Salivary Proteome and Intact N-Glycopeptides Analysis Reveal Specific Signatures in Periodontitis.

Periodontitis is a prevalent oral inflammatory disease that can result in tooth loss and is closely linked to type 2 diabetes (T2D). In this study, we analyzed the salivary proteome and intact N-glycopeptides (IGPs) of individuals with mild-moderate, severe, aggressive periodontitis, and periodontitis with T2D, including those treated with antidiabetic drugs, to identify specific signatures associated with the disease. Our results revealed that salivary proteins and glycoproteins were altered in all periodontitis groups (PRIDE ID: 1-20230612-72345), with fucose- and sialic acid-containing N-glycans showing the greatest increase. Additionally, differentially expressed proteins were classified into 9 clusters, including those that were increased in all periodontitis groups and those that were only altered in certain types of periodontitis. Interestingly, treatment with antidiabetic drugs reversed many of the changes observed in the salivary proteome and IGPs in T2D-related periodontitis, suggesting a potential therapeutic approach for managing periodontitis in patients with T2D. Consistent with MS/MS results, the expression of salivary IGHA2 and Fucα1-3/6GlcNAc (AAL) was significantly increased in MP. These findings provide new insights into the pathogenesis of periodontitis and highlight the potential of salivary biomarkers for diagnosis, prognosis, and monitoring of disease progression and treatment response.

Mesenchymal stem cell-derived extracellular vesicles for treatment of bone loss within periodontitis in pre-clinical animal models: a meta-analysis.

BACKGROUND: Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) represent an effective and promising strategy for periodontitis, although studies remain pre-clinical. Herein, a meta-analysis was conducted to assess the efficacy of MSC-EVs in animal models of periodontitis. METHODS: The PubMed, Web of Science, and Embase electronic databases were searched up to Dec 2022 to retrieve preclinical studies examining the use of MSC-EVs for periodontitis treatment. Meta-analyses and sub-group analyses were performed to assess the effect of MSC-EVs on Bone Volume/Total Volume (BV/TV) or the distance between the cementoenamel junction and alveolar bone crest (CEJ-ABC) in pre-clinical animal models of periodontitis. RESULTS: 11 studies published from Mar 2019 to Oct 2022 met the inclusion criteria. Overall, MSC-EVs contributed to periodontal bone regeneration in the inflammatory bone loss area due to periodontitis, as represented by a weighted mean difference (WMD) of 14.07% (95% CI = 6.73, 21.41%, p < 0.001) for BV/TV and a WMD of -0.12 mm (95% CI= -0.14, -0.11 mm, p < 0.001) for CEJ-ABC. However, sub-analysis suggested that there was no significant difference in CEJ-ABC between studies with bioactive scaffolds and studies without bioactive scaffolds (p = 0.60). CONCLUSIONS: The present study suggests that MSC-EVs may represent an attractive therapy for the treatment of inflammatory bone loss within periodontitis.

PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose.

BACKGROUND: Oxidative stress mediated by hyperglycemia damages cell-reparative processes such as mitophagy. Down-regulation of mitophagy is considered to be a susceptible factor for diabetes mellitus (DM) and its complications. However, the role of mitophagy in DM-associated periodontitis has not been fully elucidated. Apoptosis of human gingival epithelial cells (hGECs) is one of the representative events of DM-associated periodontitis. Thus, this study aimed to investigate PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy activated in the process of high glucose (HG)-induced hGECs apoptosis. METHODS: For dose-response studies, hGECs were incubated in different concentrations of glucose (5.5, 15, 25, and 50 mmol/L) for 48 h. Then, hGECs were challenged with 25 mmol/L glucose for 12 h and 48 h, respectively. Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL), caspase 9 and mitochondrial membrane potential (MMP). Subsequently, autophagy was evaluated by estimating P62, LC3 II mRNA levels, LC3 fluorescent puncta and LC3-II/I ratio. Meanwhile, the involvement of PINK1-mediated mitophagy was assessed by qRT-PCR, western blotting and immunofluorescence. Finally, hGECs were transfected with shPINK1 and analyzed by MMP, caspase 9 and annexin V-FITC apoptosis. RESULTS: The number of TUNEL-positive cells and caspase 9 protein were significantly increased in cells challenged with HG (25 mmol/L) for 48 h (HG 48 h). MMP was impaired both at HG 12 h and HG 48 h, but the degree of depolarization was more serious at HG 48 h. The autophagy improved as the amount of LC3 II increased and p62 decreased in HG 12 h. During this process, HG 12 h treatment induced PINK1-mediated mitophagy. PINK1 silencing with HG 12 h resulted in MMP depolarization and cell apoptosis. CONCLUSIONS: These results suggested that loss of the PINK1 gene may cause mitochondrial dysfunction and increase sensitivity to HG-induced apoptosis of hGECs at the early stage. PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose.

