【作者】 鲁维飞；

【导师】 王艳玲； 杨国宇；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2013， 博士

【摘要】 Rspo基因家族是新近发现的一个基因家族,包括4个家族成员,通过与胞膜蛋白FZD和LRP5/6相互作用而激活Wnt/β-catenin (β连环蛋白)信号途径,对细胞增殖分化、个体发育以及肿瘤的生长转移等进行调控。Rspo基因家族分布广泛,在不同物种中具有很高的保守性。Rspo基因尤其是Rspo3基因,通过基因敲除手段研究其功能的时候,敲除了Rspo3基因的小鼠不能正常发育成活体,揭示了Rspo3基因在动物个体发育中起着极为关键的作用,但同时为进一步研究基因功能带来很大的障碍。通过对猪Rspo3基因的研究,对猪的新品种选育、疾病防治等都有重大的意义。本试验利用PCR的方法,克隆得到了猪Rspo3基因,检测了该基因的组织表达分布,并利用pET28a作为载体成功在大肠杆菌BL21中得到表达。为了进一步在真核细胞内研究Rspo3基因功能,构建了不同启动子组合的真核表达载体,并对不同载体的表达效率进行了筛选。1.利用电子克隆技术,设计了猪Rspo3基因的扩增引物,以猪的肝脏提取的总RNA为模板,利用RT-PCR成功获取了猪的Rspo3基因,经过测序确认,猪Rspo3的开放阅读框有828bp,编码275个氨基酸,其中包含56个酸性氨基酸和62个碱性氨基酸,蛋白质分子量为31.32KDa,等电点为：9.94,并对猪Rspo3的三维结构进行了预测,蛋白分子三维尺寸为： X：44.486Y：45.211z：47.410(单位：10-10米),属于球状蛋白质。Rspo3基因在猪各个组织表达分布检测表明Rspo3基因除了在脾和脂肪组织中几乎很少表达外,其它常见组织中均有表达,其中在脑和睾丸表达丰度最高；2.将猪Rspo3基因整合到pET28a原核表达载体上,在Rspo3基因前添加了6×His-Tag,便于后期检测和纯化,利用大肠杆菌BL21作为宿主菌,优化后的猪Rspo3基因在大肠杆菌BL21中的表达条件为：温度26℃时,0.5mg/ml的1PTG浓度,诱导时间为2h。经SDS聚丙烯酰胺凝胶电泳和Western blot检测,确认成功表达了融合蛋白,分子量为35.19kDa；3.用酶切连接技术将Rspo3基因与10个不同启动子组合的真核表达载体(P1-P10)进行连接时,Rspo3自身所带的NcoI酶切位点成为正常的酶切连接反应的障碍。利用重叠延伸PCR的方法,先对猪Rspo3基因自身携带的NcoI酶切位点进行了同义突变,测序结果证实了突变获得了成功,除了突变位点,其它序列保持了不变,编码的氨基酸序列没有改变。利用亚克隆的方法,将突变后的Rspo3基因从T载体上NcoI和Nhe1双酶切后与P1-P10相连,经PCR、酶切和测序均验证正确,成功的构建了携带Rspo3基因的10个不同启动子组合的真核表达载体；4.在PK15细胞上的转染试验结果表明,GFP蛋白表达的效率最高的P5质粒,相对于对照组转染peGFP-N1质粒提高了458.9%,到第14天还能观测到GFP荧光,P6质粒的GFP表达效率仅次于P5质粒。用Q-PCR检测Rspo3效率P5质粒和P6质粒显著高于其它组,考虑到误差因素和检测目的分子的差异,最合适PK15细胞平台的启动子组合是P5质粒所携带的CMV(Human)+HTLV RU5’(Viral)+CMV(Human)；5.对鸡成纤维细胞DF1、犬肾细胞MDCK和人源的Hela细胞,也进行了不同启动子组合的筛选,对不同细胞转染结果通过荧光显微镜检测、GFP荧光吸光值测定和表达持续时间测定,在DF1细胞和MDCK细胞中表达效率最高的是P6质粒,启动子组合为启动子组合为CMV(Human)+HTLV RU5’(Viral)+Sv40(Viral).而在Hela细胞上表达效率最高的是P5质粒,启动子组合为CMV(Human)+HTLV RU5’(Viral)+CMV(Human)。综上所述,本试验成功的克隆得到猪Rspo3基因,首次对该基因的不同组织表达分布进行了检测,在大肠杆菌BL21也成功获得了蛋白表达。构建了携带Rspo3基因不同启动子组合的真核表达载体,并对不同启动子组合的质粒在4种不同的细胞系上表达效率筛选,为进一步研究Rspo3的基因功能,促进其在理论研究及畜牧业中实际应用奠定了基础。更多还原

