CAJ | 학술논문

构建家蝇天蚕素-人溶菌酶(Mdc-hly)融合基因,实现Mdc-hly基因在大肠杆菌中的表达.通过RT-PCR分别扩增出家蝇天蚕素和人溶菌酶的成熟肽基因序列,再利用Gene-SOEing 技术构建融合基因,将融合基因克隆至pET32a表达载体,转化E. coli BL21(DE3),经IPTG诱导得到高效表达,融合蛋白分子量约为38 kD.Western blotting杂交证实了表达蛋白的抗原活性.成功构建了融合其因并进行了原核表达,为进一步的生物活性研究打下基础.
구건가승천잠소-인용균매(Mdc-hly)융합기인,실현Mdc-hly기인재대장간균중적표체.통과RT-PCR분별확증출가승천잠소화인용균매적성숙태기인서렬,재이용Gene-SOEing 기술구건융합기인,장융합기인극륭지pET32a표체재체,전화E. coli BL21(DE3),경IPTG유도득도고효표체,융합단백분자량약위38 kD.Western blotting잡교증실료표체단백적항원활성.성공구건료융합기인병진행료원핵표체,위진일보적생물활성연구타하기출.
The DNA fragment encoding the mature peptide of Musca domestica cecropin and human lysozyme were obtained by RT-PCR. The Mdc-hly gene was constructed in a Mdc-linker-hly format with the standard 15-amino acid linker (Gly_4Ser)_3 by Gene-SOEing, and the final full length product was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21(DE3). The expression fusion protein Mdc-hly-Trx were about 38 kD and Western blotting analysis confirmed that the 38 kD protein was the fusion protein, because it was specifically recognized by mouse anti-His monoclonal antibody. The fusion gene Mdc-hly was successfully constructed and expressed, the results of which could provide foundation for further study of its biological activity.