【作者】 刘艳；

【导师】 孟红；

【作者基本信息】 济南大学 ， 基础医学， 2015， 硕士

【摘要】 目的:本课题采用生物信息学方法、选用不同表达载体克隆表达EV71 2C蛋白,最终获得高纯度、性状均一、可正确折叠的可溶性蛋白,为蛋白质的晶体筛选奠定基础。方法:1.EV71生物信息学分析从NCBI数据库下载EV71(200804株)的2C的基因序列和氨基酸序列,采用Protparam软件、Protean软件、Top Pred 2服务器、Pfam软件、SMART软件分别对EV71 2C蛋白的基本理化性质、二级结构、疏水性、核心结构域等信息进行初步预测,根据氨基酸的疏水性分析结果和功能结构域分布,设计6个不同长度的氨基酸片段。2.重组EV71 2C截短片段基因克隆以EV71全长基因组A12质粒为模板,设计引物:2C-91aa-Bam HI-5、2C-118aa-Bam HI-5、2C-256aa-Xho I-3T、2C-296aa-Xho I-3T、2C-316aa-Xho I-3T。对上述截取的6个2C片段进行PCR扩增,将扩增后获得的DNA片段分别连接至带有His标签的p Gl01载体和带有MBP促溶标签的psj8载体上,构建12个重组质粒:6个包含p Gl01载体-2C截短片段的重组质粒和6个psj8载体-2C截短片段的重组质粒。酶切鉴定、测序正确后,将重组质粒转化至E.coli BL21(DE3)后获得工程菌。3.EV71 2C截短蛋白表达纯化、晶体普筛将上述工程菌液进行扩大培养至OD600在0.6-0.8之间,0.6m M浓度IPTG、20℃低温条件下诱导表达16h。收集菌液进行超声破碎,低温高速离心收集上清液。将p Gl01载体-2C截短蛋白的上清液加样到镍离子亲和层析柱上,使用含有咪唑的缓冲液洗脱目的蛋白。将psj8载体-2C截短蛋白的上清液加样到MBP Trap?HP柱,使用含有咪唑和10m M麦芽糖的缓冲液洗脱融合有MBP标签的目的蛋白。采用SDS-PAGE电泳对超声破碎菌液(CE)、高速离心上清(S)、流过镍柱蛋白样品(FL)、重悬缓冲液冲洗镍柱后的蛋白样品(W)、洗脱缓冲液冲洗镍柱后的蛋白样品(ELU)鉴定蛋白表达及可溶性情况。选择CE、ELU条带单一、清晰、量大的可溶psj8载体-2C截短蛋白M1和M4进一步采用凝胶过滤亲和层析柱进行纯化,采用坐滴法对纯化得到的2C蛋白进行晶体普筛。结果:1.EV71 2C截短片段设计根据生物信息学分析结果,本课题共截取了2C蛋白的6个基因片段。片段1为2C的91aa-256aa,包含完整ATP酶结构域和RNA解旋酶结构域N端。片段2为2C的91aa-296aa,包含完整ATP酶结构域、RNA解旋酶结构域N端、AWS domain。片段3为2C的91aa-316aa,包含完整ATP酶结构域、完整RNA解旋酶、AWS domain。片段4为2C的118aa-256aa,包含完整ATP酶结构域和RNA解旋酶中间片段,是最小片段。片段5为2C的118aa-296aa,包含完整ATP酶结构域、RNA解旋酶结构域中间片段和AWS domain。片段6为2C的118aa-316aa,包含完整ATP酶结构域、RNA解旋酶结构域C端、AWS domain。2.重组EV71 2C截短片段基因克隆PCR扩增获得了EV71 2C蛋白6个截短片段,并分别连接到p Gl01载体和psj8载体上,成功构建了p Gl01载体-2C截短片段、psj8载体-2C截短片段共12个重组质粒。鉴定正确的质粒成功转化至E.coli BL21(DE3),获得工程菌。3.EV71 2C截短蛋白表达纯化、晶体普筛SDS-PAGE结果显示:含有p Gl01表达载体的2C蛋白和pjs8载体的2C蛋白在E.coli BL21(DE3)中均有表达,蛋白表达的分子量大小与预期一致。p Gl01载体-2C截短蛋白难溶。相反,带有MBP促溶标签的psj8载体-2C蛋白可溶。凝胶过滤层析结果显示峰值高尖,表明M1和M4蛋白纯度较高、性状均一。晶体普筛尚未发现衍射良好的晶体。结论:1.psj8载体-2C蛋白M1(91aa-256aa)和M4(118aa-256aa)两个片段比其他片段的截短蛋白的性状更加稳定均一。2.MBP促溶标签提高了2C蛋白的可溶性。更多还原

