重组CD13单链抗体/蝎毒素AGAP原核表达质粒的构建

Study on construction of recombinant prokaryotic expression plasmid of AGAP and CD13scFv

目的:构建pRSETA/AGAP/CD13scFv重组表达质粒,为重组CD13单链抗体/蝎毒素AGAP融合蛋白的表达奠定实验基础。方法:利用RT-PCR法从东亚钳蝎尾腺中获得AGAP目的基因,并与克隆质粒TOPO载体连接转入DH5α菌,用EcoR I和Hind III将目的基因从克隆质粒上切下并与同样双酶切后的表达质粒pRSETA连接;在含CD13单链抗体(CD13scFv)的pSecTag2HygroC/CD13-1/RFP质粒中获得CD13scFv目的基因,用EcoR I、Xho I双酶切并与同样双酶切后的pRSETA/AGAP连接转入DH5α菌,提取重组质粒进行酶切及测序鉴定。结果:目的基因AGAP与表达质粒pRSETA连接,成功构建了pRSETA/AGAP重组质粒;目的基因CD13scFv再与该重组质粒连接,成功构建了pRSETA/AGAP/CD13scFv。结论:AGAP目的基因能经RT-PCR法获得,并能成功与表达质粒连接,构建pRSETA/AGAP;目的基因CD13scFv能由pSecTag2HygroC质粒中获得,并成功与pRSETA/AGAP连接,得pRSETA/AGAP/CD13scFv重组质粒。为该重组质粒体外融合蛋白的表达及今后研究CD13scFv和蝎毒素AGAP协同性杀伤CD13阳性肿瘤细胞提供了良好的实验基础及理论依据。

Objective： To construct recombinant prokaryotic expression plasmid pRSETA/AGAP/CD13scFv that contributes to the expression of fusion protein AGAP/CD13scFv. Methods： The AGAP gene was obtained from Buthus martensii Karsch using RT-PCR, then it was connected to the cloning from the recombinant cloning plasmid, and connected to the expression plasmid pRSETA that was digested by the same enzymes. Subsequently, CD13scFv was amplified from plasmid pSecTag2HygroC with PCR and inserted into vector pRSETA/AGAP. At last, the recombinant plasmid was identified by PCR, enzyme digestion analysis and sequencing. Results： The recombinant expression plasmid pRSETA/ AGAP/CD13scFv was constructed successfully. Conclusion： The objective gene AGAP can be obtained from Buthus martensii Karsch using RT-PCR. CD13scFv can be amplified from plasmid pSecTag2HygroC with PCR. And the recombinant prokaryotic expressing plasmid pRSETA/AGAP/CD13scFv are successfully constructed. The construction of pRSETA/AGAP/CD13scFv has a great constribution not only to the exDression of fusion nrotein, but also to the theranv of CD13 nositive tumors.