湘油15 PGIP9蛋白基因的克隆和蛋白序列结构分析及原核表达

运用生物信息学软件对湘油15 PGIP9基因的核苷酸、蛋白氨基酸序列进行分析,并对其蛋白结构进行预测.结果表明:湘油15 PGIP9编码区CDS长1011 bp,编码336个氨基酸的开放阅读框,分子量为37.5kDa,等电点为7.9.N端1～22个氨基酸是信号肽,且这一区域疏水性较强,具有5个潜在的N-糖基化位点.N端和C端还各具有4个参与二硫键形成的半胱氨酸残基.二级结构显示有11个α-螺旋,14个β-延伸和25个无规则卷曲.中心LRR结构域由6个串联的LRR基序组成.随后将PGIP9的CDS序列亚克隆到原核表达载体pET -32a(+)中,构建pET - 32a - PGIP9重组表达质粒,并转化E.coil BL21(DE3),25℃,终浓度为0.2 mmol/L和0.5 mmol/L的IPTG诱导2h,都成功的表达了融合蛋白pET - 32a - PGIP9,其分子量约为52kDa,发现主要以包涵体形式存在.没有可溶形式的蛋白表达.

Cloning,Sequence and Structure Analysis of Polygalacturonase Inhibiting Protein Gene（PGIP 9） of Brassica napus Xiangyou 15 and Prokaryotic Expression

Ribonucleotide and amino acid sequence of Xiangyou 15 PGIP were analyzed,the protein structure were predicted by biological information software.CDS of Xiangyou 15 PGIP 9 code area was 1 011 bp,which encoded 336 amino acid of open reading frame with a molecular mass of 37.5 kDa and isoelectric point of 7.9.1~22 amino acids were predicted to the N-terminal signal peptide with stronger hydrophobic region.The deduced amino acids sequence contained 5 potential N-glycosylation sites and 4 cysteine residues on N-terminal and C-terminal respectively,which involved in constituting the disulfide linkages.Secondary structure displayed it contained 11 α-helices,14 β-extension and 25 random coils.The center LRR structural domain was composed of 6 tandem LRRs motifs.Later,CDS of PGIP 9 was subencoded into the prokaryotic expression vector pET-32a（＋）constructing recombinant expression plasmid pET-32a-PGIP9 and was transformed into Escherichia coli BL21（DE3）.The fusion protein pET-32a-PGIP9 was successfully expressed under final concentration 0.2 mmol/L and 0.5 mmol/L IPTG at 25℃ inducing for 2 h.The molecular weight of fusion protein was 52 kDa,it mainly appeared as inclusion bodies,not appeared as soluble protein.