人血管抑素A K(1-3)基因的克隆表达、纯化及活性鉴定

目的 克隆人Angiostatin K(1-3)基因,获得有活性的重组人血管抑素蛋白,为进一步开发应用奠定基础.方法 以人新鲜肝脏组织为材料,通过RT-PCR得到人Angiustatin基因的AK(1-3)片段.构建重组质粒pET30a-Angiostatin,转化表达菌Rosetta(DE3),对转化子进行诱导表达,利用Ni-NTA亲和层析纯化目的 产物,并验证其活性.结果 获得了人Angiostatin基因AK(1-3)片段的正确序列,表达和纯化了人血管抑素蛋白,表达量占菌体总蛋白的30%,纯化后证明表达产物具有较高纯度(达到90%).结论 Anginstatin K(1-3)可在原核融合蛋白表达载体中表达,且得到有活性的目的 蛋白.

The Cloning expression, purification and activity of human angiostatin K (1-3) gene

Objective To clone the sequence of human Angiostatin cDNA and obtain the protein of recombinant angiostatin for further development. Methods Fresh human liver tissure was used for the extraction of total RNA and amplified the Angiostatin eDNA through RT-PCR method . After the recombinant plasmid pET30a-Angiostatin was constructed and confirmed, it was transduced into Rosetta (DE3). Then the transformation was used for fermentation and induced expression.The protein was identified by Western Blot and purified by Ni-NTA affinity chromatography. Results The sequence of human Angiostatin cDNA was identical with genehank. Angiostatin(K1-3) was expressed and purified. The target protein made up 30% of the total bacterial protein. The purity is above 90%. Conclusion Anginstatin K (1-3) can be reached using fusion vector pET30a. The product have the biological activity.