摘要
目的体外拼装适合在乳酸链球菌表达的人粒-巨噬细胞集落刺激因子(granulocyte-macrophagecolonystimulatingfactor,hGM-CSF),并构建载体pNCSF。方法根据hGM-CSF的氨基酸序列及乳酸链球菌偏好的密码子,将hGM-CSF基因外显子的全长碱基序列用DNAWORKS程序设计成16条互补的寡核苷酸片段,并且分别在第1条和第16条寡核苷酸片段上分别设计PstⅠ和BamHⅠ酶切位点,将相同终浓度的寡核苷酸混合进行PCR反应,完成hGM-CSF基因拼接,得到目的基因片段,对寡核苷酸片段进行拼装和扩增,产物稀释后进一步纯化扩增,在Tag酶存在的条件下对产物两末端加T,然后TA克隆于pGEMTeasy载体中,经酶切鉴定得到阳性克隆并经测序验证。然后亚克隆于含有P59启动子、USP45蛋白信号肽的pNBC1000载体。结果通过PCR得到420左右目的DNA片段,获得了hGM-CSF和pNCSF阳性克隆,其DNA测序结果与设计序列完全一致。结论运用基因拼装及克隆技术成功拼装了hGM-CSF基因及构建了载体pNCSF,为乳酸链球菌表达重组hGM-CSF奠定了基础。
[Objective] To assemble hGM-CSF gene expressed in Lactoeoecus lactis in vitro and construct its vector pNCSF. [Methods] Based on the amino acid sequence of hGM-CSF and the protein expression preferable eodon of Laetoeoeeus laetis, the entire human hGM-CSF gene eondon sequence was designed to 16 complementary oligonueleotides instructed by DNAWORKS program on line, and restriction enzyme sites of Pst Ⅰ and BamH Ⅰ were added in number 1 and 16 oligonueleotide respectively. All the oligonueleotides were mixed in the same concentration to obtained by Polymerase chain reaction （PCR）. Then, the targeted sequence was gel-purifed, amplified one more time, added T to 3＇ end in the presence of Taq polymerase and dATP, and cloned to pGEM T easy vector. Furthermore, the targeted gene was subeloned to the vector pNBCIO00 which contains P59 promoter, USP45 signal peptide. The positive colonies were confirmed by restriction enzyme excision and sequencing. [Result] The expected DNA fragment about 420 bp was obtained and targeted gene were obtained after Polymerase chain reaction （PCR）. The positive elonies of hGM-CSF gene and pNCSF were obtained. DNA sequencing showed that the positive colonies were finally verified by sequencing which were totally in line with the designed coding sequence of hGM- CSF gene. [Conclusion] With the technology of gene cloning and gene assembly, hGM-CSF gene was successfully synthesized in vitro and the vector pNCSF was successfully constructed, which laid a foundation for expression of recombinant hGM-CSF in Laetoeoeeus laetis.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2007年第1期9-12,共4页
China Journal of Modern Medicine
基金
国家自然科学基金项目(No:30570839)
广东省自然科学基金资助项目
No05004735