【Abstract】 Purpose: Bio-informatics methods and different expression vectors were used to express the EV71 2C protein. Soluble proteins with high purity, uniform quality and corrected folding were obtained for further crystal screening.Methods:1. Analysis of bioinformatics characteristics: EV71(200804 strain) 2C protein amino acid and gene sequence were downloaded from the NCBI database. Softwares such as Protparam, Protean, Top Pred 2 service, Pfam and SMART were used to analyze the basic characteristics and secondary class structure, hydrophilicity and core domains as well. Six different lengths of fragments were determined and designed according to the analysis of hydrophobic property and distribution of core domain.2. Gene clone of the recombinant 2C truncated fragments of EV71: EV71 full-length genome, A12 plasmid, was used as a template. The corresponding primers were designed as follows: 2C-91aa-Bam HI-5, 2C-118aa-Bam HI-5, 2C-256aa-Xho I-3T, 2C-296aa-Xho I-3T,2C-316aa-Xho I-3T. The six intercepted 2C fragments were amplified and then ligated onto p Gl01 and psj8 vectors, respectively. Twelve recombinant plasmids comprised of p Gl01vector-2C truncated fragments and psj8 vector-2C ones were constructed successfully which named as P1 to P6 and M1 to M6 respectively. Following the identification of enzyme digestion and exact sequencing, the engineering bacteria obtained after transforming into E.Coli BL21(DE3) and inducing by IPTG.3. Expression, purification and crystal screening of EV71 truncated 2C protein: The engineering bacteria were cultivated to the optical density value between 0.6 to 0.8, the0.6m M IPTG was used to induce the plasmid expression at 20℃ for 16 hours. After the bacteria were collected, ultra-sonication and high-speed centrifugation were performed at low temperature to obtain supernatant. The crude extracts of p Gl01-2C truncated proteins were loaded onto the nickel ion affinity chromatography column for a basic purification. The samples were eluted by elution buffer containing imidazole. The crude extracts of psj8-2C ones were loaded onto the MBP Trap?HP column and then eluted by elution buffer containing imidazole and 10 m M maltose. The samples were collected as follows: I. The ultrasonic broken bacteria(CE). II. High speed centrifugal supernatant(S). III. Protein samples flowed through the nickle column(FL). IV. Protein samples washed by suspension buffer(W). V. Protein samples eluted by elution buffer(ELU). SDS-PAGE electrophoresis was used for identifying and analyzing the expression as well as solubility of proteins. The targeted proteins on SDS-PAGE with the single and distinct bands of CE and ELU were figured out as M1 and M4, and then these protein isolates were further purified by gel filtration affinity chromatography. The final purified proteins were screened for crystals growing using sitting drop method.Results:1. The designation of EV71 2C fragments: Six fragments were intercepted according to the bioinformatics analysis. Core domains such as ATPase and RNA helicase were included all truncated ones. AWS domain was also designed in the truncated ones. Unless the above described, some special domains such as N terminals and C terminals including middle amino acid region were distributed among different fragments. For the detailed information please see table 2.2. Heterologous expression of the recombinant 2C truncated fragments of EV71: Six fragments of EV71 2C were amplified by PCR successfully and then ligated onto p Gl01 vector and psj8 vector respectively. Twelve recombinant plasmids comprised of six p Gl01-2C and six psj8-2C plamids were successfully constructed. The engineering bacteria were obtained following transforming into E.coli BL21(DE3) and inducing by IPTG.3. Purification and crystal screening of EV71 truncated 2C protein:According to the SDS-PAGE results, the 2C proteins containing p Gl01 and psj8 vector were all expressed in E.coli BL21(DE3). The molecular weight is conformed to expected values. The p Gl01-2C proteins were hard dissolved, while the psj8-2C proteins fused with maltose binding proteins were of solubility. The result of gel filtration affinity chromatography showed that the truncated proteins with fragment M1 and M4 are of higher purity and homogeneity. Unfortunately, we could not see a crystal growing by fine screening.Conclusions:In the present study, gene clone, heterologous expression and purification were successfully performed. Twelve recombinants of 2C intercepted fragments were constructed. Proteins with high purity, homogeneity and solubility were obtained. We conclude that the truncated proteins of psj8 vector-2C: M1 and M4(91aa-256 aa and 118aa-256aa) are more stable than the other ones used in this study. Additionally, maltose binding protein plays a critical role in improving the solubility of 2C protein.更多还原