The therapeutic role of baicalein in combating experimental periodontitis with diabetes via Nrf2 antioxidant signaling pathway.

BACKGROUND AND OBJECTIVE: Oxidative stress has been suggested as an important pathogenic factor contributing to chronic periodontitis with diabetes mellitus (CPDM). Previous studies have revealed the potential therapeutic properties of baicalein (BCI) in oxidative stress-related diseases; however, the antioxidant effects of BCI on therapy for individual with CPDM remain largely unexplored. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in cellular defence against oxidative stress. In this study, we aim to determine whether BCI prevents diabetes-related periodontal tissue destruction by regulating Nrf2 signaling pathway. MATERIAL AND METHODS: Human gingival epithelial cells (hGECs) were challenged with high glucose (HG, 25 mmol/L) and/or lipopolysaccharide (LPS, 20 µg/mL). Reactive oxygen species (ROS) were detected by fluorescence-activated cell sorting. The changes of antioxidant-related genes, including Nrf2, catalase (Cat), glutamate-cysteine ligase catalytic subunit (Gclc), superoxide dismutase 1 (Sod1), and superoxide dismutase 2 (Sod2), were quantified by real-time PCR. The localization of phospho-Nrf2 (pNrf2, S40) in the nucleus was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). PNrf2 and total form of Nrf2 were determined using western blot. The above indicators together with mitochondrial membrane potential (MMP) were further investigated in hGECs pre-treated with different concentrations of BCI (0.01, 0.1, or 0.5 µg/mL) before stimulated with HG plus LPS (GP). Finally, the role of BCI in activating Nrf2 signaling pathway and relieving the alveolar bone absorption was examined in the CPDM model of Sprague Dawley rats. CPDM rats were oral gavaged with BCI (50, 100, or 200 mg/kg daily). The pNrf2 was detected by immunohistochemistry, and the alveolar bone absorption was examined by microcomputed tomography. RESULTS: Our results showed that ROS were significantly increased in both groups of HG and LPS, with the strongest generation in the GP group. In terms of ROS-related gene expression, we found that the mRNA levels of Nrf2, Cat, Gclc, Sod1, and Sod2 were significantly decreased in HG and LPS groups. In consistent with the strongest induction of ROS in GP group, the gene expression in GP group was further decreased as compared to those of HG and LPS groups. Also, the expression of pNrf2 exhibited the same trend with the expression of those antioxidant genes. However, the generation of ROS and the loss of mitochondrial membrane potential induced by GP were abolished by pre-treatment with different concentrations of BCI (0.01, 0.1, or 0.5 µg/mL). Interestingly, we observed that BCI promoted the nucleus translocation of pNrf2, as well as the gene expression levels of pNrf2 and its target genes (Cat, Gclc, Sod1, and Sod2). Finally, in the CPDM animal model, we found that BCI (at concentrations: 50, 100, and 200 mg/kg) markedly increased the number of pNrf2-positive cells in periodontal tissue and mitigated the alveolar bone loss. CONCLUSIONS: Our data revealed a potential role for clinic application of BCI under CPDM conditions, suggesting a new therapeutic drug for CPDM patients.

Integrin ß1 Gene Therapy Enhances in Vitro Creation of Tissue-Engineered Cartilage Under Periodic Mechanical Stress.

