CAJ | 학술논문

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.
목적:극륭감합다태인용균매N단-Exendin-4기인병진행원핵표체화순화.방법:통과중조PCR기술장인용균매N단74개안기산적기인서렬여Exendin-4다태기인서렬상련접,기간인입일단유응혈매화이태기태매식별위점조성적련접서렬.이감합기인hLYZ(N74)-Ex4여질립pET-32a(+)구건원핵표체체,전화대장간균BL21(DE3)병유도표체.표체단백경Ni-NTA친화층석순화、Western blotting감정;투석복성후,이장격매절할병회수목적다태.결과:중조질립pET-32a/hLYZ(N74)-Ex4구건정학,목적단백주요이포함체형식존재,37℃유도4h、IPTG농도위0.6 mmol/L시표체량최고,약점균체단백총량적30%.Western blotting검측현시중조단백위단일청석조대.중조단백경장격매절할후,회수득도고순도적감합다태.결론:성공구건hLYZ(N74)-Ex4감합기인적원핵표체질립,고효원핵표체병획득고순도목적단백.
Aim:To clone, express and purify chimeric peptide of human lysozyme N-terminal frag-ment/exendin-4. Methods: Human lysozyme N-terminal fragment (1-74aa)and exendin-4 gene se-quences were linked by using recombinant PCR. The linker sequence between the lysozyme fragment and exendin-4 contained a site recognized by thrombin and another site by dipeptidyl peptidase Ⅳ(DPP Ⅳ). The hLYZ(N74)-Ex4 gene was inserted into prokaryotic expression vector pET-32α(+), trans-formed into E. coli BL21 (DE3) and induced with IPTG. The expressed products were purified by Ni-NTA affinity chromatography and dialysis, identified by western blotting and then digested with enterki-nase to release the chimeric peptide. Results: DNA sequence analysis showed that the recombinant plas-mid pET-32α/hLYZ(N74)-Ex4 was constructed correctly. The target protein was expressed mainly in in-clusions and reached a maximum level of about 30% of total somatic proteins after induction with 0.6 mmol/L IPTG at 37℃ for 4 h. Western blotting analysis suggested the recombinant protein was a clear band with expected molecular weight. The purified recombinant protein was cleaved into two fragments by enterokinase, the Trx-His tag and the chimeric peptide hLYZ(N74)-exendin-4. Conclusion:The pro-karyotic expression vector for hLYZ(N74)- exendin-4 fusion gene was successfully constructed, and the recombinant protein was highly expressed in E. coli. , which laid a foundation for their subsequent func-tional study.