【作者】 王晶；

【导师】 郭华荣；

【作者基本信息】 中国海洋大学 ， 生物工程（专业学位）， 2014， 硕士

【摘要】 尽管国内外学者们在对虾细胞建系上进行了大量的探索和不懈的努力，但对虾细胞培养技术目前仍处于原代培养水平，永生性细胞系一直未成功建立。而对虾永生性细胞系的建立对于研究对虾病毒的致病机理和制备病毒疫苗等具有重要科学意义。在哺乳动物细胞中，致癌基因的过表达可成功实现细胞的永生性转化。翻译控制肿瘤蛋白（TCTP）是一种重要的细胞生长相关蛋白，具有促进细胞生长、抗凋亡和重编程作用。因此，本文拟克隆凡纳滨对虾的TCTP基因，构建真核表达载体，并通过脂质体法将其转染到对虾原代培养细胞中，过表达，分析其在对虾细胞的永生性转化中的作用，为对虾细胞的永生性转化奠定技术基础。本文首先通过RT-PCR技术成功克隆得到凡纳滨对虾TCTP基因编码框的cDNA全长，长度为507bp，编码168个氨基酸。以报告质粒pCX-eGFP为模板，PCR扩增得到加强型绿色荧光蛋白（eGFP）基因编码框。然后，以BamHI和EcoRI双酶切将TCTP基因定向插入真核表达质粒pcDNA3.1/V5-HisA，构建得到pcDNA3.1-TCTP重组质粒；以KpnI和BamHI双酶切将eGFP基因定向插入到pcDNA3.1-TCTP重组质粒，构建得到eGFP融合蛋白表达载体质粒pcDNA3.1-eGFP/TCTP。通过脂质体法将融合蛋白表达载体质粒pcDNA3.1-eGFP/TCTP转染到哺乳动物细胞（Plat-GP）中，24小时后在荧光显微镜下可观察到明显的绿色荧光表达，表明所构建表达质粒在Plat-GP细胞中可有效表达。但是，通过脂质体法将该表达质粒转染到凡纳滨对虾类淋巴组织的原代培养细胞中，在荧光显微镜下却观察不到绿色荧光，表明该表达质粒在对虾细胞中不能有效表达。提取96小时转染后对虾原代培养细胞的基因组DNA和总RNA，以eGFP特异性引物为引物，PCR检测eGFP基因的导入和表达情况。发现，以基因组DNA为模板可以扩增出eGFP特异性片段，但是以总RNA反转录得到的cDNA为模板却不能扩增出eGFP特异性片段。这表明，所转染表达质粒pcDNA3.1-eGFP/TCTP已成功导入对虾细胞，但没有成功转录，因而导致所转染对虾细胞中没有绿色荧光表达。表达质粒pcDNA3.1-eGFP/TCTP的启动子为细胞肥大病毒启动子（CMV），该表达质粒的以上转染实验表明，CMV在哺乳动物细胞中为强启动子，但是在对虾细胞中却不能有效驱动基因的表达。因此，本文随后又克隆了凡纳滨对虾TCTP基因启动子（Ptctp），并将其插入表达质粒pcDNA3.1-eGFP/TCTP中，分析了该启动子在对虾细胞中的转录活性。所克隆启动子Ptctp的长度为605bp，位于TCTP基因编码区上游-13至-617bp处，通过KpnI和BamHI双酶切将该启动子插入载体pcDNA3.1-eGFP/TCTP中CMV之后，eGFP基因之前，构建得到表达载体pcDNA3.1-Ptctp-eGFP/TCTP。然后通过脂质体法将该表达质粒转染到凡纳滨对虾类淋巴组织原代培养细胞中，24小时后在荧光显微镜下观察到明显的绿色荧光。以上结果表明，凡纳滨对虾TCTP基因启动子可有效启动外源基因在对虾细胞中的转录和表达。提取96h转染后对虾原代培养细胞的基因组DNA和总RNA，以eGFP特异性引物为引物，PCR检测eGFP基因的导入和表达情况。发现均可扩增得到eGFP特异性片段，在DNA和RNA水平上进一步验证了荧光观察的结果。最后，为避免融合蛋白中eGFP对TCTP生物学功能的影响，本文又构建了含有对虾TCTP启动子Ptctp但不含eGFP的非融合蛋白表达质粒pcDNA3.1-Ptctp-TCTP。并将两种表达质粒pcDNA3.1-Ptctp-TCTP和pcDNA3.1-Ptctp-eGFP/TCTP分别通过脂质体法转染到凡纳滨对虾类淋巴组织原代培养细胞中，过表达，分析其在对虾细胞永生性转化中的作用。结果显示，转染了以上2种表达质粒的对虾细胞的生长状态明显好于未转染对虾细胞，表现为细胞存活率变大，形状相对粗短；而两个不同表达质粒转染细胞之间的细胞形态和存活率未呈现明显的差异，表明eGFP的存在未对TCTP的折叠和构象产生明显的影响。转染TCTP基因的对虾细胞以良好的生长状态可维持40天以上，但是由于染菌的问题，未能最终得到永生性转化对虾细胞。以上研究结果，为今后开展对虾细胞的永生性转化研究以及对虾功能基因研究奠定了技术基础。更多还原

