四川白鹅 CD4基因胞外去信号肽区的可溶性表达及纯化
Soluble Expression and Purification of Extracellular Region without Signal Peptide of Sichuan White Goose CD4 Gene

中文摘要：根据GenBank公布的四川白鹅T细胞表面分子 CD4基因序列设计特异性引物,扩增其胞外去信号肽区基因,将其构建成原核重组表达质粒goCD4-pET-32a(+),并在大肠杆菌原核表达系统中进行可溶性表达并纯化,为进一步应用与研究提供基础。通过从成年四川白鹅胸腺组织中提取RNA,经反转录合成cDNA为模板扩增 CD4基因胞外去信号肽区,并构建原核表达载体goCD4-pET-32a(+)。在大肠杆菌 Rosetta (DE3) pLysS系统中进行原核表达,通过改变表达温度、培养基、诱导剂IPTG浓度、诱导时间以及抗性浓度等条件,最终获得可溶性表达并经NI-NTA亲和层析获得纯化蛋白。结果显示,鹅 CD4胞外区蛋白只有在16 ℃的TB培养基中目的蛋白his-goCD4可表达,鉴定结果与预期相符,诱导表达成功且多以不可溶的包涵体形式存在。研究发现改变IPTG浓度对表达产物存在形式影响较大,最终筛选出以0.4 mM的IPTG条件作为可溶性表达的最佳诱导浓度。NI-NTA亲和层析获得纯化蛋白并经Western-blot验证。
英文摘要：CD4 molecule is a glycoprotein that locates in surface of T lymphocytes, and this protein is closely related to the antigen that recognizing and activating T lymphocytes. To obtain the recombinant protein of goose CD4 using prokaryotic expression system, specific primers for the amplification of the goose CD4 extracellular region without signal peptide were designed based on the cDNA of goose CD4 (GenBank accession: JX902315), and the recombinant plasmid [goCD4-pET-32a(+)] containing the amplicon was successfully constructed and transformed into Rosetta(DE3) followed by IPTG induction. After detailed optimization of induction temperature, concentration of IPTG, and induction time, the best condition of expression was confirmed. Subsequently, the recombinant protein was purified by NI-NTA, followed by analyzing the target band by SDS-PAGE and Western-blot. The results showed that the highest level of protein expression was obtained when the recombinant bacteria was induced by IPTG at the concentration of 0.4 mmol·L -1 at 16 ℃ over night. The recombinant protein (64 kDa) was mainly existed in the supernatant of bacterial lysate, and the band of purified recombinant protein was correct. The result of Western-blot showed that the soluble recombinant protein could be recognized by specific his-tag antibody, indicating the successful expression of targeted protein in prokaryotic expression system [pET-32a (+)/Rosetta (DE3) pLysS]. 2015,34(6): 875-879 收稿日期：2015-04-02 DOI：10.11984/j.issn.1000-7083.20150121 分类号：Q959.7;Q78 基金项目：国家自然科学基金项目(31201891); 四川省科技厅青年基金项目(2012JQ0040); 四川省教育厅重点项目(12ZA107); 教育部高等学校博士学科点专项科研基金项目(20125103120012); 水禽疫病防控四川省青年科技创新研究团队项目(2013TD0015) 作者简介：程蓓蓓,女,硕士研究生,研究方向:禽类分子免疫学,E-mail:19910825cbb@sina.com *通讯作者：陈舜,女,博士,副研究员,研究方向:禽类分子免疫学,E-mail:sophia_cs@163.com 参考文献： 刘爽, 胡宝成. 2005. 原核系统可溶性表达策略[J]. 生物技术通讯, 16(2): 172-175. 张姝, 王敏, 韩梅琳, 等. 2009. 酵母提取物诱导重组大肠杆菌合成HrpNEcc蛋白的研究[J]. 中国农业科学, 42(11): 3380-3387. 张雪莲, 魏双施, 邵建伟. 2014. 东北白鹅 CD4基因的克隆及其胞外区的表达与抗血清的制备[J]. 畜牧兽医学报, 45(4): 639-646. 朱红裕, 李强. 2006. 外源蛋白在大肠杆菌中的可溶性表达策略[J]. 过程工程学报, 6(1): 150-155. Bannwarth M, Schulz GE. 2003. The