【摘要】 利用同尾酶法构建人胰高血糖素样肽-2类似物{PP-h[Gly2]GLP-2(1-35)}基因串联体重组质粒,实现目的基因在大肠杆菌中高效表达。通过PCR扩增获得PP-h[Gly2]GLP-2(1-35)基因片段,利用同尾酶BamHⅠ和BclⅠ产生相同的粘性末端,经连接、转化、筛选等操作构建其基因串联体。经DNA测序分析,成功构建PP-h[Gly2]GLP-2(1-35)的二、四、六、八拷贝基因串联体。SDS-PAGE显示,各重组菌经D-乳糖诱导后均表达目的融合蛋白,其相对分子质量与预期结果相符,目的肽含量并非随串联个数增加而明显增加,改变融合伙伴后,目的肽的表达量从0.106 g/L提高到0.372 g/L,为大量制备和纯化PP-h[Gly2]GLP-2(1-35)奠定了基础。更多还原

【Abstract】 The multimers gene of Human Glucagon-like Peptide-2 Analog {PP-hGLP-2(1-35)} was constructed by isoaudamers and expressed high-efficiently in Eschericha.coli BL21(DE3).The PP-hGLP-2(1-35) gene was cloned by PCR,and then the multimers were constructed by a pair of isoschizomers,BamHⅠ(G^GATCC)and BclⅠ(T^GATCA),which could produce the same cohesive end(GATC).When they cut its target DNA sequence,their digested fragments could be ligated by T4 ligase and could not be excised by BamHⅠor BclⅠagain.The recombinant plasmids containing different copies of interest gene(multimers) were constructed after ligation,transformation,screening and so on.The recombinant plasmids containing 2,4,6,8 copies of interest gene(multimers) were successfully constructed respectively and analyzed by DNA sequencing.All the recombinant plasmids containing multimers could express the target protein by D-Lactose and their relative molecular mass met the expectation.The concentration of target peptide was not always increasing obviously with the number of multimers,but the concentration of target peptide increased from 0.106 g/L to 0.372 g/L,when the fusion parter was changed.It benefits for mass preparation and purification of PP-hGLP-2(1-35).更多还原