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蝎毒镇痛活性肽Bm K Ang M1是从东亚钳蝎(Buthus martensii Karsch)蝎毒中分离得到的一种新型长链蝎毒素,其镇痛活性强且毒性低,有望开发成镇痛新药。本文将Bm K Ang M1基因转入毕赤酵母(Pichia pastoris)GS115,筛选得到甲醇利用缓慢型(Muts)和快速型(Mut+)的重组子;采用实时荧光定量PCR方法,检测了Mut+重组子中Bm K Ang M1基因的拷贝数,筛选出含单拷贝Bm K Ang M1基因的Mut+重组子;在相同培养条件下,比较了含单拷贝Bm K Ang M1基因的Muts和Mut+重组子表达Bm K Ang M1的水平。结果表明,Muts重组子中Bm K Ang M1基因转录水平是Mut+重组子的2.7倍,Muts重组子中Bm K Ang M1蛋白表达量是Mut+重组子的1.5倍。因此,Muts重组子比Mut+重组子具有更强的Bm K Ang M1表达能力。
Scorpion venom analgesic active peptide Bm K Ang M1 is a new scorpion venom isolated from buthus martensii Karsch scorpion venom. It has strong analgesic activity and low toxicity and is expected to be developed into a new analgesic drug. In this study, the Bm K Ang M1 gene was transferred into Pichia pastoris GS115, and Muts and Mut + were screened. Real-time PCR was used to detect Bm K Ang Ml gene copy number, Mutant recombinants with single copy of Bm K Ang M1 gene were screened; Muts and Mut + recombinants containing a single copy of Bm K Ang Ml gene were compared under the same culture conditions to express Bm K Ang M1 level. The results showed that the transcription level of Bm K Ang M1 gene in Muts was 2.7-fold that of Mut + and that of Bm K Ang Ml in Muts was 1.5-fold that of Mut +. Therefore, Muts recombinants have stronger BmK Ang M1 expression than Mut + recombinants.