目的构建牙龈卟啉单胞菌胶原酶基因prtC原核表达系统并鉴定其表达产物的抗原性和免疫反应性,确定牙周损伤与其局部PrtC水平之间的关系.方法采用高保真PCR从牙龈卟啉单胞菌ATCC 33277 和47A-1株扩增出全长prtC基因片段,T-A克隆后测序.采用pET32a质粒和大肠杆菌BL21DE3株构建prtC基因原核表达系统,并用不同浓度的IPTG诱导目的重组蛋白rPrtC的表达.采用Western blot检测rPrtC的抗原性和免疫反应性.建立ELISA,检测人慢性牙周炎龈下菌斑标本中的PrtC水平.结果牙龈卟啉单胞菌ATCC 33277 和47A-1株prtC基因核苷酸序列完全相同.与报道的相应序列比较,克隆的prtC基因核苷酸和氨基酸序列相似性分别为98.46% 和99.07%.在不同浓度的IPTG诱导下, rPrtC的产量高达细菌总蛋白的50%左右.rPrtC能与牙龈卟啉单胞菌全菌抗体结合,并能有效地诱导家兔产生特异性抗体.91.39%的人慢性牙周炎龈下菌斑标本中可检出PrtC,重度牙周炎标本中PrtC水平明显高于轻度牙周炎(P＜0.05).结论本研究成功地构建了牙龈卟啉单胞菌prtC基因高效原核表达系统.rPrtC具有良好的抗原性和免疫反应性,可作为研制牙龈卟啉单胞菌疫苗和血清学检测试剂盒的候选抗原.人慢性牙周炎的牙周破坏程度与局部PrtC水平密切相关.%Porphyromonas gingivalis is a specific causative agent of human chronic periodontitis. This anaerobe produce collagenase PrtC encoded by prtC. To constructed the prokaryotic expression system for the prtC gene from P.gingivalis so as to be used to explore antigenicity and immunoreactivity of prtC gene product as well as the relationship between the local level of PrtC and the periodontitis damage, the entire length of prtC genes fragment from the ATCC-33277 and 47A-1 strains of P.gingivalis was amplified by PCR. After T-A cloning and sequencing, the prokaryotic expression system for prtC was constructed by using pET32a plasmid and E.coli BL21DE3 strain. The expression of the target recombinant PrtC protein was induce with different concentrations of IPTG. Western blot assay was used to detect the antigenicity and immunoreactivity of PrtC protein, and ELISA assay was used to detect the PrtC level in the subgingival plaque samples of patients with chronic periodontitis. The experimental results showed that the nucleotide sequences of the prtC genes from ATCC-33277 and 47A-1 strains of P.gingivalis were entirely identical, and the sequence similarities of nucleotides and amino acids were 98.46% and 99.07% respectively. Under the induction of different concentration of IPTG, the output of recombinant expressed product PrtC may reach up to 50% of the total bacterial proteins. It was also proved that the recombinant PrtC could bind with antibody against the whole cell of P.gingivalis, and could induce the production of specific antibodies in rabbit. In 91.39% of the subgingival plaque samples, the PrtC could be detectable, in which the level of positive rates of detection was higher in severe cases of chronic periodontitis than that in the mild cases. So far, a prokaryotic expression system for the prtC gene from P.gingivalis with high expression efficiency was successfully constructed in the present study, and the expressed product PrtC possesses well antigenicity and immunoreactivity, suggesting the possibility to be used as the candidate antigen for developing the serological kit and P.gingivalis vaccine.
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