中华大蟾蜍mcl-1基因克隆及其重组蛋白在原核体系中的诱导表达

Bufo gargarizans mcl-1 cloning and its prokaryotic recombinant protein expression

以蟾蜍（Bufo）为基原的常用中药材有蟾酥、蟾衣、蟾皮、干蟾等，具有强心、麻醉、解毒、止痛、抗菌、增强免疫力及抗肿瘤等药理作用，按溶解性可将其成分划分为脂溶性甾族类和水溶性吲哚生物碱类。体内外实验已证明由蟾皮制成的水溶性注射液——华蟾素具有良好的抗肿瘤效果。

MCL-1 is encoded by myeloid cell leukemia-1 gene （mcl-1）, which is one of the anti-apoptotic members of bcl-2 cell apoptotic gene superfamily. ChanSu is made of dorsal secretions of several Bufo species, commonly used in the prescriptions of traditional Chinese medicine for treating many diseases including cancer. To clarify if mcl-1 is expressed in the dorsal skin of B. gargarizans, the PCR （polymerase chain reaction） was performed with its dorsal skin first strand cDNA as the template and a pair of specific primers of mcl-1, and PCR products were cloned into the pGM-T vector. DNA sequencing indicated that the ORF length was 639 bp encoding 212 amino acid residues, and the homology of 44%-95% with the MCL-1 of several other animals. For the further studies on MCL-1 biological functions during the oncogenesis and preparation of its antibody, the prokaryotic expression construct of pET-28b-mcl-1 was prepared which was confirmed by DNA sequencing, and its recombinant protein expression （0.02% wet weight） in E. coli BL21 （DE3） strain was confirmed by SDS-PAGE and Western blotting.