摘要
目的 以构建的pET28a为表达载体,探讨人 IFN-α2b基因在pET系统中的表达。方法 合成引物,通过PCR扩增人 IFN-α2b基因并引入点突变,在不改变天然 IFN-α2b氨基酸序列的前提下,使原 IFN-α2b基因4个大肠杆菌稀有密码子改变为偏爱密码子,在起始密码子后,编码前16个氨基酸的碱基序列均成为大肠杆菌的偏爱密码子。将PCR产物次级克隆人pET28a载体,构建重组表达载pET28a-IFN-α2b。筛选正确的重组表达质粒pET28a-IFN-α2b转入感受态表达菌BL21(DE3)中,IPTG诱导表达,粗提并检测 IFN-α2b活性。结果 重组质粒pET28a-IFN-α2b在宿主菌 BL21(DE3)中表达出的rhIFN-α2b以可溶性蛋白形式存在于粗提上清液中,与原来 pBV889系统表达的干扰素相比,具有更高的生物学活性。结论 应用pET表达系统可表达出具有更高生物学活性的rhIFN-α2b蛋白。
Objective To study the expression of rhIFN-α2b gene in pET system using constructed vector.Methods Amplify human IFN-α2b gene by PCR using synthesized primers, and change the 4 rare codons into biased ones of E. coli by introducing site-directed mutagenesis. In this way, all the base sequences encoding the first 16 amino acids after the start codon were changed into E. coli biased, however, the ami-no acid sequence identical to natural IFN-α2b was unchanged. Subclone the PCR product into pET28a vector. Transform the recombinant plasmid pET28a- IFN-α2b to competent E. coli strain BL21(DE3) and express under induction of EPTG. The expressed product was primarily purified and detected for IFN-α2b activity. Results The expressed rhIFN-α2b existed in the form of soluble protein, and showed high biological activity compared with that expressed in pBV889 system. Conclusion pET system can be used for the expression of rhIFN-α2b protein with higher biological activity.
出处
《中国生物制品学杂志》
CAS
CSCD
2003年第3期147-150,共4页
Chinese Journal of Biologicals
