原核表达载体pKpL5的构建及应用研究

Construction of Prokaryotic Expressing Plasmid pKpL5

目的构建基因工程生产用高效原核融合表达质粒载体。方法在原核表达质粒pKpL4起始密码下游嵌入人工合成的六聚组氨酸编码区、16s RNA3端互补区、凝血酶识别位点编码区接头,构建成原核表达质粒pKpL5,并将人生存素单体、双体及利什曼原虫LACK抗原基因编码区分别克隆入pQE32、pKpL4和pKpL5进行表达,SDS-PAGE电泳分析各目的蛋白的表达量。结果人生存素单体、双体及利什曼原虫LACK抗原基因读码框在pQE32和pKpL4质粒载体内均不表达,而在pKpL5内,其目的蛋白表达量分别占细菌总蛋白量的5.1%,19%和35%。结论pKpL5可用作通用原核高效表达质粒载体表达各种目的基因。

Objective In order to construct the prokaryotic expressing plasimad for gene engineering product. Methods The 6×His adaptor, 16s RNA 3＇-end pairing region adaptor and thrombin adaptor were inserted at the downstream of ATG of the prokaryotic expressing plasimad pKpL4 to design pKpL5, then, the encoding region of human survivin single, human survivin .double and LACK antigens of Leishmania donovvani were expressed in pKpL5, pKpL4 and pQE32, and the target protein was analyzed by SDS-PAGE. Resuits The encoding regions of human survivin single, human survivin double and LACK antigens of Leishmania donovvani were not expressed when cloned in pKpL4 and pQE32; the target protein expression levels were 5.1% , 19% and 35% respectively in total E. coli protein when cloned in pKpLS. Conclusion The data suggested that pKpL5 could be used as tool expression plasmid to produce recombinant protein of general encoding region.