CAJ | 학술논문

目的：采用杆状病毒-昆虫细胞表达系统获取可溶性 His-Tat-Vp3融合蛋白,并观察其对细胞增殖和凋亡的影响。方法：构建重组质粒 pFastBacTM1-TAT-VP3,转化DH10Bac 感受态细胞,获得杆状病毒质粒,转染 Sf9昆虫细胞,得到重组杆状病毒,纯化并扩增该病毒。用最适滴度的病毒刺激 Sf9细胞以诱导 His-Tat-Vp3蛋白表达,经 SDS-PAGE和 Western bolt鉴定,获得分子质量约21 kDa 的融合蛋白。采用 MTT 实验与流式细胞技术检测500、1000、2000 nmol/L的融合蛋白抑制肿瘤细胞(HeLa细胞)增殖及促凋亡的活性。结果：通过昆虫表达系统成功表达及纯化出Tat-Vp3融合蛋白,其中1000、2000 nmol/L融合蛋白组可显著抑制肿瘤细胞的增殖,并提高细胞的凋亡率。结论：成功构建 His-TAT-VP3融合蛋白昆虫系统表达载体,在昆虫表达系统中可诱导性可溶性高表达,所获融合蛋白能显著抑制 H eLa细胞的增殖,并促进其凋亡。
목적：채용간상병독-곤충세포표체계통획취가용성 His-Tat-Vp3융합단백,병관찰기대세포증식화조망적영향。방법：구건중조질립 pFastBacTM1-TAT-VP3,전화DH10Bac 감수태세포,획득간상병독질립,전염 Sf9곤충세포,득도중조간상병독,순화병확증해병독。용최괄적도적병독자격 Sf9세포이유도 His-Tat-Vp3단백표체,경 SDS-PAGE화 Western bolt감정,획득분자질량약21 kDa 적융합단백。채용 MTT 실험여류식세포기술검측500、1000、2000 nmol/L적융합단백억제종류세포(HeLa세포)증식급촉조망적활성。결과：통과곤충표체계통성공표체급순화출Tat-Vp3융합단백,기중1000、2000 nmol/L융합단백조가현저억제종류세포적증식,병제고세포적조망솔。결론：성공구건 His-TAT-VP3융합단백곤충계통표체재체,재곤충표체계통중가유도성가용성고표체,소획융합단백능현저억제 H eLa세포적증식,병촉진기조망。
Objective:A Bac-to-Bac baculovirus expression system was designed to obtain His-Tat-Vp3 soluble fusion pro-tein,and verify its pro-apoptotic function and activity. Methods:The plasmid pFastBac1-TAT-VP3 was constructed and transformed into DH10 BacTM E. coli for the construction of baculovirus plasmid. Then Sf9 insect cells were transfected with the plasmid to obtain recombinant baculovirus,which was purified and amplified. Sf9 insect cells were stimulated with the vi-rus with the optimum titer in order to induce the expression of the fusion protein. The output of target protein with molecu-lar mass of about 21 kDa was identified by SDS-PAGE and Western blot. Finally,MTT assay and flow cytometry apoptosis assay were used to detect the inhibition of tumor cell (HeLa)proliferation and pro-apoptotic activity with 500,1 000,2 000 nmol/L fusion protein. Results:The Tat-Vp3 fusion protein was successfully expressed and purified by Bac-to-Bac Baculovir-us Expression System,in which 1 000,2 000 nmol/L of fusion protein group was found to be able to provide a high inhibi-tive effect on the proliferation of tumor cells,as well as to enhance pro-apoptotic activity. Conclusions:The fusion protein ex-pression vector His-TAT-VP3 is successfully constructed based on the Bac-to-Bac Baculovirus Expression System,through which a soluble recombinant protein is induced. The obtained fusion protein can significantly inhibit the proliferation of Hela cells and induce apoptosis. This finding lays an important foundation for further research targeting pro-apoptotic agents.