绿色微囊藻抗HIV凝集素MVL蛋白的原核表达及纯化

目的原核表达并纯化绿色微囊藻抗HIV凝集素MVL蛋白,为进一步研究其生物学活性及其应用奠定基础。方法以绿色微囊藻Microcystis viridis NIES-103基因组DNA为模板,PCR扩增MVL基因阅读框序列,并克隆至pET-30b(+)载体中,构建重组表达质粒pET-30b-MVL,经PCR、测序鉴定后,转化感受态E.coliBL21(DE3),IPTG诱导表达。表达的蛋白采用Ni-NTA亲和柱层析纯化及复性。结果重组表达质粒pET-30b-MVL序列完整,插入的基因片段全长345bp,与GenBank中公布的MVL基因序列一致。重组菌的最佳诱导表达条件为:诱导起始浓度A600=0.5左右,IPTG浓度0.5mmol/L,诱导温度37℃,诱导时间4h,诱导培养基采用LB。以此条件诱导,在相对分子质量约20000处可见目的蛋白表达条带,表达量可占菌体总蛋白的47%,且表达蛋白主要以可溶性形式存在。纯化的重组蛋白纯度达95%以上,质谱鉴定为甘露糖结合凝集素。结论已成功地原核表达并纯化了绿色微囊藻抗HIV凝集素MVL蛋白。

Prokaryotic Expression and Purification of Microcystis viridis Lectin Protein against HIV

Objective To express Microcystis viridis lectin(MVL)protein against HIV in prokaryotic cells, purify the expressed product and lay a foundation of further study on its biological activity and application. Methods The open reading frame (ORF)sequence of MVL gene was amplified by PCR using the genomic DNA of Microcystis viridis NIES-103 as a template and cloned into vector pET-30b (+). The constructed recombinant plasmid was identified by PCR and sequencing, then transformed to competent E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by Ni2+-NTA affinity chromatography then re-naturalized. Results The gene fragment at a full-length of 345 bp was inserted into recombinant plasmid pET30b-MVL, with an identical sequence to that of MVL gene reported in GenBank. The condition for induction of recombinant E. coli and expression of target protein was optimized as follows: the recombinant E. coli at an original concentration (A600)of about 0.5 was induced with 0. 5 mmol / L IPTG in LB medium at 37℃ for 4 h. The expressed product under the optimized condition, with a relative molecular mass of about 20 000, mainly existed in a soluble form and contained 47% of total somatic protein. The purified recombinant protein reached a purity of more than 95% and was identified as mannose-bound MVL by mass spectrography. Conclusion MVL protein against HIV was successfully expressed in prokaryotic cells and purified.