[Establishment and content validity assessment of evaluation index system for development value of traditional Chinese medicine preparations in medical institutions].

The development of traditional Chinese medicine(TCM) preparations as an incubator for new drugs in medical institutions has flourished, while an evaluation index system remains to be established for comprehensively assessing the development value of these prescriptions. This study established an item pool through literature research, employed the Delphi method to determine the content of evaluation indexes, and adopted the superiority chart to determine the weight of each index. Two-level evaluation index system for the development value of TCM preparations in medical institutions was established, which included 7 first-level items and 36 se-cond-level items, demonstrating scientific validity. The first-level items(weight) were inheritance(10.61%), effectiveness(23.22%), safety(22.71%), innovation(13.21%), economy(10.00%), suitability(8.57%), and accessibility(11.68%). The top three second-level items in terms of weight distribution were adverse reaction monitoring(6.73%), evidence of therapeutic effect(5.71%), and clinical response rate(4.75%). The bottom three second-level items were production advantages(0.86%), medicinal dosage(0.48%), and medicinal smell or taste(0.18%). The content validity of the established system was assessed, which revealed that the index system was reliable, with the overall and average content validity indexes of 0.47 and 0.90, respectively. Furthermore, the established evaluation index system was used to evaluate six TCM preparations in a city-level hospital of TCM in Sichuan Province, which demonstrated that the system had operability. The results indicate that the evaluation index system is scientific, reliable, and operable, providing a reference for developers to selectively develop TCM preparations in medical institutions. In practical application, the system can be adjusted regarding the index weights according to actual conditions.

[Guideline for clinical comprehensive evaluation of Chinese patent medicine (2022 version)].

Currently,the research or publications related to the clinical comprehensive evaluation of Chinese patent medicine are increasing,which attracts the broad attention of all circles. According to the completed clinical evaluation report on Chinese patent medicine,there are still practical problems and technical difficulties such as unclear responsibility of the evaluation organization,unclear evaluation subject,miscellaneous evaluation objects,and incomplete and nonstandard evaluation process. In terms of evaluation standards and specifications,there are different types of specifications or guidelines with different emphases issued by different academic groups or relevant institutions. The professional guideline is required to guide the standardized and efficient clinical comprehensive evaluation of Chinese patent medicine and further improve the authority and quality of evaluation. In combination with the characteristics of Chinese patent medicine and the latest research achievement at home and abroad,the detailed specifications were formulated from six aspects including design,theme selection,content and index,outcome,application and appraisal,and quality control. The guideline was developed based on the guideline development requirements of China Assoication of Chinese medicine. After several rounds of expert consensus and public consultation,the current version of the guideline has been developed.

Transcriptomic study of the mechanism of anoikis resistance in head and neck squamous carcinoma.

BACKGROUND: Normal epithelial cells rapidly undergo apoptosis as soon as they lose contact with the extracellular matrix (ECM), which is termed as anoikis. However, cancer cells tend to develop a resistance mechanism to anoikis. This acquired ability is termed as anoikis resistance. Cancer cells, with anoikis resistance, can spread to distant tissues or organs via the peripheral circulatory system and cause cancer metastasis. Thus, inhibition of anoikis resistance blocks the metastatic ability of cancer cells. METHODS: Anoikis-resistant CAL27 (CAL27AR) cells were induced from CAL27 cells using the suspension culture approach. Transcriptome analysis was performed using RNA-Seq to study the differentially expressed genes (DEGs) between the CAL27ARcells and the parental CAL27 cells. Gene function annotation and Gene Ontology (GO) enrichment analysis were performed using DAVID database. Signaling pathways involved in DEGs were analyzed using Gene Set Enrichment Analysis (GSEA) software. Analysis results were confirmed by reverse transcription PCR (RT-PCR), western blotting, and gene correlation analysis based on the TCGA database. RESULTS: GO enrichment analysis indicated that the biological process (BP) of the DEGs was associated with epidermal development, DNA replication, and G1/S transition of the mitotic cell cycle. The analysis of cellular component (CC) showed that the most significant up-regulated genes were related to extracellular exosome. KEGG Pathway analysis revealed that 23 signaling pathways were activated (p-value ≤ 0.05, FDR q-value ≤ 0.05) and 22 signaling pathways were suppressed (p-value ≤ 0.05, FDR q-value ≤ 0.05). The results from the GSEA indicated that in contrast to the inhibition of EGFR signaling pathway, the VEGF signaling pathway was activated. The VEGF signaling pathway possibly activates STAT3 though induction of STAT3 phosphorylation. Gene correlation analysis revealed that the VEGFA- STAT3-KLF4-CDKN1A signal axis was not only present in head and neck squamous carcinoma (HNSCC) but also two other epithelial-derived carcinomas that highly express VEGFA, including kidney renal clear cell carcinoma (KIRC) and ovarian serous cystadenocarcinoma (OV).