BACKGROUND/AIMS: Periodic mechanical stress activates integrin ß1-initiated signal pathways to promote chondrocyte proliferation and matrix synthesis. Integrin ß1 overexpression has been demonstrated to play important roles in improving the activities and functions of several non-chondrocytic cell types. Therefore, in the current study, we evaluated the effects of integrin ß1 up-regulation on periodic mechanical stress-induced chondrocyte proliferation, matrix synthesis and ERK1/2 phosphorylation in chondrocyte monolayer culture, and evaluated the quality of tissue-engineered cartilage constructed in vitro under periodic mechanical stress combined with integrin ß1 up-regulation. METHODS AND RESULTS: Our results revealed that under periodic mechanical stress, pre-treatment with integrin ß1-wild type vector significantly enhanced chondrocyte proliferation and matrix synthesis and promoted ERK1/2 phosphorylation in comparison to mock transfectants. Furthermore, when chondrocytes were seeded in PLGA scaffolds, more accumulated GAG and type II collagen tissue were detected after Lv-integrin ß1 transfection compared with sham controls exposed to periodic mechanical stress. In contrast, in the Lv-shRNA-integrin ß1 group, the opposite results were observed. CONCLUSION: Our findings collectively suggest that in addition to periodic mechanical stress, integrin ß1 up-regulation in chondrocytes could further improve the quality of tissue-engineered cartilage.

Genomic surveillance reveals low-level circulation of two subtypes of genogroup C coxsackievirus A10 in Nanchang, Jiangxi Province, China, 2015-2023.

Introduction: In recent years, coxsackievirus (CV) A10 has been associated with increasing sporadic hand, foot, and mouth disease (HFMD) cases and outbreaks globally. In addition to mild symptoms such as pharyngitis and herpangina, CVA10-related complications or even fatality can occur. Currently, systematic phylogenetic studies of CVA10 are limited. Methods: In this study, we first explored the epidemiological and genetic characteristics of CVA10 in Nanchang, an inland southeastern city of China, based on the HFMD surveillance network from 2015-2023. Results: Among 3429 enterovirus-positive cases, 110 (3.04%) were associated with CVA10, with a male-to-female ratio of 1.62. The median age of the CVA10 patients was 2.3 years (interquartile range, IQR 1.0-4.0), with 94.55% (104/110) of the patients aged less than 5 years. Phylogenetic analyses using the full-length VP1, 5'UTR, P1, P2, P3 sequences and near full-length genomes indicated that CVA10 strains (n = 57) isolated in Nanchang belonged to genogroup C; two strains identified in 2017 belonged to C1 subtypes clustered with strains from Vietnam, Madagascar, France and Spain; and the others belonged to C2 subtypes interdigitating with CVA10 isolates from mainland China, the United States and Australia. Through extensive analysis, we identified a rare F168Y mutation in epitope 4 of VP1 in a Madagascar strain of genogroup F and a Chinese strain of genogroup C. Based on Bayesian evolutionary analyses, the average nucleotide substitution rate for the VP1 gene of CV10 strains was 3.07×10-3 substitutions/site/year. The most recent common ancestor (tMRCA) of genogroup C was dated 1990.84, and the tMRCA of CVA10 strains from Nanchang was dated approximately 2003.16, similar to strains circulating in other regions of China, suggesting that the viruses were likely introduced and cryptically circulated in China before the establishment of the HFMD surveillance network. Recombination analysis indicated intertypic recombination of the Nanchang strain with the genogroup G strain in the 3D region. Discussion: Given the shifting dominance of viral genotypes and frequent recombination events, the existing surveillance system needs to be regulated to enhance genomic surveillance efforts on a more diverse spectrum of genotypes in the future.

Adjunctive Diode Laser Therapy and Probiotic Lactobacillus Therapy in the Treatment of Periodontitis and Peri-Implant Disease.

