【作者】 陈小佳；

【导师】 林剑；

【作者基本信息】 暨南大学 ， 遗传学， 2002， 硕士

【摘要】 利用计算机生物软件DNASIS2.5辅助分析，设计了20条上游引物和1条下游引物，在不改变hbFGF氨基酸序列的前提下，对重组人碱性成纤维细胞生长因子cDNA序列的5’端TIR区域的碱基进行改造。通过PCR定点突变技术，选用大肠杆菌偏好的密码子，适当降低G+C含量，增加标准自由能变化ΔG~0，从而改变mRNA的TIR二级结构，使其更有利于mRNA的翻译表达，并由此构建了非融合rhbFGF重组载体系列pET-JN。将这二十种重组子分别转化大肠杆菌BL21(DE3)pLysS，进一步筛选得到16株表达量不等的菌株，其中有绝大部分表达率≥10％，且有5株可溶性rhbFGF的表达较高。随机选取一株rhbFGF表达率约20％并有相当可溶性蛋白表达的菌株进行大规模发酵、纯化。表达产物经SDS-PAGE电泳分析、免疫分析、活性检测，表明与hbFGF具有相同N端氨基酸序列，相同的免疫特性和生物学活性。研究结果说明采用计算机辅助分析对rhbFGF原核高效表达体系的筛选有一定的积极性。更多还原

【Abstract】 Computer software-assisted analysis is more and more important for the research of Genetics and Molecule Biology to design, screen and assess experiments. To explore high expression prokaryote system of exogenous hbFGF, twenty modified upstream primers and one downstream primer were designed by DNASIS 2.5 software and then were employed to adjust the translation initiation region (TIR) , reduce G+C content and raise A G?through nucleotide change. Using pET-3c as vector, the cloned rhbFGF genes were expressed in BL21(DE3) plysS. Most rhbFGF were expressed in E.coli ≥ 10% of total cellular protein. And 5 recombinants have much soluble rhbFGF. One of these recombinants by random chose was fermented. The target protein from the supernatant of bacteria lysate was purified. The immunology character was detected by antibody in western blot. The bioactivity of the purified rhbFGF was identical with the standard bFGF. The result showed this rhbFGF had the same amino acid sequence, bioactivity and immunology character with nature hbFGF. The conclusion indicted the computer software-assisted analysis in genetics and molecular biology has good prospect.更多还原