IFITM3 inhibits virus-triggered induction of type I interferon by mediating autophagosome-dependent degradation of IRF3.

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that can be induced by viral infection and interferons (IFNs). It inhibits the entry and replication of many viruses, which are independent of receptor usage but dependent on processes that occur in endosomes. In this study, we demonstrate that IFITM3 plays important roles in regulating the RNA-virus-triggered production of IFN-ß in a negative-feedback manner. Overexpression of IFITM3 inhibited Sendai virus-triggered induction of IFN-ß, whereas knockdown of IFITM3 had the opposite effect. We also showed that IFITM3 was constitutively associated with IRF3 and regulated the homeostasis of IRF3 by mediating the autophagic degradation of IRF3. These findings suggest a novel inhibitory function of IFITM3 on the RNA-virus-triggered production of type I IFNs and cellular antiviral responses.

[Research on symmetrical optical waveguide based surface plasmon resonance sensing with spectral interrogation].

Surface plasmon resonance (SPR) sensors with spectral interrogation can adopt fiber to transmit light signals, thus leaving the sensing part separated, which is very convenient for miniaturization, remote-sensing and on-site analysis. Symmetrical optical waveguide (SOW) SPR has the same refractive index of the-two buffer media layers adjacent to the metal film, resulting in longer propagation distance, deeper penetration depth and better performance compared to conventional SPR In the present paper, we developed a symmetrical optical, waveguide (SOW) SPR sensor with wavelength interrogation. In the system, MgF2-Au-MgF2 film was used as SOW module for glucose sensing, and a fiber based light source and detection was used in the spectral interrogation. In the experiment, a refractive index resolution of 2.8 x 10(-7) RIU in fluid protocol was acquired. This technique provides advantages of high resolution and could have potential use in compact design, on-site analysis and remote sensing.

Prevalence of human papillomavirus infection in oral squamous cell carcinoma: a case-control study in Wuhan, China.

High risk forms of the human papilloma virus (HPV) are generally accepted as necessary causative agents for cervical cancer. Recently, a possible relation between HPV and oral squamous cell carcinoma (OSCC) has also been noticed. The present study was conducted to investigate the prevalence of HPV infection in OSCCs in Wuhan city. DNA samples were collected from fresh tissues in 200 patients with OSCC and 68 normal controls. The polymerase chain reaction and direct sequencing were used to identify the HPV types in the samples. The prevalence of HPV of all types in the OSCC group was higher than in the control group (55/200 vs 2/68, OR=11.5, 95% CI=2.6-50.2). HPV16 and HPV18 were the main types detected, with HPV6 was the only low-risk type identified. High-risk HPV types HPV16 and HPV18 are prevalent in OSCC patients and may participate in the development of OSCC with traditional risk factors, tobacco and alcohol, possibly exerting synergistic effects. The results of multinomial logistic regression showed that those who smoked, consumed alcohol and with HPV infection have the highest risk of developing oral cancer (OR=13.3, 95% CI=3.1-56.8). Adjusted for age, smoking and alcohol use, HPV infection was independently associated with oral squamous cell carcinoma.

The effect of antigen size on the immunogenicity of antigen presenting cell targeted DNA vaccine.

Directing antigens to antigen presenting cells (APCs) has been demonstrated to be an efficient strategy to enhance immune responses induced by DNA vaccination. Fusion of antigens to cytotoxic T-Lymphocyte antigen 4 (CTLA4), a ligand of B7 molecules on the surfaces of APCs with strong binding affinity, enhanced the immunogenicities of antigens in various degrees. To investigate the relationship between antigen size and the immunogenicity of CTLA4 fusion DNA vaccine, we constructed CTLA4 targeted fusion anti-caries DNA vaccines containing different size of antigens. In vivo and in vitro experiments showed that CTLA4 fusion with smaller antigen induced stronger humoral immune responses and had higher affinity to B7-expressed cells than fusion with larger antigen. In conclusion, antigen size is one of the important factors regulating the potency of humoral immune response induced by CTLA4 targeted DNA vaccines.

