蓖麻蚕微孢子虫孢壁蛋白NpSWP12基因的克隆分析与原核表达

Cloning,analysis,and prokaryotic expression of a gene coding for the spore wall protein NpSWP12 of Nosema philosamiae

目的克隆并表达蓖麻蚕微孢子虫孢壁蛋白NpSWP12。方法根据家蚕微孢子虫基因组数据库信息设计简并引物进行PCR扩增,克隆得到蓖麻蚕微孢子虫孢壁蛋白NpSWP12基因。利用生物信息学软件对NpSWP12的基因与蛋白序列进行分析与结构功能预测。将该蛋白基因克隆到原核表达载体pET-30a(+)中,转化表达载体至大肠埃希菌BL21(DE3)菌株,经IPTG诱导后进行SDS-PAGE与Western blot检测。结果蓖麻蚕微孢子虫孢壁蛋白NpSWP12基因长687bp,编码228个氨基酸残基(基因登录号:KT287071),预测蛋白质分子质量单位为26.6ku,等电点(pI)为5.96。蓖麻蚕微孢子虫孢壁蛋白NpSWP12基因结构为单外显子结构,编码的蛋白中α螺旋占77.19%。该编码蛋白含有1个BAR结构域(Bin/Aphiphysin/Rvs domain),属BAR结构域蛋白质超家族成员。NpSWP12与家蚕微孢子虫孢壁蛋白NbSWP12核苷酸序列序列相似性为95.2%,氨基酸序列相似性为96.9%。表达载体NpSWP12/pET-30a(+)转入BL21(DE3)菌株,IPTG诱导,得到的重组蛋白rNpSWP12与理论蛋白分子质量大小(30ku)相符。Western blot鉴定。结论成功克隆了蓖麻蚕微孢子虫孢壁蛋白NpSWP12基因,构建的NpSWP12/pET-30a(+)重组原核表达载体表达预期分子质量的融合蛋白,该蛋白能被相应抗体识别,为进一步研究NpSWP12的细胞定位和功能奠定了基础。

Objective To clone and express the spore wall protein NpSWP12 of Nosema philosamiae Methods The NpSWP12 gene of N. philosamiae was cloned with degenerate primers （NpSWP12F/NpSWP12R） designed based on ge- nomie data on N. bornbycis. The NpSWP12 gene and the protein it codes for were analyzed bioinformatically and the gene ＇s function was predicted using bioinformatics software such as DNAstar, GSDS, NPS@, and NetNGlyc 1.0 Server. The gene sequence of NpSWP12 coding for a mature peptide was cloned into the prokaryotic expression vector pET-30a （＋）. The expression vector was transformed into an Escherichia coli BL21 （DE3） strain and expression was induced with iso- propylβ-D-1-thiogalactopyranoside （IPTG, final concentration 1 retool/L）. The recombinant protein was detected using SDS-PAGE and Western blotting. Results The sequence of the NpSWP12 gene of N. philosamiae had 687 nucleotides coding for a polypeptide of 228 amino acids with a molecular weight of 26.6 ku and an isoelectric point of 5.96 （GenBank No. KT287071）. The structure of the NpSWP12 gene was a single exon. The alpha helix accounted for 77.19% of the protein. The NpSWP12 protein contained one Bin/amphiphysin/Rvs （BAR） domain and belonged to the BAR superfami- ly. The NpSWP12 of N. philosamiae shares a high sequence similarity with N. bombycis NpSWP12, as indicated by a nucleotide sequence similarity of 95.2% and an amino acid sequence similarity of 96.9~/oo. The NpSWP12/pET-30a（ ＋ ） expression vector was constructed and transformed into E. coli BL21 （DE3）. Expression was induced with 1 mmol/L IPTG for 6 hours at 37？ C. The molecular weight of the recombinant protein rNpSWP12 was consistent with the theoret- ical value （30 ku）. The results were confirmed by Western blot analysis. Conclusion The NpSWP12 gene of N. phi-losamiae was successfully cloned. A prokaryotic expression plasmid, NpSWP12/pET-30a（＋）, that expressed the fusion protein rNpSWP12 was successfully constructed. The fusion protein was recognized by anti-His-tag antibodies. This re- search has laid the foundation for further study of the cellular localization and functions of NpSWP12.