Cloning and expression of Porphyromonas gingivalis pgmA gene encoding outer membrane protein associated with fimbria / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To clone pgmA gene of Porphyromonas gingivalis, to construct the expression vector of the gene and to identify immunity of the fusion protein.</p><p><b>METHODS</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>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.</p>