[1]李启英,项颖,余维倩,等.Luffin-β-KDEL-uPAcs融合毒素的构建及其抗胃癌SGC-7901细胞的活性研究[J].陆军军医大学学报(原第三军医大学学报),2013,35(08):774-778.
 Li Qiying,Xiang Ying,Yu Weiqian,et al.Construction, expression and purification of prokaryotic fusion protein luffin-β-KDEL-uPAcs and its cytotoxic effect on gastric carcinoma cell line SGC-7901[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(08):774-778.
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Luffin-β-KDEL-uPAcs融合毒素的构建及其抗胃癌SGC-7901细胞的活性研究(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
35卷
期数:
2013年第08期
页码:
774-778
栏目:
论著
出版日期:
2013-04-30

文章信息/Info

Title:
Construction, expression and purification of prokaryotic fusion protein luffin-β-KDEL-uPAcs and its cytotoxic effect on gastric carcinoma cell line SGC-7901
作者:
李启英项颖余维倩王莉黄德鸿唐显军张曼张文军杨涛肖春燕
重庆市肿瘤研究所生物治疗、血液肿瘤科
Author(s):
Li Qiying Xiang Ying Yu Weiqian Wang Li Huang Dehong Tang Xianjun Zhang Man Zhang Wenjun Yang Tao Xiao Chunyan
Department of Biotherapy & Heamoonclogy, Chongqing Cancer Institute, Chongqing, 400030, China
关键词:
Luffin-β尿激酶纤溶酶原激活剂裂解位点KDEL驻留信号序列融合毒素杀瘤活性
Keywords:
luffin-β urokinase plasminogen activator KDEL (Lys-Asp-Glu-Leu) fusion protein cytotoxic effect
分类号:
R394-33;R73-362;R735.2
文献标志码:
A
摘要:
目的   构建含尿激酶型纤溶酶原激活剂(urokinase plasminogen activator,uPA)裂解位点(uPAcs)和KDEL(Lys-Asp-Glu-Leu)驻留信号序列的丝瓜毒素(Luffin-β)基因的原核载体,表达并纯化其融合毒素蛋白Luffin-β-KDEL-uPAcs(LKP),并探讨融合毒素蛋白LKP抗胃癌SGC-7901细胞的活性。   方法   RT-PCR两步法克隆Luffin-β基因,引物延伸法构建Luffin-β-KDEL-uPAcs融合基因并亚克隆至原核表达载体pET-32a(+)中,诱导其表达融合蛋白Trx-EK-Luffin-β-KDEL-uPAcs(TELKP)并纯化TELKP,肠激酶(enterokinase,EK)切割TELKP后,纯化与回收目的毒素蛋白LKP,SDS-PAGE对LKP蛋白予以检测鉴定,高效液相色谱法(HPLC)对其进行纯度检测。采用cell counting kit-8 (CCK-8)、RT-PCR、Western blot等方法,体外检测毒素蛋白LKP经uPA酶裂解后释放Luffin-β的抗胃癌SGC-7901细胞的活性。   结果   成功诱导重组载体pET-32a (+)/Luffin-β-KDEL-uPAcs表达相对分子质量约48.8×103含载体表达标签(Trx)的融合免疫毒素TELKP,EK酶切该蛋白获含290个氨基酸,相对分子质量约31.8×103的目的蛋白LKP。SDS-PAGE检测鉴定表明,LKP蛋白与预期大小一致,其纯度达98.8%。CCK-8、RT-PCR、Western blot等法检测显示,LKP经uPA酶体外裂解可释放具杀瘤活性的Luffin-β小分子毒素。   结论   成功克隆到Luffin-β-KDEL-uPAcs融合基因,并将其构建于原核表达载体pET-32a(+)中,且诱导该载体表达了相对分子质量约31.8×103的融合毒素LKP。LKP毒素经uPA酶体外裂解能释放具杀瘤活性的Luffin-β小分子毒素。
Abstract:
Objective   To construct a prokaryotic expression vector carrying a fusion gene containing tandemly ligated luffin-β, KDEL (Lys-Asp-Glu-Leu) and urokinase plasminogen activator cleavage site (uPAcs), to express and purify the fusion protein, and to investigate the cytotoxic effect of the fusion protein on gastric carcinoma cell line SGC-7901.    Methods   Luffin-β gene was cloned by two-step reverse transcriptase-polymerase chain reaction (RT-PCR). The fusion gene of luffin-β-KDEL-uPAcs was obtained by primer extension, and was inserted into pET-32a(+) vector to construct a recombinant vector pET-32a(+)/luffin-β-KDEL-uPAcs. The fusion protein Trx-EK-luffin-β-KDEL-uPAcs (TELKP) was expressed under induction, purified, and digested by enterokinase (EK) to obtain luffin-β-KDEL-uPAcs (LKP). The LKP protein was purified and its purity was determined by high performance liquid chromatography (HPLC). Cell counting kit-8 (CCK-8), RT-PCR and Western blotting were employed to detect the cytotoxic effect of LKP on gastric carcinoma cell line SGC-7901 after uPA cleavage.    Results   The recombinant vector pET-32a(+)/luffin-β-KDEL-uPAcs could successfully express the fusion protein TELKP (48.8×103), and the LKP protein (31.8×103) could be obtained by digesting the fusion protein TELKP with EK. The purity of the LKP protein was about 98.8%. The results of CCK-8, RT-PCR and Western blotting revealed that immunotoxin luffin-β, which had cytotoxic effect on tumor cells, could be released from the LKP protein cleaved by uPA in vitro.    Conclusion   The fusion gene luffin-β-KDEL-uPAcs and its prokaryotic expression vector are successfully constructed. Recombinant fusion protein LKP (31.8×103) is successfully prepared. The immunotoxin luffin-β, which possesses cytotoxic effect on tumor cells, could be released from the LKP protein cleaved by uPA in vitro.

相似文献/References:

[1]项颖,黄德鸿,唐显军,等.VEGF受体单抗与Luffin双靶融合毒素的构建及其对非小细胞肺癌细胞的抑制作用[J].陆军军医大学学报(原第三军医大学学报),2013,35(18):1921.
 Xiang Ying,Huang Dehong,Tang Xianjun,et al.Construction of double-targeting fused immunotoxin KDRscFv-uPAcs-Luffin-β-KDEL and its cytotoxic effect on non-small-cell lung carcinoma cells[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(08):1921.

更新日期/Last Update: 2013-04-22