[A novel spectral surface plasmon resonance 2-D sensing technique and its applications in DNA microarrays].

The authors have previously proposed a novel refractive index two-dimensional sensing technique named "parallel scan spectral surface plasmon resonance imaging". In the technique, with a line-shaped light illumination, an image acquired with CCD detector could provide both SPR wavelength information and one-dimensional spatial distribution, and then provide one-dimensional distribution of refractive index with further calculation. Thus, two-dimensional distribution of refractive index of the entire sensing area can be obtained with one-dimensional optical line parallel scan. The technique offers advantages of both high sensitivity and high throughput, and could have potential applications in microarray analysis. In the present paper, the authors improve the data processing methods of the technique. The authors use the refractive index of air as a reference to get over the problem of precision of the incident angle. The authors also sense a manually dotted Legionella pneumophila mip DNA probe array with this technique and prove the feasibility of sensing microarrays by this highly sensitive and label-free technique. The relation between the equivalent refractive indices and the concentrations of the dotted Legionella pneumophila mip DNA probes is obtained, which has important reference value for further study.

Fusion of antigen to chemokine CCL20 or CXCL13 strategy to enhance DNA vaccine potency.

DNA vaccination is a promising method to induce specific immune responses. However, it remains challenging to enhance DNA vaccine potency. Chemokines were used as adjuvants to improve the efficacy of DNA vaccination. Herein, we fused murine chemokine CCL20 or CXCL13 to a model antigen green fluorescent protein (GFP). The fusion DNA vaccines enhanced specific anti-GFP immune responses in mice compared with vector pEGFP-N1. Co-immunization with both of chemokine-GFP fusion constructs induced the significantly highest level of humoral immune responses. CCL20-GFP or CXCL13-GFP fusion DNA vaccine induced predominant IgG2a or IgG1 response respectively. However, co-immunization with both of these fusion DNA vaccines induced a predominant IgG2a response. Therefore, fusing chemokine CXCL13 or CCL20 to antigen provides new attractive strategy to enhance the immunogenicity of DNA vaccine and modulate immune responses.

Enhanced efficacy of CTLA-4 fusion anti-caries DNA vaccines in gnotobiotic hamsters.

AIM: To evaluate the comparative immunogenicity and protective efficacy of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) fusion anti-caries DNA vaccines pGJA-P/VAX1, pGJA-P, and non-fusion anti-caries DNA construct pGLUA-P in hamsters. In addition, the ability of CTLA-4 to target pGJA-P/VAX1-encoding antigen to dendritic cells was tested in vitro. METHODS: All DNA constructs contain genes encoding the A-P regions of a cell surface protein (PAc) and the glucan binding (GLU) domain of glucosyltransferases (GTFs) of cariogenic organism Streptococcus mutans. Human dendritic cells were mixed with the CTLA-4-Ig-GLU-A-P protein expressed by pGJA-P/VAX1-transfected cells and analyzed by flow cytometry. Gnotobiotic hamsters were immunized with anti-caries DNA vaccines by intramuscular injection or intranasal administration. Antibody responses to a representative antigen PAc were assayed by ELISA, and caries protection was evaluated by Keyes caries scores. RESULTS: A flow cytometric analysis demonstrated that CTLA-4-Ig-GLU-A-P protein was capable of binding to human dendritic cells. pGJA-P/VAX1 and pGJA-P induced significantly higher specific salivary and serum anti-PAc antibody responses than pGLUA-P. Significantly fewer caries lesions were also observed in hamsters immunized with pGJA-P/VAX1 and pGJA-P. There was no significant difference in the anti-PAc antibody level or caries scores between pGJA-P/VAX1 and pGJA-P-immunized groups. CONCLUSION: Antigen encoded by CTLA-4 fusion anti-caries DNA vaccine pGJA-P/VAX1 could specifically bind to human dendritic cells through the interaction of CTLA-4 and B7 molecules. Fusing antigen to CTLA-4 has been proven to greatly enhance the immunogenicity and protective efficacy of anti-caries DNA vaccines.

[The immunocontraceptive effects of Lagurus lagurus and Mus musculus zona pellucida 3 DNA vaccine].

