的生长曲线图，根据曲线走势确定细胞进入指数生长期阶段，再根据刘冰洁等^[16]所述公式计算细胞群体倍增时间。

1.4.3. 核型分析

取指数生长期的供试细胞，根据Mitsuhashi^[17]所述方法进行细胞固定制片，其中，低渗溶液KCl的浓度为0.6%。使用Nikon ECLIPSE E800生物显微镜进行观察并拍照，挑选染色体清晰且分散比较好的细胞样本进行染色体计数(样本数≥100)，根据Yeh等^[18]所述方法进行统计分析。使用Photoshop(Ver. 13.0.1)中的“计数工具”对各细胞系的染色体条数进行统计，每个细胞系统计的样本数(n)不少于100个细胞。使用SPSS(Ver. 19)对供试细胞系染色数量进行频数分析并绘制直方图以及正态曲线。使用SPSS(Ver. 19)对供试细胞系染色体数量进行t检验，确定它们是否存在显著差异(P＜0.05)。

3. 讨论

虽然经过30年的发展，IC-BEVS的各种技术不断进步，表达了多种蛋白，表达产量也得到了提高。但也有一些因素制约了IC-BEVS的进一步发展和应用，如昆虫细胞内N端糖基化途径与哺乳动物细胞不同。昆虫细胞内缺乏哺乳动物细胞所具备的N-乙酰葡萄糖氨转移酶Ⅱ、半乳糖基转移酶/N-乙酰氨基半乳糖转移酶、α-2, 3-唾液酸转移酶和α-2, 6-唾液酸转移酶等延长N-糖链的糖基转移酶^[19-20]，导致表达的部分哺乳动物蛋白无活性，限制了IC-BEVS在生产药用蛋白领域的应用。Palomares等^[21]对黑脉金斑蝶(Danaus plexippus)细胞系DpN1的糖基化研究发现，DpN1表达的重组蛋白糖基化程度较粉纹夜蛾(T. ni)细胞系Tn5B1-4更加完全，并认为它可以代替常见工程细胞系进行复杂糖基化蛋白的生产，因此，建立更多的蝶类细胞系和克隆株，开展外源基因在蝶类细胞中的表达特性研究很有意义。作为新建蝶类细胞系，达摩凤蝶细胞中外源蛋白的糖基化修饰水平尚未可知，本研究对达摩凤蝶细胞系RIRI-PaDe-2及其克隆株RIRI-PaDe-2-C6的外源蛋白表达特性进行研究，希望获得外源基因表达水平高的细胞系，为下一步达摩凤蝶细胞系的糖基化研究奠定基础。

从结果可以看出：通过单细胞克隆技术确实能够获得较原细胞系RIRI-PaDe-2表达水平更高的克隆株RIRI-PaDe-2-C6，但与IC-BEVS常用的宿主细胞系Sf 9相比其表达水平仍较低。相同试验条件下Sf 9表达重组β-Gal和SEAP的最高酶活性分别为(4.132±0.942) U·mL^-1和(4.196±0.923)×10^-2 U·mL^-1，较RIRI-PaDe-2-C6分别高出19和35倍，所以RIRI-PaDe-2-C6还无法作为IC-BEVS的工程细胞株用于生产。笔者推测造成这种结果的主要原因是达摩凤蝶并不是AcMNPV的天然宿主。自然界中杆状病毒具有高度的宿主特异性，一种病毒往往只能感染亲缘性较近的少数几种昆虫，但这种宿主特异性是相对的，每种病毒有一个原始宿主，但也往往可以感染若干替代宿主。一般来说，替代宿主对病毒的感受性较原始宿主低^[22]。AcMNPV的宿主域较广，能感染多种鳞翅目昆虫。正是这个原因使AcMNPV成为IC-BEVS最常用的载体而被用于构建重组病毒，在其敏感型宿主细胞内能够大量表达外源蛋白。由此可见，重组病毒表达载体的选择是影响外源蛋白表达水平的重要因素。目前已知有多种方法可用于提高外源蛋白在IC-BEVS的表达水平，如共表达伴侣蛋白^[23-24]、开发转基因细胞^[25]、延长转染后宿主细胞寿命以及延缓重组蛋白降解^[26]等。这些基于细胞和病毒改造的生物技术为外源蛋白在IC-BEVS中高效表达提供了可能，但达摩凤蝶细胞系及其克隆株能否通过上述方法提高外源基因表达水平还待进一步研究。

4. 结论

综上所述，达摩凤蝶细胞细胞系RIRI-PaDe-2及其克隆株RIRI-PaDe-2-C6均能表达β-半乳糖苷酶和分泌型碱性磷酸酶，通过单细胞克隆方法获得的克隆株RIRI-PaDe-2-C6对外源蛋白的表达水平与原细胞系RIRI-PaDe-2有一定区别，特别是对重组β-Gal的表达水平显著高于原细胞系RIRI-PaDe-2(P < 0.05)。在生物学特性方面，克隆株RIRI-PaDe-2-C6与原细胞系RIRI-PaDe-2-C6也存在明显差异，研究结果为后续改进驯化宿主细胞系和外源基因表达提供了可能和基础。

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Expression of Two Reporter Genes in Clonal Cell Line RIRI-PaDe-2-C6 Developed from Papilio demoleus Linnaeus (Lepidoptera: Papilionidae)