禽鹦鹉热嗜性衣原体PmpD基因N端区的克隆与表达

Cloning and prokaryotic expression of N-domain of PmpD gene of Chlamydophila psittaci

为探讨禽鹦鹉热嗜性衣原体PmpD基因的生物学功能,本实验根据禽类鹦鹉热嗜性衣原体Cal-10株全基因序列设计引物,用PCR法扩增禽嗜性衣原体PmpD基因N端区,将特异性目的基因片段克隆入PBS-TII载体,经测序后确认为目的基因,并提交GenBank。试验扩增的禽鹦鹉热嗜性衣原体Cal株PmpD基因与禽鹦鹉热衣原体6BC株的PmpD基因和氨基酸序列的相似性均达99%,与流产衣原体成员嗜流产衣原体S26/3株、嗜豚鼠衣原体(GPIC)、嗜猫衣原体(Fe/C-56)核苷酸相似性分别为89%、82%和77%。PmpD基因N端区基因亚克隆到pET-32a(+)载体上,重组表达载体转化BL21(DE3)表达宿主菌,以IPTG诱导该重组菌,结果表达了约63 ku大小的融合蛋白;分别用禽鹦鹉热嗜性衣原体多克隆抗体、猪、羊流产嗜性衣原体多克隆抗体与表达出的重组蛋白进行Western blotting检测,结果表明该蛋白与禽源衣原体多克隆抗体呈阳性反应,与猪、羊源衣原体多克隆抗体呈阴性反应,显示该重组蛋白具有种属特异性。本研究首次表达了禽类鹦鹉热嗜性衣原体PmpD基因的N端区的重组蛋白,为进一步研究其生物学功能提供依据。

In order to explore the function of the PmpD gene in preventing and diagnose of the avian,the N-domain of the PmpD gene was cloned by PCR method based on the whole gene sequence of the avian Chlamydophila psittaci CAL-10 strain.Specific target gene was cloned into the PBS-TII vector and identified by the sequence analysis.The target gene and deduced peptide sequence were compared to the N-domain gene sequence of the characterized C.psittaci 6BC strain.They were found to be 99% identical to the N-domain gene sequence that of C.psittaci 6BC strain.Compared to other C.abortus strain the similarity of the gene sequence and peptide arranged form 77% to 89%.Approximately 63 ku recombinant fusion protein was expressed after the N-domain was subcloned into pET-32a vector,the recombinant vector was transformed into the host bacteria BL21（DE3）and the recombinant fusion protein,63 kD,was highly expressed after induction with IPTG.The recombinant protein could react specifically with polyclonal antibody of C.psittaci by Western-blot analysis,but it could not react with polyclonal antibody originated form swine C.abortus and sheep C.abortus.The results showed that the PmpD have species-specificity.In conclusion,the N-domain was successfully cloned and expressed in prokaryotic vector,the current study might play a key role in exploring the fundamental biological function.