【Abstract】 Although many efforts have been done in the shrimp cell culture, up to date, noimmortalized shrimp cell line has been successfully established and only primaryshrimp cell culture can be obtained and maintained successfully. Continuous shrimpcell line can provide us a useful tool for the study of the biology and pathogenicmechanism of shrimp virus as well as for the preparation of shrimp virus vaccine. Ithas been reported that over-expression of exogenous oncogenes in mammalian cellscan successfully induce the immortalization transformation of the cells and establish acontinuous cell line. Translationally controlled tumor protein (TCTP) is reported asgrowth-related protein and plays a key role in several cellular processes including cellgrowth, apoptosis and cell reprogramming. Therefore, the main purposes of this studyare to clone the TCTP gene from Litopenaeus vannamei and construct the eukaryoticexpression vector, and to analyze the role of over-expressed TCTP on theimmortalization transformation of shrimp primary cell culture by lipofection method.The obtained data will lay a technical foundation for the immortalizationtransformation of shrimp primary cells.In the present study, we firstly successfully cloned the full-length cDNA of theopen reading frame (ORF) of TCTP gene from Litopenaeus vannamei by RT-PCR,named as Lv-TCTP. The obtained Lv-TCTP fragment is507bp in size, encoding168amino acids. Second, using the plasmid pCX-eGFP as a template, the ORF ofenhanced green fluorescent protein (eGFP) was amplified by PCR. Then Lv-TCTPgene was directional inserted into the eukaryotic expression vectorpcDNA3.1-V5/HisA flanking with BamHI and EcoRI to construct pcDNA3.1-TCTPrecombinant plasmid, followed by the directional insertion of eGFP gene into the recombinant plasmid pcDNA3.1-TCTP flanking with KpnI and BamHI to constructthe eGFP fusion protein expression vector of pcDNA3.1-eGFP/TCTP.The fusion protein expression vector plasmid pcDNA3.1-eGFP/TCTP was thentransformed into the mammalian cells (Plat-GP) by lipofection method. Twenty fourhours later, obvious green fluorescence was observed in the transformed Plat-GP cellsunder the inverted fluorescence microscope, indicating that the constructed expressionplasmid of pcDNA3.1-eGFP/TCTP can be expressed effectively in plat-GP cells.When the same expression plasmid was transformed into the primary culture cellsderived from the lymphoid-like tissues (Oka organ) of shrimp Litopenaeus vannameiby the same lipofection method, however, no green fluorescence was observed in thetransformed shrimp cells, showing that the above-mentioned expression plasmid wasnot be expressed effectively in shrimp cells. The genomic DNA and total RNA werethen extracted from the transformed shrimp primary cells at96hours aftertransfection, respectively, and PCR amplification was performed to investigate theintroduction and transcription of foreign eGFP gene in the shrimp cells usingeGFP-specific primers. The obtained results showed that eGFP-specific fragmentcould be amplified using genome DNA as a template, but not using the cDNAderived from total RNA as a template, indicating that the expression plasmidpcDNA3.1-eGFP/TCTP had been successfully introduced into the shrimp cells, butnot been transcribed successfully. This is in agreement with the results of greenfluorescence observation in the transfected shrimp cells.The promoter of the expression plasmid pcDNA3.1-eGFP/TCTP iscytomegalovirus promoter (CMV), the results from the transformation experimentsusing this expression plasmid showed that the activity of CMV promoter is strong inmammalian cells, but weak in shrimp cells. Therefore, we cloned the promoter regionof TCTP gene from Litopenaeus vannamei (Ptctp) which was605bp in size, located inthe3’UTR of Lv-TCTP gene (at the position from-3to-617). Then the Ptctpwasdirectionally inserted into the expression vector of pcDNA3.1-eGFP/TCTP at theposition between CMV and eGFP flanking with BamHI and EcoRI and the expressionvector plasmid pcDNA-Ptctp-eGFP/TCTP was successfully construct. Then the expression vector plasmid of pcDNA-Ptctp-eGFP/TCTP was transformed into thecultured shrimp primary cells by lipofection method to analyze the transcriptionalactivity of Ptctpin shrimp cells. After24hours, green fluorescence signal could bedetected under the fluorescence microscope. The obtained results showed that, thePtctp promoter from Litopenaeus vannamei can effectively activate the transcriptionand expression of exogenous genes in shrimp cells. And also, the genomic DNA andtotal RNA were then extracted from the transformed shrimp primary cells at96hoursafter transfection, respectively, and PCR amplification was performed to investigatethe introduction and transcription of foreign eGFP gene in the shrimp cells usingeGFP-specific primers. The obtained results showed that eGFP-specific fragmentscould be amplified from the above-mentioned two templates, indicating that theexpression plasmid pcDNA3.1-eGFP/TCTP had been successfully introduced into andtranscribed in the shrimp cells. This is also in agreement with the results of greenfluorescence observation in the transformed shrimp cells.In order to examine the effect of fused eGFP protein on the biological function ofTCTP, a non-fusion protein expression vector pcDNA3.1-Ptctp-TCTP, whichexpressed TCTP alone, containing shrimp promoter Ptctpbut not eGFP gene, wasconstructed. Plasmids of pcDNA-Ptctp-eGFP/TCTP and pcDNA3.1-Ptctp-TCTP weretransformed into the primary cultured shrimp lymphoid cells respectively and theeffects of over-expressed eGFP/TCTP and TCTP on the immortalizationtransformation of shrimp cells were compared. The obtained results indicated that,compared with the control group, the shrimp cells transformed with theabove-mentioned two expression plasmids exhibited better cellular status with highercell survival rate and relative stubby shape, indicating an effect on cell transformation;but no obvious difference in cellular morphology and survival rate was observed inthe shrimp cells transformed with pcDNA-Ptctp-eGFP/TCTP andpcDNA3.1-Ptctp-TCTP, inferring that the fused eGFP did not affect the fold andconformation of TCTP. And also, the transformed shrimp cells could be maintained over40days in a healthy status. However, we failed to get the immortalized shrimpcells due to microorganism contamination.Taken together, all the obtained data in the present study have laid a technicalfoundation for the future work on the immortalization transformation of shrimpprimary cells and biological functions of shrimp genes.更多还原