人纤溶酶原K1-3基因的克隆、表达和产物的纯化及抗癌活性分析

纤溶酶原K1－3功能区是近年发现的血管生成抑制因子，具有抑制肿瘤生长和转移的活性。以人血管生成抑制素cDNA为模板用PCR技术扩增了K1－3功能区的基因，DNA序列分析后克隆至质粒pPIC9上获得重组质粒pPIC9K13转化毕节酵母GS115，用PCR和G418法筛选高拷贝转化子，进行摇瓶发酵。SDS－PAGE和Western blot分析结果证实K1－3基因已在GS115分泌表达，并具有免疫活性。选用30L和80L罐进行高密度发酵，甲醇诱导48h细胞密度达到OD250－300，比摇瓶提高5－6倍，表达量150－200mg/L，发酵上清液经Streamline SP离子交换及Phenyl-Sephorose疏水层析纯化，在SDS－PAGE上显示一条带，纯度96％，动物实验证明纯化产物具有抗血管生成和抗肿瘤的活性。

Cloning and Expression of Kringle 1-3 Gene of Human Plasminogen and the Purification and Bioactivity of Its Expressed Product

Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.