Periodontal and peri-implant diseases are plaque-induced infections with a high prevalence, seriously impairing people's quality of life. The diode laser has long been recommended as adjunctive therapy in treating periodontitis. However, the optimal combination of usage mode (inside or outside periodontal pocket) and application regimen (single or multiple sessions of appointment) has not been described in detail. Meanwhile, probiotic Lactobacillus is regarded as a potential adjuvant in the management of the peri-implant disease. Nonetheless, a detailed protocol for an effective probiotic application is lacking. This article aims to summarize two clinical protocols. For periodontitis, the optimal collaboration of laser usage mode and application regimen was identified. Regarding peri-implant mucositis, a combined therapy containing professional topical use and home administration of probiotic Lactobacillus was established. This updated laser protocol clarifies the relationship between the treatment mode (inside or outside the periodontal pocket) and the number of laser appointments, further refining the existing diode laser therapy. For inside pocket irradiation, a single session of laser treatment is suggested whereas, for outside pocket irradiation, multiple sessions of laser treatment provide better effects. The improved probiotic Lactobacillus therapy resulted in the disappearance of swelling of the peri-implant mucosa, a reduced bleeding on probing (BOP), and an obvious reduction and good control of plaque and pigmentation; however, probing pocket depth (PPD) had limited improvement. The current protocol should be regarded as preliminary and could be further enhanced.

Inhibitory effects of a water-soluble jujube polysaccharide against biofilm-forming oral pathogenic bacteria.

Oral diseases caused by infectious pathogens raises significant concerns in public health. In the light of side effects of current antibiotics therapy and growing drug resistance of pathogenic bacteria, natural products have become attractive alternatives for antibiotics agents in dental practice. This current study investigated the effects of polysaccharides extracted from Zizyphus jujuba Mill. on three major oral biofilm-forming pathogenic bacteria including caries-inducing Streptococcus mutans, lesions-causing MRSA, and periodontitis-related Porphyromonas gingivalis, as well as general oral microbiota. Our results demonstrated that jujube polysaccharide prepared in this study was mainly composed by galacturonic acid with an average molecular weight 242 kDa, which were further characterized for structural features by FT-IR spectra and NMR spectroscopy analysis. This jujube polysaccharide was shown to exhibit remarkable inhibitory effects against all the tested oral bacterial pathogens through various mechanisms including growth inhibition, biofilm prevention and disruption, intervention of bacterial infection (adhesion and invasion), attenuation of cytotoxicity, modulation of excessive inflammatory response of LPS-stimulated and MRSA-infected macrophages as well as positive regulation of oral microbiota. The present study paves the way to explore jujube polysaccharides for the prevention and treatment of oral infectious diseases. Graphic Abstract.

Surveillance, Epidemiology and Impact of EV-A71 Vaccination on Hand, Foot, and Mouth Disease in Nanchang, China, 2010-2019.

After the first national-scale outbreak of Hand, foot, and mouth disease (HFMD) in China, a national surveillance network was established. Here we described the epidemiology and pathogenic profile of HFMD and the impact of EV-A71 vaccination on pathogen spectrum of enteroviruses in the southeastern Chinese city of Nanchang during 2010-2019. A total of 7,951 HFMD cases from sentinel hospitals were included, of which 4,800 EV-positive cases (60.4%) were identified by real-time RT-PCR. During 2010-2012, enterovirus 71 (EV-A71) was the main causative agent of HFMD, causing 63.1% of cases, followed by 19.3% cases associated with coxsackievirus A16 (CV-A16). Since 2013, the proportion of other enteroviruses has increased dramatically, with the sub genotype D3 strain of Coxsackievirus A6 (CV-A6) replacing the dominance of EV-A71. These genetically diverse native strains of CV-A6 have co-transmitted and co-evolved in Nanchang. Unlike EV-A71 and CV-A16, most CV-A6 infections were concentrated in autumn and winter. The incidence of EV-A71 infection negatively correlated with EV-A71 vaccination (r = -0.990, p = 0.01). And severe cases sharply declined as the promotion of EV-A71 vaccines. After 2-year implementation of EV-A71 vaccination, EV-A71 is no longer detected from the reported HFMD cases in Nanchang. In conclusion, EV-A71 vaccination changed the pattern of HFMD epidemic, and CV-A6 replaced the dominance of EV-A71 over time.

Complement 3 mediates periodontal destruction in patients with type 2 diabetes by regulating macrophage polarization in periodontal tissues.

