Qing Liu-Ting, Qu Xiao-Ling
College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2002 Jul;34(4):405-10.
The coding sequence of abrin-a A-chain (ABRaA) gene was obtained by RT-PCR and cloned into the expression vector pET28b. The mature ABRaA has been highly expressed in the cytoplasm of Escherichia coli by 1 mmol/L IPTG induction, and the yield of the soluble recombinant protein was 4 mg/L of induced culture. The recombinant ABRaA was purified to be homogeneity. The biological activities of expressed ABRaA were demonstrated in vitro. It strongly inhibited the protein biosynthesis of rabbit reticulocyte lysates, with an IC(50) of 0.08 nmol/L. It also depurinated 28 S rRNA through cleaving at the A4324 site in rat liver ribosomes by its N-glycosidase activity. These data suggested that the recombinant ABRaA could be used for the preparation of immunotoxins as a potential cancer chemotherapeutic agent.
通过逆转录聚合酶链反应（RT-PCR）获得相思子毒素-a A链（ABRaA）基因的编码序列，并将其克隆到表达载体pET28b中。成熟的ABRaA通过1 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导在大肠杆菌细胞质中高效表达，可溶性重组蛋白的产量为4 mg/L诱导培养物。重组ABRaA被纯化至均一性。表达的ABRaA的生物学活性在体外得到证实。它强烈抑制兔网织红细胞裂解物的蛋白质生物合成，半数抑制浓度（IC50）为0.08 nmol/L。它还通过其N-糖苷酶活性在大鼠肝脏核糖体的A4324位点切割使28 S核糖体RNA脱嘌呤。这些数据表明，重组ABRaA可作为一种潜在的癌症化疗药物用于制备免疫毒素。
