摘要
目的构建多拷贝脑钠肽(BNP)基因重组表达质粒,并探讨BNP基因的拷贝数与其表达水平的相关性。方法通过人工合成和PCR技术扩增BNP基因,将其克隆至载体pCW111中,构建表达质粒pBNP11,并通过亚克隆构建含有不同数量表达盒的正向串联表达质粒,分别转化大肠杆菌DH5α和BL21(DE3),经温度诱导表达,SDS-PAGE分析,并经激光扫描测定目的蛋白的表达量。结果测序及酶切鉴定证明1～3拷贝BNP重组表达质粒构建正确,重组蛋白在大肠杆菌DH5α和BL21(DE3)中均可获得表达,表达量分别为菌体总蛋白的8.12%、9.94%、和9.18%。结论已构建了多拷贝BNP的重组表达质粒,BNP的表达水平随BNP拷贝数的增加而升高,但并未成倍增加。
Objective To construct the recombinant expression vector for multi-copy B-type natriuretic peptide （BNP） gene and explore the relationship between the gene copy number and expression level. Methods BNP gene was amplified by PCR using synthetic primers and cloned into vector pCW111 to construct recombinant plasmid pBNP11, based on which a series of recombinant plasmids containing muhi-copy expression cassettes were constructed and transformed to E. coli DHSct and BL21 （DE3） separately for expression under thermal induction. The expressed product was identified by SDS-PAGE and determined for expression level by laser scanning. Results Both sequencing and restriction analysis proved that the recombinant plasmids containing 1-3 copy expression cassettes were constructed correctly. Recombinant BNP protein was expressed in both E. coli DH5ct and BL21 （DE3）. The expressed products of recombinant plasmids containing 1, 2 and 3 copy expression cassettes contained 8. 12%, 9. 94% and 9. 18% of total somatic protein respectively. Conclusion The recombinant expression vector for muhi-copy B-type BNP gene was successfully constructed. The expression level of BNP increased, but not in proportion, with the increasing gene copy number.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第12期1062-1065,共4页
Chinese Journal of Biologicals
基金
深圳大学科研启动基金资助项目(200711)
关键词
脑钠肽
质粒拷贝数
表达盒
原核表达
B-type natriuretic peptide（BNP）
Plasmid copy number
Expression cassette
Prokaryotic expression