OBJECTIVES: Diabetes aggravates the risk and severity of periodontitis, but the specific mechanism remains confused. Complement 3 (C3) is closely related to complications of type 2 diabetes (T2DM). In the present study, we concentrated on whether C3 mediates the development of periodontitis in T2DM. MATERIALS AND METHODS: Levels of C3 in blood and gingival crevicular fluid (GCF) of patients were measured first. A C3-knockout diabetic mouse model was established, real-time PCR, Western blotting and histological investigation were performed to evaluate the progress of periodontitis. Microcomputed tomography (micro-CT) and TRAP staining were performed to detect alveolar bone resorption. Immunofluorescence was performed to detect polarization of macrophages. RESULTS: Our data showed that C3 levels were elevated in the blood and GCF of T2DM patients compared with non-diabetic individuals. Increased C3 was closely related to the upregulation of inflammatory cytokines including interleukin (IL)-1, IL-6 and tumour necrosis factor-alpha (TNF-α), as well as the decline of the bone volume density (BMD) and bone volume over total volume (BV/TV) of the alveolar bones in diabetic mice. The deletion of C3 inhibited inflammatory cytokines and rescued the decreased BMD and BV/TV of the alveolar bones. C3-mediated polarization of macrophages was responsible for the damage. CONCLUSION: T2DM-related upregulation of C3 contributes to the development of periodontitis by promoting macrophages M1 polarization and inhibiting M2 polarization, triggering a pro-inflammatory effect on periodontal tissues.

Baicalin inhibits toll-like receptor 2/4 expression and downstream signaling in rat experimental periodontitis.

Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis (P. gingivalis) for 4weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200mg/kg/day) or vehicle for 4weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1ß, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis.

Induction of immune response and prevention of alveolar bone loss with recombinant Porphyromonas gingivalis peptidylarginine deiminase.

OBJECTIVE: Treatments for periodontitis are not absolutely perfect, and a vaccine against Porphyromonas gingivalis (P. gingivalis) could become a valuable adjunct therapy for periodontitis. DESIGN: In this study, a vaccine of peptidylarginine deiminase (PAD) from P. gingivalis was evaluated in P. gingivalis-induced murine lesion and periodontitis models. The prevention of alveolar bone loss analysis determined by micro-computed X-ray tomography (micro-CT), and histological assays. Furthermore, the induction of immune response of mouse anti-PAD done with ELISA and Western Blot analysis. RESULTS: Compared with animal immunization with incomplete Freund's adjuvant (IFA) alone, PAD group significantly inhibited (P<0.05) bone resorption. ELISA and Western Blot showed that PAD induced response involving immunoglobulin G1 (Ig G1) predominantly. CONCLUSIONS: These results suggest that PAD could be a candidate antigen for a vaccine against P. gingivalis infection.

[Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells].

PURPOSE: The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs). METHODS: DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection (MOI) and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package. RESULTS: DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80±108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 µmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01). CONCLUSIONS: The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells. Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).

[Cloning and expression of peptidylarginine deiminase of Porphyromonas gingivalis].

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 degrees centigrade, 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.

[Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density. RESULTS: DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed. CONCLUSION: The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.

[Construction of prokaryotic expression vector of FimA gene from Porphyromonas gingivalis, fusion expression and purification in E. coli BL21(DE3)pLyS].

OBJECTIVE: To clone the FimA gene of fimbriae from Porphyromonas gingivalis (P. gingivalis) and to construct prokaryotic expression vector which was induced in E.coli BL21(DE3)pLyS in the form of fusion protein expression and to identify, purify the product of its expression. METHODS: To clone the FimA gene of fimbriae from P. gingivalis and to construct prokaryotic expression vector pET15b-FimA vector which was transformed into the competent cells of BL21(DE3)pLyS. The expression of fusion protein was induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). With anti-6xHis Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co(2+)-NTA affinity chromatography. RESULTS: Cloned FimA gene sequences and inserted into expression vector of the FimA sequences were related to the sequence in GenBank database showed 100% homology. IPTG induced and then identified by Western blot showed a fragment of 4.1 x 10(4) has been expressed. Co(2+)-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein. CONCLUSION: The recombinant prokaryotic expression vector of pET15b-FimA was constructed and was expressed and purified successfully in E. coli BL21 (DE3)pLyS. This study laid the experimental foundation to further prepare for monoclonal antibodies of fimbriae of P. gingivalis and to develop the subunit protein vaccine of prevention of periodontitis.