CAJ | 학술논문

目的将甲型流感病毒M2蛋白胞外区(M2e)基因与人血清白蛋白(HSA)基因融合,以构建高效分泌表达的毕赤酵母菌株.方法通过RT-PCR扩增HSA基因,构建pPICZα-HSA质粒,将人工合成的M2e序列与pPICZα-HSA质粒分别经BstBI 和KpnI 酶切消化后连接,构建真核表达载体pPICZα-HSA/M2e,线性化后电转化毕赤酵母X-33.用PCR法筛选Zeocin抗性阳性的克隆,SDS-PAGE和Western印迹法筛选高表达HSA/M2e菌株.结果经RT-PCR法克隆的HSA基因序列与GenBank登录的cDNA序列一致,构建的pPICZα-HSA/M2e真核表达载体,经电转化获得Zeocin抗性阳性的克隆.SDS-PAGE和Western印迹分析证实了甲醇诱导的培养基上清中含有HSA/M2e,分子量约为68 kD.结论成功构建了分泌表达重组HSA/M2e融合蛋白的毕赤酵母菌株,为进一步研究其生物学活性奠定了基础.
목적 장갑형류감병독M2단백포외구(M2e)기인여인혈청백단백(HSA)기인융합,이구건고효분비표체적필적효모균주.방법 통과RT-PCR확증HSA기인,구건pPICZα-HSA질립,장인공합성적M2e서렬여pPICZα-HSA질립분별경BstBI 화KpnI 매절소화후련접,구건진핵표체재체pPICZα-HSA/M2e,선성화후전전화필적효모X-33.용PCR법사선Zeocin항성양성적극륭,SDS-PAGE화Western인적법사선고표체HSA/M2e균주.결과 경RT-PCR법극륭적HSA기인서렬여GenBank등록적cDNA서렬일치,구건적pPICZα-HSA/M2e진핵표체재체,경전전화획득Zeocin항성양성적극륭.SDS-PAGE화Western인적분석증실료갑순유도적배양기상청중함유HSA/M2e,분자량약위68 kD.결론 성공구건료분비표체중조HSA/M2e융합단백적필적효모균주,위진일보연구기생물학활성전정료기출.
Objective To fuse the gene of M2e with that of HSA in order to construct a P. pastoris yeast strain with high-efficient expression of HSA/M2e. Methods The gene of HSA was amplified by RT-PCR and the plasmid of pPICZα-HSA was constructed. M2e artificial synthesized was digested with Eco81I and KpnI, then ligated to the same sites of pPICZα-HSA and obtained the expression vector pPICZα-HSA/M2e. The recombinant vector was linearized and introduced into P.pastoris X-33 by electroporation. Pichia pastoris secreting HSA/M2e were obtained to extract genomic DNA of transformed X-33 and perform PCR. SDS-PAGE and Western blot was used to screen high expressed that HSA/M2e engineering bacteria and to identify HSA/M2e in culture supernatant induced by methanol. Results The sequence of HSA obtained by RT-PCR was identical with that published on GenBank. Recombinant expression vector was constructed. SDS-PAGE and Western blot analysis showed that HSA/M2e was successfully expressed in the culture medium with an apparent molecular weight of 68 kD.Conclusions Pichia pastoris yeast strain with high-efficient expression of HSA/M2e is successfully constructed, providing a basis for further study of its bioactivity.