十二指肠钩虫ASP-2基因密码子优化及在大肠杆菌中高效表达

目的:获得十二指肠钩虫(Ancylostoma duodenale)分泌蛋白2(mAd-ASP-2)编码基因,构建其高效表达的大肠杆菌表达体系?方法:以RT-PCR的方法得到克隆了编码mAd-ASP-2的成熟肽全基因序列,克隆入原核表达载体pET-22b(+)中,组装成表达载体pET-22b-mAd-ASP-2?利用大肠杆菌偏爱密码子和GenScript rare codon analysis 软件获得优化密码子优化的mAd-ASP-2*基因序列并人工合成该基因,将其克隆入原核表达载体pET-22b(+),组装成表达载体pET-22b-mAd-ASP-2*?将这2个表达载体分别转化入大肠杆菌表达菌株Rosetta-gami-2(DE3)中,经IPTG诱导表达?结果:同样条件下,密码子优化的mAd-ASP-2*基因比优化前能够获得较高的表达,且以可溶形式存在?利用His Trap HP亲和柱获得了纯化的重组蛋白mAd-ASP-2*,确定了纯化重组蛋白咪唑洗脱液最佳浓度为200 mmol/L?Western blot分析结果进一步显示,该融合蛋白可与鼠抗His-tag抗体发生特异性结合,说明表达蛋白为目的蛋白?结论:通过密码子优化实现了十二指肠钩虫ASP-2的高效表达,为进一步研究Ad-ASP-2的功能和以其作为血清学诊断抗原或保护性疫苗奠定了基础?

A study on codon optimization and expression of mature Ancylostoma-secreted protein 2 of Ancylostoma duodenale in Escherichia coli

Objective:To obtain the cDNA encoding Ancylostoma-secreted protein 2 of Ancylostoma duodenale(mAd-ASP-2)and construct the expression system of mAd-ASP-2 in E.coli. Methods:The cDNA encoding of the mature Ancylostoma-secreted protein 2 of Ancylostoma duodenale(mAd-ASP-2)was cloned by RT-PCR. The mAd-ASP-2 was inserted into the pET-22b(+)vector to construct prokaryotic expression plasmid pET-22b-mAd-ASP-2. An optimized codon mAd-ASP-2 gene,designated mAd-ASP-2*,was designed and synthesized based on optimization of synonymous codon bias of E. coli,without modification of amino acid sequence,and inserted into pET-22b(+)to construct prokaryotic expression plasmid pET-22b-mAd-ASP-2*. The two expression plasmids were transformed into E. coli Rosetta-gami-2(DE3)cells. Results:SDS-PAGE analysis showed that the expected recombinant mAd-ASP-2 fusion protein was expressed in E. coli Rosetta-gami-2(DE3)cells induced by isopropyl-3-thiogalactopyranoside(IPTG),and the mAd-ASP-2* with the optimized codon was more highly expressed than the original mAd-ASP-2 in the soluble fraction of E. coli cell lysates. The recombinant mAd-ASP-2* fusion protein was purified using His Trap HP affinity column. Western blot analysis showed that the recombinant mAd-ASP-2* protein was combined with mouse anti-His-Tag. So the expressed protein was definitely confirmed to be the target protein. Conclusion:The expression system of mAd-ASP-2 in E. coli was constructed successfully,which provided a fundamental basis for further studies on subunit vaccine for prevention of hookworm infections.