CAJ | 학술논문

目的从家蝇三龄幼虫中提取总RNA,先利用RT-PCR扩增编码天蚕素Cecropin的cDNA序列,克隆入T载体pUCm-T并测定其序列.然后以pUCm-T/Cecropin为模板,通过PCR方法扩增Cecropin成熟肽cDNA序列,并将该序列的N-端大肠杆菌稀有密码子GGA点突变为GGC,C-端终止密码子TAA前加进一个天冬酰胺(Asn)密码子AAC,命名为mCecropin.mCecropin基因序列克隆至融合表达载体pGEX-4T-1中.酶切分析和测序鉴定后,将阳性重组子质粒转入不同的大肠杆菌宿主细胞中进行融合表达.经SDS-PAGE分析,选用E. Coli BL21(DE3)作为表达宿主菌.表达的融合蛋白GSTmCecropin通过GSTrap亲合柱纯化后用凝血酶酶切GST标签,mCecropin经HiTrap柱进一步纯化后,具有较强的抗菌活性.
목적 종가승삼령유충중제취총RNA,선이용RT-PCR확증편마천잠소Cecropin적cDNA서렬,극륭입T재체pUCm-T병측정기서렬.연후이pUCm-T/Cecropin위모판,통과PCR방법확증Cecropin성숙태cDNA서렬,병장해서렬적N-단대장간균희유밀마자GGA점돌변위GGC,C-단종지밀마자TAA전가진일개천동선알(Asn)밀마자AAC,명명위mCecropin.mCecropin기인서렬극륭지융합표체재체pGEX-4T-1중.매절분석화측서감정후,장양성중조자질립전입불동적대장간균숙주세포중진행융합표체.경SDS-PAGE분석,선용E. Coli BL21(DE3)작위표체숙주균.표체적융합단백GSTmCecropin통과GSTrap친합주순화후용응혈매매절GST표첨,mCecropin경HiTrap주진일보순화후,구유교강적항균활성.
In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the cDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E. coli was changed to the favorable codon GGC,and a Asn codon AAC was added in front of the stop coden TAA in the C- terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E. coli cells and the fusion protein was expressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E. coli BL21(DE3) cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.