牙龈卟啉单胞菌肽酰精氨酸脱亚氨酶的克隆与表达

Cloning and expression of peptidylarginine deiminase of Porphyromonas gingivalis

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。

PURPOSE：To clone the peptidylarginine deiminase（PAD） gene of Porphyromonas gingivalis（P.gingivalis） and to be expressed in E.coli under the best conditions.METHODS： With P.gingivalis strains ATCC33277 genomic DNA as a template,PCR was applied to obtain gene PAD which was then inserted into linear cloning vector PMD-18-T to construct clone recon.Recombinant PMD18-T-PAD was cloned and analyzed with PCR and restriction endonuclease,and PET-28a expression vector was digested by Xhol and Ncol,their products were linked to construct expression plasmid PET-28a-PAD under certain connection system.The recombinant expression plasmid PET-28a-PAD which had been confirmed correctly was transformed to E.coli competent cells BL21 and induce the expression of PAD with IPTG of different density and time.With His Tag monoclonal antibody as the first antibody,the expressed fusion protein was characterized by Western blot.RESULTS：DNA sequencing showed that the fragment was same as the sequence published in NCBI.Under the condition of 37℃,0.5mmol/L IPTG,250r/min shaking for 6 hours,the PAD could be highly expressed.CONCLUSIONS：The PAD is successfully cloned and expressed in E.coli which can be further uesd to study the immunity of PAD and the homologues antibody preparation.