通过乙型肝炎病毒核心基因5'端的修饰使核心抗原在大肠杆菌中高效表达

为了使乙型肝炎病毒核心抗原(HBcAg)在大肠杆菌中获得高效表达,本文首次采用一种新方法对核心基因前区进行改造与修饰,即用限制性内切酶Taq I从核心基因内部5′端切开,去除核心基因起始信号ATG及ATG 5′端上游的全部前核心区(Precore),再化学合成一段既包含核心基因起始信号又具有多种功能的DNA片段。将两者拼接重组到表达载体pUC9上,转化受体菌,成功地获得高效表达HBcAg的菌株。用ELISA法检测,表达滴度为1:80000。表达产量占菌体总蛋白的16％。其菌体裂解液经免疫电镜观察,可见到成堆聚集的典型HBcAg颗粒。抗原单体分子量约为22000道尔顿,双体为44000。与目前国内外所普遍采用的方法比较,本文的方法有许多明显的优点,可用于其它基因改造。

HIGH LEVEL EXPRESSION OF HBcAg IN E.coli BY NIODIFICATION OF THE 5' END OF HBc GENE

HBcAg is an important reagent in clinical diagnosis, epidemiology and research works. In order to get high expression of HBcAg, we made some modification of the structure of 5' end of HBc-gene by a novel method. The Bam H I-digested fragment containing the whole HBc-gene was prog-ressively cleaved by using Taq I to remove the initiation codon ATG of HBcAg and the secondary structure-like a stem with a loop before gene C which can obstruct the expression of HBcAg. A special piece of DNA se quence about 17 bp in length containg the ATG and a polylinker was chemically synthesized by DNA-synthesizer. After ligation of this DNA piece to the HBc fragment without the initiation codon, the whole fragment was inserted into the polylinker of the plasmid pUC9 under the control of the Lac-promotor. The recombinant plasmid was traasformed into the host E.coli JM103. Extremely high expression of HBcAg was obtained. The ELISA titer of the HBcAg expressed is 1:80000, the specific protein of HBcAg is 16 percent of total bacterial protein. Under the immune electron microscopy, the expressed HBcAg is typical particles of HBcAg but in an aggregated form. Its morphology and size are as same as HBcAg derived from liver. The molecular weights of HBcAg in the product are about 22 000 Daltons as monomer and 44 000 Daltons as dimer. These results showed that the key of high expression of HBcAg is to remove the second-ary structure in the precore region.