中文摘要：

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—macrophage colony stimulatingfactor，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pGEM T easy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。

英文摘要：

[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.