摘要
设计引物以pCDPT2质粒(含PEA全序列)为模板亚克隆获得PEA功能区Ⅰa及功能区Ⅱ基因片段(1100 bp); 提取人染色体DNA作为模板,利用一对设计引物扩增获得了人组蛋白H4基因片段(316 bp);将两基因片段连接,构建了融合基因中间载体pMD-18T-PEA-H4;融合基因经NcoⅠ及XhoⅠ酶切,以正确阅读框架插入相同酶切的pET-28A中,经酶切分析及PCR扩增检测,筛选到阳性克隆,其质粒测序结果表明成功地构建了毒性基因缺失的PEA与人组蛋白H4融合基因的原核表达载体,为进一步表达并获得大量的融合蛋白奠定了基础。
A 1100 bp PEA fragment which contain structural domain Ⅰa and Ⅱ was amplified using cloned pCDPT2 plasmid as template by PCR. Human genome DNA was extracted then as template, human histone H4 gene(346 bp) was amplified by PCR. The two fragment was melted and linked with pMD-18T victor. Then for correct reading code frame, the recombinant plasmid pMD-18T-PEA-H4 was cleaved with NcoⅠ/XhoⅠand cloned into the pET-28c expression victor. Positive clone was identified successfully.
出处
《黑龙江八一农垦大学学报》
2002年第3期60-63,共4页
journal of heilongjiang bayi agricultural university
