CAJ | 학술논문

目的通过基因体外拼装(PBGA)获得带有BamHI和Pst Ⅰ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Pst Ⅰ酶切位点,通过DNASTAR 6.0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,bFGFI -bFGF22.(2)采用基因拼装技术,通过PCR反应,拼接1～ 22段寡核苷酸片段,合成带有设定内切酶位点的rhbFGFcDNA.(3)构建rhbFGF TA克隆载体.纯化上步拼接rhbFGF基因片段,产物与PMD1 8-T VECTOR进行连接反应.连接产物转化于E.coli TOP10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Pst Ⅰ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-PM D18重组质粒.
목적 통과기인체외병장(PBGA)획득대유BamHI화Pst Ⅰ내절매위점적중조인감성성섬유생장인자(rhbFGF)cDNA,병이용TA극륭기술구건함rhbFGF기인편단적중조질립.방법 (1)근거기지적hbFGF안기산서렬병결합유산련구균단백표체밀마자래설계괄우유련균내표체적hbFGF기인서렬,분별재5′화3′단설계BamHI화Pst Ⅰ매절위점,통과DNASTAR 6.0장상술목적기인서렬설계성22개가이상호중첩적과핵감산편단,bFGFI -bFGF22.(2)채용기인병장기술,통과PCR반응,병접1～ 22단과핵감산편단,합성대유설정내절매위점적rhbFGFcDNA.(3)구건rhbFGF TA극륭재체.순화상보병접rhbFGF기인편단,산물여PMD1 8-T VECTOR진행련접반응.련접산물전화우E.coli TOP10감수태세포,도판、사선、배양후제질립,매절감정병측서.결과 (1)체외병장대유BamHI화Pst Ⅰ내절매매절위점적rhbFGFcDNA경2％경지당전영험증；(2)구건료재체hrbFGF-PMD18,경매절감정화측서증실감기서렬완전정학.결론 이용PBGA화TA극륭기술가성공구건rhbFGF-PM D18중조질립.
Objective To obtain the sequence of recombinant human basic fibroblast growth factor (rhbFGF) with endonuclease sites of BamHI and Pst Ⅰ by PCR based gene assembly and construct the recombining vector of rhbFGF gene by TA cloning technique.Methods The rhbFGF gene sequence which was designed for lactococcus with endonuclease sites of BamHI and Pst Ⅰ was divided into 22 oligonucleotides by DNASTAR 6.0 (bFGFl-bFGF22).The 22 oligonucleotides were spliced by PCR based gene assembly 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 cuuing and sequencing.Results The rhbFGFcDNA synthesized by PCR based gene assembly with endonuclease sites of BamHI and Pst Ⅰ was verified by 2％ agarose electrophoresis.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.