重组豹蛙酶的制备及其生物学特性分析  

Preparation and characterization of recombinant ranpirnase

作　　者：汪楠[1] 唐小军[1] 熊四平[1] 郑峰[2] 张慧林[3] 高畅[1] 李文杰[1] 董华[3] 朱进[1,2] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室,病理学系,江苏南京210029 [2]南京军区军事医学研究所,江苏南京210002 [3]南京医科大学附属妇幼保健院妇产科,江苏南京210024

出　　处：《南京医科大学学报（自然科学版）》2013年第8期1034-1038,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：南京市科技发展项目(ZKX12025)

摘　　要：目的:通过构建豹蛙酶原核表达载体pColdⅡ-Rap,表达、纯化豹蛙酶并研究其生物学活性。方法:合成豹蛙酶全基因序列,克隆于原核表达载体pColdⅡ中,鉴定正确后转化至大肠杆菌BL21,用SDS-PAGE和Western blot验证豹蛙酶的表达;大量表达重组豹蛙酶融合蛋白(Rap-His),通过MTT法分析Rap-His对乳腺癌细胞增殖的影响。结果:成功构建了pColdⅡ-Rap原核表达载体,经优化表达、纯化的条件,获得纯度较高的Rap-His;MTT法检测结果表明,该融合蛋白在320、160μg/ml时对乳腺癌细胞MDA-MB-231增殖的抑制率在加药后4 d达到93.49%。结论:本研究制备的重组Rap-His融合蛋白能够抑制乳腺癌细胞MDA-MB-231增殖。这为进一步与肿瘤特异抗体偶联,构建肿瘤特异性靶向药物的研究奠定了基础。Objective：To construct a prokaryotic expression vector pCold-Ⅱ-Rap and to optimize the expression conditions of ranpirnase（Rap） protein and to determine the protein activity.Methods：Recombinant pCold-Ⅱ-Rap vector was constructed,and positive clone was selected and transformed into E.coli.BL21.The recombinant protein expression was induced by IPTG.The Rap recombinant protein was purified,and detected by SDS-PAGE and Western blot.The RNase activity of Rap was analyzed by MTT.Results：The results showed that the purified ranpirnase was obtained by optimizing conditions of expression and purification,and degradation of tRNA could be detected by ranpirnase in vitro.The fusion protein can inhibit the proliferation of breast cancer cell MDA-MB-231,the inhibition ratios were 93.49% at the 4th day.Conclusion：The recombinant ranpirnase would be applied as a potential therapeutic drug,which could conjugated with a tumor specific antibody or antibody fragment.

关 键 词：豹蛙酶 融合蛋白 细胞增殖抑制 

分 类 号：Q786[生物学—分子生物学]