CAJ | 학술논문

　　根据半滑舌鳎(Cynoglossus semilaevis Güther)类胰岛素生长因子-Ⅰ(IGF-I)基因的cDNA序列,设计引物扩增编码IGF-I成熟肽序列,此序列由210个碱基组成,包括B-C-A-D 4个功能域。利用PCR方法将扩增片段克隆到原核表达载体 pET-28a 上,得到重组质粒,将重组质粒导入到大肠杆菌 BL21( DE3)后经 IPTG 诱导产生 N端含6个组氨酸的融合蛋白。以SDS-PAGE电泳检测和SigmaScan软件分析获得的多肽,结果表明： IGF-I融合蛋白大小为11.4 kD, IPTG诱导3 h时目的蛋白表达量最高,占细菌总蛋白的58.5%,1 L菌液表达目的蛋白40.7 mg,主要以包涵体形式存在。利用Western-blotting方法验证融合蛋白可特异性的被6×His抗体识别。诱导表达后的菌液沉淀经6 mol/L盐酸胍变性、Ni2+离子亲和柱纯化和尿素梯度复性,获得大小为11.4 kD的纯化蛋白。细胞增殖实验表明, IGF-I融合蛋白可显著促进乳腺癌细胞MDA231细胞的增殖,具有生物学活性。本研究通过原核表达技术获得了体外重组的具有生物活性的半滑舌鳎IGF-I融合蛋白,结果可为半滑舌鳎生长调控技术研究提供基础资料。
　　근거반활설탑(Cynoglossus semilaevis Güther)류이도소생장인자-Ⅰ(IGF-I)기인적cDNA서렬,설계인물확증편마IGF-I성숙태서렬,차서렬유210개감기조성,포괄B-C-A-D 4개공능역。이용PCR방법장확증편단극륭도원핵표체재체 pET-28a 상,득도중조질립,장중조질립도입도대장간균 BL21( DE3)후경 IPTG 유도산생 N단함6개조안산적융합단백。이SDS-PAGE전영검측화SigmaScan연건분석획득적다태,결과표명： IGF-I융합단백대소위11.4 kD, IPTG유도3 h시목적단백표체량최고,점세균총단백적58.5%,1 L균액표체목적단백40.7 mg,주요이포함체형식존재。이용Western-blotting방법험증융합단백가특이성적피6×His항체식별。유도표체후적균액침정경6 mol/L염산고변성、Ni2+리자친화주순화화뇨소제도복성,획득대소위11.4 kD적순화단백。세포증식실험표명, IGF-I융합단백가현저촉진유선암세포MDA231세포적증식,구유생물학활성。본연구통과원핵표체기술획득료체외중조적구유생물활성적반활설탑IGF-I융합단백,결과가위반활설탑생장조공기술연구제공기출자료。
We designed a pair of primers based on the full-length cDNA sequence of IGF-I from Cynoglossus se-milaevis Güther to clone the mature peptide domain of the IGF-I gene. The mature peptide consisted of 70 amino acids, including four functional domains B-C-A-D. The mature peptide fragment was subcloned into the prokary-otic expression vector pET-28a to construct a recombinant plasmid that was transformed into E. coli BL21 (DE3). Induction of the plasmid by IPTG yielded a special fusion polypeptide containing His 6 at the N-terminus. SDS-PAGE analysis revealed that the IGF-I polypeptide had a molecular weight of 11.4 kD. The recombinant IGF-I protein proximately accounted for 58.5%of the whole bacterial protein 3 h post induction with IPTG. Thus, 1 L of broth would yield 40.7 mg of the target protein. Western-blotting analysis suggested the fusion polypeptide exhibited antigenicity to His 6 antibodies. The IPTG-induced bacterial precipitate was denaturalized using 6 mol/L guanidine HCl, purified using Ni-NTA affinity chromatography, and annealed by gradient dialysis in urea, thereby yielding the purified protein. The recombinant IGF-I protein promoted the proliferation of breast cancer MDA231 cells, suggesting it is biologically active. Our results provide a basis and suggested methods for regulating the growth of farmed Cynoglossus semilaevis Güther.