人碱性成纤维生长因子重组质粒的构建

Construct the recombinant plasmid of recombinant human basic fibroblast growth factor

目的通过基因体外拼装（PBGA）获得带有BamHI和PstI内切酶位点的重组人碱性成纤维生长因子（rhbFGF）cDNA，并利用TA克隆技术构建含rhbFGF基因片段的重组质粒。方法（1）根据己知的hbFGF氨基酸序列并结合乳酸链球菌蛋白表达密码子来设计适于乳链菌内表达的hbFGF基因序列，分别在5’和3’端设计BamHI和PstI酶切位点，通过DNASTAR6．0将上述目的基因序列设计成22个可以相互重叠的寡核苷酸片段，bFGFl-bFGF22。（2）采用基因拼装技术，通过PCR反应，拼接I～22段寡核苷酸片段，合成带有设定内切酶位点的rhbFGFcDNA。（3）构建rhbFGFTA克隆载体。纯化上步拼接rhbFGF基因片段，产物与PMD18-TVECTOR进行连接反应。连接产物转化于E．coliTOP10感受态细胞，涂板、筛选、培养后提质粒，酶切鉴定并测序。结果（1）体外拼装带有BamHI和PstI内切酶酶切位点的rhbFGFcDNA经2％琼脂糖电泳验证；（2）构建了载体hrbFGF—PMD18，经酶切鉴定和测序证实碱基序列完全正确。结论利用PBGA和TA克隆技术可成功构建rhbFGF．PMD18重组质粒。

Objective To obtain the sequence of recombinant human basic fibroblast growth factor （rhbFGF） with endonuelease sites of BamHl and Pst I by PCR based gene assembly and construct the re- combining vector of rhbFGF gene by TA cloning technique. Methods The rhbFGF gene sequence which was designed for lactoeoccus with endonuclease sites of BamHI and Pst I was divided into 22 oligonucleoti- des by DNASTAR 6.0 （ bFGFl-bFGF22 ）. The 22 oligonucleotides were spliced by PCR based gene assem- bly to get the rhbFGF eDNA with endonuclease sites of BamHI and Pst I ~ The PCR product was inserted into the PMD18-T VECTOR. The recombining vector were converted to the competent E. coli TOP10. The clones generated from LAB were analyzed by miniprep isolation from LAB host. They were identified by the restriction enzyme cutting and sequencing. Results The rhbFGFcDNA synthesized by PCR based gene assembly with endonuclease sites of BamHI and Pst I was verifiedby 2% agarose eleetrophoresis. The recombining vector of rhbFGF gene by TA cloning technique was identified by enzyme digestion and gene sequencing. Conclusions The TA cloning vector of recombining hbFGF with endonuclease sites of BamHI and Pst I was constructed successfully.