Guo Chunhe, Huang Yumao, Zheng Hongyu, Tang Liyun, He Jun, Xiang Linsheng, Liu Dehui, Jiang Houquan
College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, P.R. China.
Exp Ther Med. 2012 Dec;4(6):1063-1068. doi: 10.3892/etm.2012.719. Epub 2012 Sep 24.
To express the antimicrobial peptide cecropin D in Pichia pastoris and determine the activity of the expressed product, four oligonucleotide fragments were synthesized in accordance with the available cecropin D sequences and a codon bias suitable for Pichia pastoris. Sequence fragments were phosphorylated, annealed, linked and cloned into the expression vector pGAPZαA and the yeast α-mating factor signal peptide was used as the signal sequence. The P. pastoris SMD1168 cells were transformed by electroporation using the constructed recombinant plasmid pGAPZαA-cecropin D. We were able to demonstrate by PCR that the cecropin D sequence had integrated into the P. pastoris genome. The expressed and secreted product was identified using Tricine-SDS-PAGE. Antibacterial activity was demonstrated using an agarose diffusion test and turbidimetry. The molecular mass of the recombinant cecropin D was estimated to be 3,900 Da. The recombinant cecropin D exhibited antibacterial activity for both Gram-positive and Gram-negative bacteria, suggesting that cecropin D was successfully expressed in P. pastoris. This approach holds great promise for antibacterial drug development.
为了在毕赤酵母中表达抗菌肽天蚕素D并测定表达产物的活性，根据现有的天蚕素D序列和适合毕赤酵母的密码子偏好性合成了4条寡核苷酸片段。将序列片段进行磷酸化、退火、连接并克隆到表达载体pGAPZαA中，以酵母α-交配因子信号肽作为信号序列。使用构建的重组质粒pGAPZαA-天蚕素D通过电穿孔法转化毕赤酵母SMD1168细胞。通过PCR我们能够证明天蚕素D序列已整合到毕赤酵母基因组中。使用Tricine-SDS-PAGE鉴定表达并分泌的产物。使用琼脂糖扩散试验和比浊法证明抗菌活性。重组天蚕素D的分子量估计为3900 Da。重组天蚕素D对革兰氏阳性菌和革兰氏阴性菌均表现出抗菌活性，表明天蚕素D在毕赤酵母中成功表达。这种方法在抗菌药物开发方面具有很大的前景。
