摘要
目的:构建蓝藻抗病毒蛋白(CV-N)基因原核表达载体,表达、纯化并复性其重组蛋白。方法:根据GenBank中CV-N的全基因序列,人工合成CV-N序列,进行PCR扩增。将扩增的PCR产物克隆至原核表达载体pET30a(+)上,构建重组表达载体pET30a-CV-N,测序鉴定后,转化E.coliBL21(DE3),IPTG诱导表达蛋白,SDS-PAGE和Westenblot鉴定表达产物,Ni Sepharose柱纯化目的蛋白,稀释法复性蛋白。结果:重组质粒pET30a-CV-N测序结果显示,插入片段为303 bp,与GenBank中的CV-N基因序列完全相同。经IPTG诱导的CV-N基因可在E.coli中高效表达;SDS-PAGE分析表明,相对分子质量11000处出现一条新条带,主要以包涵体形式存在,37℃诱导2、4、6、8 h后,表达蛋白分别占菌体蛋白的3.87%、19.10%、33.98%和31.58%。Western blot结果显示,重组蛋白能与抗His单抗特异性反应。将诱导6 h的菌体超声破碎后,Ni Sepharose柱纯化,相对分子质量11 000处显示出清晰的单一条带,且蛋白复性效果良好。结论:成功地构建了CV-N原核表达载体,表达并纯化出重组蛋白,为深入研究其抗病毒的活性奠定了基础。
Objective ： To construct the prokaryotic vector of cyanovirin-N （CV-N） gene and to express, purify and renature its recombinant protein. Methods ： The DNA coding sequence for CV-N was synthesized and amplified by PCR, the resulting PCR product was cloned into pET30a （ ＋ ） vector and sequenced. The confirmed recombinant clone pET30a（ ＋ ） -CV-N was transformed into E. coli BL21 （ DE3 ） and induced to express proteins by IPTG. The expression of the proteins was analyzed by SDS-PAGE and Western blotting and subsequently purified with Ni Sepharose column. The protein was renatured by dilution method. Results： The sequencing results of pET30a （ ＋ ）-CV-N vector demonstrated that the inserted CV-N gene（303 bp） was the same as that of indicated that the molecular weight of the expressed CV-N gene in GeneBank. The results of SDS-PAGE protein was about 11 000 and the expression rates of the total bacterial proteins after 2 h, 4 h, 6 h and 8 h induction were 3.87%, 19. 10%, 33.98%, 31.58% respectively. Western blotting analysis proved that the recombinant protein had good reactivity against monoclonal antibody anti-6 × His. The purified protein was about 11 000 and renatured successfully. Conclusion： The prokaryotic expression system of CV-N gene has been successfully constructed and the purified recombinant protein provides a basis for further research and development of CV-N as an antiviral microbicide.
出处
《医学研究生学报》
CAS
2007年第11期1139-1142,共4页
Journal of Medical Postgraduates
基金
山东省科技攻关资助项目(批准号:2005GG3202068)