【Abstract】 Rspo is a newly discovered gene family composed of four gene members (Rspol to Rspo4), which interacts with membrane proteins FZD and LRP5/6to activate Wnt/β-catenin pathway. This interaction is closely associated with cell growth and differentiation, organ development, and tumor metastasis. Rspo gene family is widely distributed in different species with high conservativeness. Among all the Rspo genes, Rspo3is indispensible for individual development of animal, which is indicated by the fact that mice with Rspo3knock-out cannot grow normally to adults. This phenomenon brings obstacles to further study gene functions of Rspo3. Among all the husbandry animals in our country, pig is especially important:studying of Rspo3gene will significantly benefit the new species development and disease control of pigs;In the current study, we used PCR to clone porcine Rspo3gene and successfully expressed this gene in E.Coli BL21strain using pET28a as the vector. To further study the function of Rspo3in eukaryotic cells, eukaryote expressing vector with different promoters was established and screened for gene expression efficiency.1.Using the technology of electronic cloning, the amplification primers for porcine Rspo3was designed. RT-PCR was used successfully to acquire the porcine Rspo3gene with total RNA from pig liver as the template. With confirmation from gene sequencing, the open reading frame of porcine Rspo3has828base pairs and encodes275amino acids, including56acid amino acids and62basic amino acids, and the molecular weight of its expressed protein is31.32kDa with a PI of9.94. The three-dimensional structure of RSPO3was also predicted by us. The protein sizes are: X:44.486Y:45.211Z:47.410(unit:angstrom) and it belongs to globular proteins. The distribution study of Rspo3indicated that it is universally distributed in every organs except spleen and fats, with highest expression level in brain and testicles.2.Porcine Rspo3gene was integrated in a pET28a prokaryotic expression vector, and6X Histine-tag was added for further detection and purification without compromising the folding and immunogenicity of RSPO3. With E.coli BL21as the recipient bacteria, the optimized expression condition was at26℃and0.5mg/ml of IPTG, with induction time of2h. Using SDS-PAGE and Western Blot, we confirmed the expression of fusion protein with the molecular weight of 35.19kDa.3.Rspo3gene was ligated with eukaryotic expression vectors with ten different promoters (P1-P10), however, the NcoI site inside Rspo3served as the major obstacle of normal enzyme-cleavage based ligation. Using SOE-PCR, we conducted samesense mutation of NcoI and confirmed the mutation by gene sequencing:the sequence is unchanged except the NcoI region, and the encoding amino acid sequence is exactly the same to the non-mutant. Mutated Rspo3gene was ligated with P1-P10after double enzyme cleavage by NcoI and NheI from T vector. With the confirmation of PCR, enzyme cleavage, and gene sequencing, successful eukaryotic expression vectors carrying Rspo3gene and10different promoters were established.4.The transfection experimental results in PK15cells demonstrated that P5plasmid has the highest expression efficiency for GFP expression:458.9%increase was observed compared with control group using peGFP-N1plasmid, and GFP fluorescence was observable even on day14post-transfection.P6plasmid has the second highest efficiency for GFP expression. With Q-PCR detection, the most suitable promoter combination for PK15cells were CMV(Human)+HTLV RU5’(Viral)+CMV(Human) carried by P5plasmid.5.Similar promoter screening was also conducted in chicken fibroblast DF1, canine kidney cell MDCK, and human ovarian cancer cell line Hela. By fluorescence microscopy detection, fluorscence absorbance test and expression lasting time measurement, we confirmed that P6plasmid has the highest expression efficiency in DF1and MDCK cells, with the promoter combinations of CMV(Human)+HTLV RU5’(Viral)+Sv40(Viral). At the same time, P5plasmid has the highest expression efficiency in Hela cells, with the promoter combinations of CMV(Human)+HTLV RU5’(Viral)+CMV(Human).In summary, we successfully cloned the porcine Rspo3gene and evaluated its distribution in different organs. The protein was successfully acquired in E.Coli BL21strain. Eukaryotic expressing vectors with different promoter-combination were established and screened in4different cell lines for expression efficiency. These studies provided the foundation for further research of Rspo3gene functions and clarification of its applicability in husbandry animals.更多还原