单核细胞增多性李氏杆菌TA分离株P60基因的克隆与表达

Cloning and expression of P60 gene of TA strain of Listeria monocytogenes

目的对单核细胞增多性李氏杆菌TA分离株入侵相关蛋白P60基因进行克隆、测序和表达。方法利用PCR方法扩增P60基因,克隆入T载体中进行测序,并亚克隆到大肠埃希菌表达载体pET28a中进行诱导表达。结果该基因全长1122bp,编码374个氨基酸,其中前25个氨基酸残基构成信号肽序列。与GenBank已经报道的5个不同分离株P60基因相比,核苷酸和推导的氨基酸序列的同源性分别在96.4%～99.6%和97.1%～99.8%之间。在推导的P60蛋白氨基酸序列中,存在9个潜在的N-联糖基化位点,5个Thr-Asn重复序列区,Thr占所有氨基酸残基的16.3%。重组菌菌体裂解物经SDS-PAGE电泳可检测到分子质量单位为45.6ku的重组蛋白,Western blot分析表明重组蛋白为特异的。经凝胶薄层扫描,重组蛋白表达量可占菌体蛋白的31.6%。结论成功克隆和表达了单核细胞增多性李氏杆菌TA分离株P60基因。

Objective To clone and express P60 gene of strain TA of Listeria monocytogenes. Methods P60 gene was amplified by PCR method and cloned into pMD18-T vector for sequencing. Then P60 gene was subcloned into prokaryotic expressing vector pET28a and transformed into host Escherichia coli strain. BL21 （DE3） for expression. Results The complete length of P60 gene was 1 122 bp, encoding 374 amino acids. The front 25 amino acids consist of signal peptide. Compared with the P60 genes of other isolates reported in GenBank, the homology of nucleotide sequence and deduced amino acid sequence were 96.4 %- 99.6 % and 97.1% - 99.8 %, respectively. There were 9 N-glycosylation sites, 5 direct repeat regions and 16.3 percent threonine in the deduced amino acid sequence. The expressed P60 protein was detected by SDS-PAGE and confirmed by western blot. It revealed that it had an expressed protein with the molecular weight of 45.6 ku, which amounts to 31.6% in the total protein of bacteria by the assaying of gel scanning. Conclusion P60 gene of strain TA of L. monocytogenes is successfully cloned and expressed in E. coll.