To investigate the immunocontraception effects of different kinds of mice and seek the relatively effective species-specific DNA vaccines, we constructed four corresponding immunocontraceptive recombinant plasmids: pcDNA3-mzp3 (pcD-M), pcDNA3-Izp3 (pcD-L), pcDNA3-aat-comp-mzp3 (pcD-ACM) and pcDNA3-aat-comp-lzp3 (pcD-ACL) using zona pellucida 3 gene of Lagurus lagurus (Lzp3) and Mus musculus (Mzp3) respectively to immunize NIH mice. With the introduction of hydrnamic transfection instead of traditional Hela cell transfection to study the genes expression in vivo, the results indicated that all four recombinants could be expressed in livers of mice; Histogram pattern of ELISA showed that all of the recombinants in mice could elicit high quantity and lasting specific anti-ZP3 antibody; Antifertility experiment showed that Mzp3 and Lzp3 groups both enhanced sterile effects (P < 0.05), especially the group of pcD-ACM had a significant difference compared with control group (P < 0.01). Histology of ovarian sections demonstrated that pcD-M and pcD-L groups had no disruption of follicular development while pcD-ACL and pcD-ACM did the opposite. The present studies proved that L. lagurus zona pellucida 3 gene (Lzp3) and M. musculus zona pellucida 3 gene (Mzp3) had immunocontraception effects and primarily presumed that they didn't possess species specificity.

[Expression pattern and level of cytotoxic T lymphocyte-associated antigen-4 targeted anti-caries plasmids in eukaryotic cells].

OBJECTIVE: To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells. METHODS: The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants. RESULTS: Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX. CONCLUSIONS: CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.

Immunogenicity of CTLA4 fusion anti-caries DNA vaccine in rabbits and monkeys.

Enhancement of mucosal and systemic immune responses is still a challenge for the application of DNA vaccine. Here, we show anti-caries DNA vaccines, pGJA-P and pGJA-P/VAX, encoding Streptococcus mutans antigens fused to cytotoxic T lymphocyte antigen-4 (CTLA4), which binds to B7 molecule expressed on the surfaces of antigen-presenting cells. Rabbits and monkeys were immunized via intranasal or intramuscular routes. The fusion vaccine induced accelerated and increased specific antibody responses in serum and saliva compared with non-fusion DNA vaccine in rabbits. Significant specific serum IgG and salivary IgA levels could be detected in fusion vaccine-immunized monkeys. Therefore, this study demonstrates that fusing antigens to CTLA4 results in enhancing immune efficacy and strongly suggests that it may represent a promising approach to prevent dental caries or other mucosal infectious diseases. These findings also suggest that CTLA4 fusion anti-caries DNA vaccine may be effective immunogen in primates.

Mucosal immunization against dental caries with plasmid DNA encoding pac gene of Streptococcus mutans in rats.

Salivary secretory immunoglobulin A (S-IgA) antibodies act as the first line of defense against dental caries by blocking of adherence of Streptococcus mutans to tooth surfaces. This study focused on finding proper mucosal immunization route and delivery system to induce higher level of specific anti-S. mutans saliva S-IgA and inhibit dental caries in animal model. By immunizing rats with an anti-caries DNA vaccine, pCIA-P, via different mucosal routes, we found that intranasal (i.n.) immunization with pCIA-P/bupivacaine DNA complexes elicited the highest specific anti-S. mutans saliva S-IgA mucosal antibody responses compared with naked DNA and other routes. Correspondingly, rats immunized with pCIA-P/bupivacaine DNA complex via i.n. displayed the least carious lesions. Our findings suggested that DNA vaccination via intranasal immunization with bupivacaine delivery system be a promising approach against dental caries.

[Enhancement of immune responses in rabbits with a targeted anti-caries DNA vaccine pGJA-P].

