| 重组抗LMP1单链抗体/鱼精蛋白截短体融合蛋白的基因构建及表达 |
| |
| 引用本文： | 黄秀兰,唐旭东,熊亮,周克元.重组抗LMP1单链抗体/鱼精蛋白截短体融合蛋白的基因构建及表达[J].湛江医学院学报,2008,26(1):1-5. |
| |
| 作者姓名： | 黄秀兰 唐旭东 熊亮 周克元 |
| |
| 作者单位： | 广东医学院附属医院儿科研究室，广东医学院生物化学与分子生物学研究所，广东医学院赣南医学院预防医学系，广东医学院生物化学与分子生物学研究所 广东医学院生物化学与分子生物学研究所，广东湛江524023，广东湛江524023，江西赣州341000，广东湛江524023 |
| |
| 基金项目： | 广东省重点学科建设项目
,
广东省湛江市科技攻关项目 |
| |
| 摘 要： | 目的体外拼装人抗EB病毒编码的潜伏膜蛋白1单链抗体(LMP1 scFv)/截断的鱼精蛋白截短体(truncatedprotamine,tP)融合基因,将其克隆至原核表达载体pET32a,并在大肠杆菌中诱导表达LMP1 scFv/tP融合蛋白。方法设计融合基因LMP1 scFv/tP,在LMP1 scFv/tP的5’和3’端设计EcoRⅠ和HindⅢ酶切位点;在DNAWORKS程序指导下设计成22个相互互补的寡核苷酸序列,经PCR扩增获得融合基因LMP1 scFv/tP,再将目的基因片段亚克隆至pMD 18-Tsi mple载体中,经过酶切鉴定和测序验证。进一步将目的基因片段克隆至原核表达载体pET32a,在大肠杆菌中诱导表达融合蛋白并经SDS-PAGE和western blot验证。结果各寡核苷酸片段经PCR扩增后可见明确的产物带,将获得的LMP1 scFv/tP融合基因构建至原核表达载体pET32a,经IPTG诱导后成功表达目的蛋白。结论成功获得LMP1 scFv/tp融合基因,并在大肠杆菌中成功诱导表达LMP1 scFv/tP融合蛋白,为该融合蛋白用于LMP1相关肿瘤的基因治疗奠定基础。
|
| 关 键 词： | LMP1 scFv/tP 基因拼装 表达 融合蛋白 |
| 文章编号： | 1005-4057(2008)01-0001-05 |
| 修稿时间： | 2007年12月8日 |
Construction and expression of the recombinant anti-LMP1 scFv/tP fusion protein |
| |
| HUANG Xiu-lan,TANG Xu-dong,XIONG Liang,ZHOU Ke-yuan.Construction and expression of the recombinant anti-LMP1 scFv/tP fusion protein[J].Journal of Guangdong Medical College,2008,26(1):1-5. |
| |
| Authors: | HUANG Xiu-lan TANG Xu-dong XIONG Liang ZHOU Ke-yuan |
| |
| Abstract: | Objective To assembe the fusion gene anti-EBV LMP1 scFv/tP in vitro and construct and express this fusion gene in an expression vector.Methods The fusion gene LMP1 scFv/tP was erected an EcoR I at the 5' end and a Hind III at the 3' end.Based on the sequence of LMP1 scFv/tP,a pair of 22 nucleotide primers that complement each other were designed by following the instruction of the DNAWORKS program.The target gene fragment of LMP1 scFv/tP was obtained by PCR,and then subcloned into the pMD 18-Tsimple vector.The positive clones were confirmed with the restriction enzyme digestion and sequence analysis.Finally,the LMP1 scFv/tP was cloned into the vector of pET32a and expressed in E coliBL21 (Rosseta).The LMP1 scFv/tP protein was detected in SDS-PAGE and western blot.Results The PCR produced exact product of the LMP1 scFv/tP gene from the construct.SDS-PAGE and Western Blot analysis displayed the detected protein that was the protein encoded by the LMP1 scFv/tP gene.Conclusion The LMP1 scFv/tP gene can be successfully constructed and expressed,which could be used for the gene therapy of the LMP1 associated tumors. |
| |
| Keywords: | LMP1 scFv/tP based gene assembly express fusion protein |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ |
