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目的:构建牙龈卟啉单胞菌外膜蛋白肽酰精氨酸脱亚氨酶(PAD)克隆表达重组子,转化于大肠杆菌BL21中,并在最适宜条件下诱导表达。方法:以牙龈卟啉单胞菌ATCC33277全基因组DNA为模板,利用PCR技术获得目的基因PAD,将扩增得到的PAD基因定向插入线性克隆载体PMD18-T Vector中,得到克隆重组子PMD18-T-PAD。经PCR和双酶切鉴定正确的克隆重组子PMD18-T-PAD与表达载体PET-28a经Xhol和Ncol双酶切后,在一定连接体系下,连接构建表达质粒PET-28a-PAD。鉴定正确的原核重组表达质粒PET-28a-PAD,转化大肠杆菌BL21感受态细胞,在不同浓度异丙基硫代-β-D-半乳糖苷(IPTG)及时间诱导下表达融合蛋白。以抗His Tag单克隆抗体为一抗,Western免疫印迹鉴定。结果:DNA测序结果表明,PAD与NCBI核酸数据库中收录的PAD序列同源性达100%;37℃,IPTG浓度为0.5mmol/L,250r/min振摇培养6h的诱导条件下,PAD可高效表达。结论:本实验成功构建了PAD的克隆表达重组子,并在大肠杆菌中表达了PAD蛋白,为进一步研究PAD的免疫学性能及相应的抗体制备奠定了基础。
Objective: To construct the recombinant Porphyromonas gingivalis peptidase arginine deiminase (PAD) recombinant expression vector and transform it into E. coli BL21 and express it under the most suitable conditions. Methods: The whole genome DNA of Pseudomonas gingivalis ATCC33277 was used as a template to obtain the target gene PAD by PCR. The amplified PAD gene was inserted into the vector PMD18-T vector to obtain the cloned recombinant PMD18-T- PAD. The correct cloned recombinant PMD18-T-PAD and PET-28a were identified by PCR and double digestion. The recombinant plasmid was digested by Xhol and Ncol and ligated to construct PET-28a-PAD under certain connection system. The recombinant prokaryotic expression plasmid PET-28a-PAD was identified and transformed into competent E. coli BL21 cells. The fusion protein was expressed under different concentrations of isopropylthio-β-D-galactoside (IPTG) and time induction. Anti-His Tag monoclonal antibody as a primary antibody, Western immunoblot identification. Results: The results of DNA sequencing showed that PAD was 100% homologous to the PAD sequence contained in the NCBI nucleic acid database. Under the conditions of 37 ℃, IPTG concentration of 0.5mmol / L and shaking culture at 250r / min for 6h, PAD was efficient expression. CONCLUSION: The cloning and expression recombinant PAD was successfully constructed in this experiment and the PAD protein was expressed in E. coli, which laid the foundation for further study on the immunological properties of PAD and the corresponding antibody preparation.