重组人干细胞因子／血小板生成素融合基因的克隆、表达及其蛋白质结构预测  

Cloning, expression and protein structure prediction of a novel SCF-TPO fusion gene

作　　者：刘楠[1] 奚永志[1] 郭斯启[1] 孙玉英[1] 袁志宏[1] 崔建武[1] 习彩霞[1] 梁飞[1] 孔繁华[1] 

机构地区：[1]军事医学科学院附属医院及国家生物医学分析中心免疫学研究室,北京100039

出　　处：《中国病理生理杂志》2005年第10期1919-1922,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.3980056);国家博士后基金资助项目

摘　　要：目的:获得重组人干细胞因子／血小板生成素(SCF-TPO)融合蛋白的高表达,预测该全新型融合蛋 白的蛋白质结构。方法:设计引物,利用RT-PCR从胎肝细胞中扩增SCF和TPO氨基端功能区片段,采用基因融合 新策略将其融合成SCF-TPO融合基因,构建pET32a／SCF-TPO融合基因重组表达载体,在E．coli BL21(DE3)plysS 中获得正确高表达,采用生物信息学方法对融合蛋白的结构特征进行模拟分析。结果:首次成功构建了pEF32a／SCF -TPO原核表达载体,并获得了融合蛋白的高表达,表达量高达40％,主要以包涵体形式表达。蛋白结构预测结果 显示,融合蛋白的等电点、抗原性及亲水性无显著改变,接头序列具有高柔性。融合蛋白除一级结构有4个氨基酸发 生改变外,原来的α螺旋和β片层无改变。结论:获得了SCF-TPO融合蛋白的高表达,结构预测融合蛋白的设计符 合要求。为进一步研究SCF-TPO融合蛋白的生物学特性奠定了基础。AIM： To obtain the high expression of recombinant human stem cell factorthrombopoitin （SCF-TPO） fusion gene and predict its structure property. METHODS：Four primers were designed according to known sequence of TPO and SCF to amplify the functional amino acid domain of TPO and SCF by RT- PCR, respectively from fetus hepatocytes. The expression plasmid pET32a/SCF- TPO was constructed by VOE gene fusion technique and expressed in BL21（DE3）plysS. The fusion protein property, such as second structure, flexibility, and hydrophilicity were predicted by DS Gene and Protscale software. RESULTS： The expression vector, pET32a/SCF- TPO was constructed and the high expression of the SCF/TPO fusion protein was obtained, with the expression amount of up to 40% of the total cellular protein. DS Gene1.5 and Protscale predict no new antigenicity in fusion protein, and the second structure and ioelectric point have no changes except four amino acids change in first structure. There are high flexibility and low hydrophilicity in the linker peptide. CONCLUSION： High expression of SCF - TPO fusion protein has been obtained and protein prediction shows that the fusion protein design is reasonable, which lay foundation for further study of biological fundation of SCF - TPO fusion protein.

关 键 词：干细胞因子 血小板生成素融合蛋白质 原核表达 蛋白质结构 

分 类 号：R392.11[医药卫生—免疫学]