CAJ | 학술논문

　　为研究中国明对虾(Fenneropenaeus chinensis)血蓝蛋白 C 末端(FcHC-C)的抗菌功能，将血蓝蛋白基因 FcHC 的2个 C 末端基因片段连接到毕赤酵母表达载体 pPIC9K 中，构建酵母表达载体pPIC9K/FcHC-C。该载体经Sal I酶切后，采用PEG法转化毕赤酵母(Pichia pastoris GS115)。转化子经过PCR鉴定后，阳性克隆通过含有G418的YPD平板筛选，获得高拷贝重组子。重组毕赤酵母利用甲醇诱导表达目的基因。经Tricine-SDS-PAGE和Western blot分析结果表明，利用酵母工程菌成功表达了血蓝蛋白C末端片段(rFcHC-C1和rFcHC-C2)。抑菌活性鉴定实验结果显示，重组蛋白rFcHC-C1和rFcHC-C2作为阴离子抗菌肽具有抗真菌和抗细菌的活性。
　　위연구중국명대하(Fenneropenaeus chinensis)혈람단백 C 말단(FcHC-C)적항균공능，장혈람단백기인 FcHC 적2개 C 말단기인편단련접도필적효모표체재체 pPIC9K 중，구건효모표체재체pPIC9K/FcHC-C。해재체경Sal I매절후，채용PEG법전화필적효모(Pichia pastoris GS115)。전화자경과PCR감정후，양성극륭통과함유G418적YPD평판사선，획득고고패중조자。중조필적효모이용갑순유도표체목적기인。경Tricine-SDS-PAGE화Western blot분석결과표명，이용효모공정균성공표체료혈람단백C말단편단(rFcHC-C1화rFcHC-C2)。억균활성감정실험결과현시，중조단백rFcHC-C1화rFcHC-C2작위음리자항균태구유항진균화항세균적활성。
Two C-terminal coding sequences of Fenneropenaeus chinensis hemocyanin FCHc were cloned into the Pichia pastoris expression vector pPIC9K to produce the pPIC9K/FcHC-C yeast expression vectors. The con-structed vectors pPIC9K/FcHC-C1 and pPIC9K/FcHC-C2 were linearized by Sal I enzyme, and transformed into Pichia pastoris GS115 using PEG-mediated transformation method. PCR identified transformants were screened by G418 selected YPD plates. The P. pastorist transfomants of pPIC9K/FcHC-C expressed the two hemocyanin C-terminal gene fragments by methanol induction. The results of Tricine-SDS-PAGE and Western blotting showed that the recombinant FcHC-C1 and FcHC-C2 peptides (rFcHC-C1 and rFcHC-C2) were expressed successfully. An antimicrobial assay showed that rFcHC-C1 and rFcHC-C2 have antifungal and antibacterial activities as anionic AMPs.