单核细胞增多性李氏杆菌野毒株InlB的分子特征及其表达和纯化研究

Molecular characteristics of InlB protein of Listeria monocytogenes wild strain and it's expression and purification

摘要:

采用PCR技术扩增单核细胞增多性李氏杆菌TA野毒株内化素B(InlB)基因,进行编码分子的序列和结构分析,并克隆入大肠杆菌表达载体pET28a中诱导表达。该基因全长1893bp,编码630个氨基酸,其中前35个氨基酸残基构成信号肽序列。在推导的InlB蛋白氨基酸序列中,从N端到C端分别包括1个α-螺旋的Cap结构域、6个富含亮氨酸的重复基序(LRR)、1个免疫球蛋白样结构域(IR)、1段B重复序列和3个串联的GW结构域,同时还存在5个潜在的N-联糖基化位点,Leu占所有氨基酸残基的10.2%。与GenBank已经报道的18个不同流行株InlB基因相比,核苷酸和推导的氨基酸序列的同源性分别在91.1%~99.6%和92.3%~99.8%之间。重组菌菌体裂解物经SDS-PAGE和Western blot分析证实该基因已经正确表达。用Ni2+亲和层析柱纯化了InlB重组蛋白。

Abstract:

Internalin B of Listeria monocytogenes TA wild strain was amplified by PCR method and sequenced, then subcloned into prokaryotic vector pET28a for expression. The complete length of InlB gene was 1893 bp, encoding 630 amino acids. The front 35 amino acids consisted of signal peptide. There were one cap domain with α-helix region, 6 leucine-rich repeat motifs, one immunoglobulin (Ig)-like domain, one B repeat sequence, 3 direct repeat GW domains and 5 N-glycosylation sites. The amino acid of leucine amounted to 10.2 percent in all amino acids of deduced sequence. Compared the InlB genes with that of other 18 isolates reported in GenBank, the identities of nucleotide sequence and deduced amino acid sequence were 91.1%～99.6% and 92.3%～99.8%, respectively. Expressed InlB protein was detected by SDS-PAGE and confirmed by western blot. Recombinant protein was successfully purified by Ni2+ affinity chromatograph column.