OBJECTIVE: To detect the immunoreactivity of targeted fusion anti-caries DNA vaccine pGJA-P in vitro, and the ability to enhance the immune responses compared with the non-targeted fusion anti-caries DNA vaccine pGLUA-P. METHODS: The CHO cells were transfected with pGJA-P and the expression of recombinant protein in cultured supernatants were detected using Western blotting. 5 to 6-month-old female Japanese rabbits were immunized with either pGJA-P or pGLUA-P via either intramuscular injection (i.m.) or intranasal route (i.n.). The sera and saliva were collected and the antibody responses were checked by ELISA. The effect of immune sera on the synthesis of water-insoluble glucan by glucosyltransferase of S. mutans was examined. RESULTS: The expressed protein could response to specific anti-GTF antibody. The antibody responses in serum generated by pGJA-P via i.m. were significantly higher than those generated by pGLUA-P (P < 0.01). The antibody responses in saliva generated by pGJA-P via i.n. were significantly higher than those generated by pGLUA-P (P < 0.01). The higher mucosal antibody response induced by pGJA-P via i.m. compared with pGLUA-P (P < 0.01) was detected. The immune sera of rabbits immunized by pGJA-P via i.m. most significantly inhibited the synthesis of water-insoluble glucan by glucosyltransferase. CONCLUSIONS: The recombinant protein expressed by pGJA-P had the immunoreactivity to anti-GTF antibody. pGJA-P could induce faster and higher specific mucosal SIgA antibody responses via i.n. or serum IgG antibody responses via i.m. compared with non-targeted DNA vaccine, pGLUA-P. High titres of specific mucosal antibodies were found in rabbits immunized with pGJA-P via i.m. The immune sera of rabbits immunized by pGJA-P via i.m. displayed the ability of inhibiting the synthesis of water-insoluble glucan by glucosyltransferase.

[Immunization with targeted fusion anticaries DNA vaccine via intramuscular route:experiment with murine].

OBJECTIVES: To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in muscular in vivo. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and fusion anticaries DNA vaccine pGLUA-P in gnotobiotic rats, and observe the kinetics of antibody responses in BALB/c mice. METHODS: (1) Twelve 28-day-old female Wistar rats were randomly divided into 2 groups of 6 rats to be injected with the plasmid pGJA-P containing the signal peptide and extracellular regions of human CTLA(4), hinge and Fc regions of human IgG, the glu sequence of gtfB gene and A-P fragment of pac gene of Streptococcus mutans or the eukaryotic expression plasmid pCI into the quadriceps muscle of thigh respectively. Three days after the rats were killed and specimens of quadriceps muscles of thigh were taken. Immunohistochemical SP staining was used to examine the in situ expression of pGJA-P. (2) Twenty-four 18-day-old female Wistar rats were randomly divided into 4 groups of 6 rats. The rats were fed with cariogenic food. During the age of 20 - 22 days cariogentic food containing broad-spectrum antibiotics was fed. Then aseptic cotton stick was used to swab the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. During the age of 24 - 26 days, S. mutans Ingbritt cultured anaerobically was swab onto the surface of teeth of the rats twice with an interval of 30 minutes. After the inoculation aseptic cotton stick was used to wipe the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. When the gnotobiotic rats were 28 days old they were injected with pGJA-P, pGLUA-P, fusion anticaries DNA vaccine against both PAc, cell surface protein antigen, and glucosyltransferase (GTF), pCI or normal saline into the quadriceps muscle of thigh respectively, 2 weeks later a booster shot was given. When the rats were 63 days old their saliva and blood samples were collected. The serum IgG and salivary IgA were assayed by using ELISA. The gnotobiotic rats were killed and their maxillary bone the mandibles were isolated. The anticaries effect was evaluated by Keyes caries scores. (3) Twenty-four 4-week-old BALB/c mice were randomly divided into 4 groups of 6 mice: to be injected with pGJA-P, pGLUA-P, pCI, or normal saline respectively into the quadriceps muscles of thigh, 2 weeks later a booster shot was given. Before the injection and every 2 weeks after the immunization specimens of saliva and blood were collected. The serum IgG and salivary IgA were assayed by using ELISA. RESULTS: (1) Recombinant protein could be detected in the quadriceps muscles of the rats immunized with pGJA-P, but not in the muscles of the rats immunized with pCI. (2) The levels of serum anti-PAc IgG (1:200 000) and anti-GTF IgG (1:58 000) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:23 000 and 1:11 000 respectively) (both P < 0.01). The levels of salivary anti-PAc IgA (1:8) and anti-GTF IgA (1:6) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:2 and 1:2 respectively) (both P < 0.01). The Keyes scores of the pGJA-P group were significantly lower than those of the pGLUA-P group and the control groups (all P < 0.01). The effective serum IgG and salivary IgA in the pGJA-P group and effective serum IgG in the pGLUA-P group all persisted to the end of the experiment. (3) Two weeks after the initial immunization the serum anti-PAc IgG level of the mice immunized with pGJA-P increased remarkably, 4 times that of the mice immunized with pGLUA-P, and 33 times those of the mice injected with pCI or normal saline. Two weeks after the booster immunization, the serum anti-PAc IgG level of the mice immunized with pGJA-P was 14 times that of the mice immunized with pGLUA-P, and 117 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG immunized with pGJA-P reached its peak 10 weeks after the initial immunization, 4 times that of the mice immunized with pGLUA-P, and 160 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG of the mice immunized with pGLUA-P reached its peak at 16 weeks, however, significantly lower than the peak of the mice immunized with pGJA-P (P < 0.01). The serum anti-Pac IgG levels of the mice injected with pCI or with normal saline were not significantly different (P > 0.05). Since the second week after the initial immunization, significant difference in the serum anti-PAc IgG level could be seen between the mice immunized with pGJA-P or the mice immunized with pGLUA-P, and between the mice immunized with pGJA-P and the mice immunized with pGLUA-P and those injected with pCI or normal saline (all P < 0.01). Six weeks after the initial immunization the salivary anti-PAc IgA level of the mice immunized with pGJA-P was 18 times those of the mice injected with pCI or with normal saline (both P < 0.01), 10 weeks after the salivary anti-PAc IgA level of the mice immunized with pGJA-P reached its peak, 24 times those of the mice immunized with pCI or normal saline without a significant difference between the latter 2 groups (P > 0.05). No effective salivary IgA response was seen in the mice immunized with pGLUA-P. CONCLUSION: pGJA-P can be expressed in vivo. Immunization with pGJA-P intramuscularly induces effective mucosal and systematic humoral responses. It is an effective DNA vaccine against dental caries.

