牙龈卟啉单胞菌菌毛相关外膜蛋白pgmA基因的克隆和表达

Cloning and expression of Porphyromonas gingivalis pgmA gene encoding outer membrane protein associated with fimbria

目的 :克隆牙龈卟啉单胞菌 (Porphyromonasgingivalis,Pg) pgm A基因 ,构建 pgm A基因表达载体 ,鉴定其表达产物的免疫性。方法 :采用高保真 PCR分别从牙龈卟啉菌 ATCC332 77和 4 7A- 1菌株中扩增 pgm A基因 ,T- A克隆后测定核苷酸序列 ,构建 p ET32 a的 pgm A表达载体 ,在 E.coli BL2 1DE3宿主菌中用不同浓度的 IPTG诱导表达 ,采用兔抗融合蛋白血清的 Western blot鉴定其免疫反应性和免疫原性 ,采用 ELISA检测 6 5株临床分离牙龈卟啉单胞菌与 Pgm A融合蛋白抗血清的反应情况。结果 :所克隆的牙龈卟啉单胞菌 ATCC332 77与 4 7A- 1菌株 pgm A基因的核苷酸序列同源性为 10 0 %,与报道的相应核苷酸序列同源性为 98.98%,氨基酸序列同源性高达99.18%。p ET32 a- pgm A- BL2 1DE3系统的 Pgm A融合蛋白表达量为细菌总蛋白的 5 0 %左右。Pgm A融合蛋白免疫家兔能获得相应抗体并与 Pgm A蛋白发生结合反应。 ELISA结果证实 92 .3%的牙龈卟啉菌临床菌株 (6 0 / 6 5 )能与Pgm A融合蛋白抗血清发生结合反应。结论 :本研究成功地构建了 Pg pgm A高效表达系统 ,所表达的 Pgm A融合蛋白有良好的免疫原性和免疫反应性 ,可作为 Pg检测试剂盒和 Pg疫苗的候选抗原。

Objective: To clone pgmA gene of Porphyromonas gingivalis,to construct the expression vector of the gene and to identify immunity of the fusion protein. Methods: The pgmA genes from ATCC 33277 and 47A-1 strains of P.gingivalis were amplified by high fidelity PCR. The nucleotide of the target DNA amplification fragments were sequenced after T-A cloning. The pET32a expression vectors inserted with pgmA gene fragments were constructed. PgmA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG with different dosages. Western blot test by using rabbit antiserum against the fusion protein was applied to determine immunity of the fusion protein. ELISA was applied to determine the immunoreaction of antibody against PgmA fusion protein and 65 strains of P.gingivalis isolates. Results: The nucleotide sequence homology of the cloned pgmA gene fragments from ATCC 33277 and 47A-1 strains was 100%. In comparison with the reported corresponding sequences,the homologies of the nucleotide sequences of the cloned pgmA gene fragments were 98.98%,while the homologies of their putative amino acid sequences were 99.18%. The expression output of PgmA fusion protein in pET32a-pgmA-BL21DE3 system was approximately 50% of the total bacterial proteins. PgmA fusion protein was able to induce rabbit to produce specific antibody that could combine with PgmA protein. 92.3% of P. gingivalis isolates (60/65) were able to react with the antibody against PgmA fusion protein. Conclusion: An expression system of P.gingivalis pgmA gene with high efficiency was established successfully. The expressed PgmA fusion protein possesse satisfied immunogenicity and immunoreactivity,which can be used as a candidate antigen in detection of P.gingivalis and possible development of corresponding vaccine.