[Immunization of rats with a targeted fusion anticaries DNA vaccine].

OBJECTIVE: To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in situ. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and pGLUA-P, a fusion anticaries DNA vaccine. METHODS: pGJA-P was administrated intramuscularly or intranasally to rats, and the expression of recombinant protein was detected by immunohistochemistry technique. Wistar rats were fed a cariogenic diet and orally infected with S. mutans, then immunized with pGJA-P or pGLUA-P via the intramuscular or intranasal route. All rats received a booster immunization 2 weeks later. At the termination of the experiment, blood and saliva samples were collected for assay of antibodies by ELISA and jaws were obtained for caries evaluation by the Keyes method. RESULTS: Recombinant protein could be detected in muscle in intramuscularly immunized rats and in nasal mucosa in intranasally immunized rats. Rats immunized intramuscularly with pGJA-P had significantly higher serum IgG levels than others (P < 0.01). Rats immunized intranasally or intramuscularly with pGJA-P had significantly higher salivary IgA levels than others (P < 0.01). Keyes scores of pGJA-P groups were significantly lower than those of pGLUA-P groups and pCI groups (P < 0.01). CONCLUSIONS: pGJA-P could be correctly expressed in vivo. pGJA-P generated increased humoral immune response and anticaries efficacy compared with pGLUA-P.

[Construction and expression in vitro of a targeted fusion anticaries DNA vaccine].

OBJECTIVE: To construct and detect the targeted anti-caries fusion DNA vaccine pGJA-P encoding the A-P fragment of pac, glu fragment of gtfB and extracellular region of the human CTLA4 and the Fc region of human Iggamma(1) gene for the targeting antigen to APC. METHODS: The extracellular region of the human CTLA4 and the Fc region of human Iggamma(1) genes were amplified by RT-PCR from human lymphocytes, and the genes were cloned into pUC(m-T) vector respectively. After sequencing, Fc region of human Iggamma(1) gene was cloned to the downstream of CTLA4 gene fragment as the recombinant plasmid pGJ. The fusion gene was then inserted into the plasmid pGLUA-P to get the recombinant plasmid pGJA-P. The CHO cells were transfected with liposome and the expression of fusion protein in cultured supernatants were detected using Western blotting. RESULTS: The plasmids pGJ and pGJA-P carried the CTLA4-Ig fusion gene and CTLA4-Ig fusion gene, A-P fragment of pac gene and glu fragment of gtfB gene respectively. The expressed protein could response to specific anti-PAc antibody. CONCLUSION: The targeted fusion anti-caries DNA vaccine pGJA-P is constructed successfully and expressed in eukaryotic cells correctly.