Study on potential anti-caries DNA vaccine pcDNA3-gtfB integration into host cell genome / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Gene vaccine security is of concern because of the possibility of insertion mutagenesis resulting in inactivation of tumor suppressor or activation of oncogene. The purpose of this study was to examine the potential of anti-caries DNA vaccine pcDNA3-gtfB integrating into the host cell genome.</p><p><b>METHODS</b>Anti-caries DNA vaccine pcDNA3-gtfB was constructed by the previous study. The gtfB gene(904-4,578 bp, genebank M17361) was cloned from Streptococcus mutans GS-5. 36 Wistar rats were divided into 2 groups: submandibular gland-targeted injection(SGT) group and control group. Rats in SGT group were injected with 100 micrograms of plasmid pcDNA3-gtfB, rats in control group with PBS solution. Genomes from submandibular gland, kidney, heart, liver, lung, and brain tissues were isolated later in 12 weeks. Genomes from different tissues were purified by low-melting agarose electrophoresis. Using the purified genomes as template, plasmid integration were examined by PCR(upper primer: 5'-ATATGGTACCATGACCGAAGCGACATCTAAGCAAGA-3', lower primer: 5'-ACTACTCGAGTTAGAACCATTGACCCTG AGCATTGC-3'). The sensitivity level of PCR was determined by adding gradient plasmid copies into genomes in control group.</p><p><b>RESULTS</b>The examination of 6 tissues failed in revealing any evidence of integration at the sensitivity level that could detect 1 copy integration in 10,000 nuclei.</p><p><b>CONCLUSION</b>The potential frequency of plasmid pcDNA3-gtfB integration into host cell genome would not exceed that of the spontaneous mutation. It was indicated that pcDNA3-gtfB was genetically safe as a promising anti-carious DNA vaccine.</p>

Detection of the transmitted strains and non-transmitted strains of Mutans streptococci by AP-PCR / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Dental caries is a transmissible infectious disease in which S. mutans plays the major role. The purpose of this study was to detect the S. mutans transmitted strains and non-transmitted strains by AP-PCR fingerprint for laying the foundation of study on the relation between bacterial properties of S. mutans and its transmission.</p><p><b>METHODS</b>Plaque samples were obtained from buccal surfaces of 20 3-4 years old children and their mothers. Caries experience was scored by dmft (DM-FT). Diet regime, breast feeding and care of the children were recorded. 200 Isolates of S. mutans were detected by S. mutans B medium and confirmed biochemically. DNA from each isolate was purified and AP-PCR fingerprinting was conducted. Amplicons were separated by electrophoresis in 1.5% agarose gels.</p><p><b>RESULTS</b>45 different patterns among the 200 isolates were found. There were 10 mothers (50%) and 15(75%) children owning one genotype while 10 mothers and 5 children owning more than one (2 mothers owning 5 types). The data showed that the mothers harbored a more heterogeneous population of S. mutans than their children. Comparisons in genotypes between children and their mothers discovered that 16(80%) children harbored the same genotypes as their mothers, indicating high transmission in the group of people. Detection of the S. mutans transmitted strains and non-transmitted strains in mothers demonstrated that 10 (50%) mothers harbored not only transmitted strains but also non-transmitted strains, suggesting that different strains had different ability of transmission.</p><p><b>CONCLUSION</b>AP-PCR was capable of detecting the S. mutans transmitted strains and non-transmitted strains. Some S. mutans genotypes had higher ability of transmission than others.</p>

An experimental study on PAc and GTF gene vaccines of Streptococcus mutans against rats caries: antibody levels in saliva and serum / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this study is to examine the levels of salivary SIgA and serum IgG induced by pcDNA3-pac and pcDNA3-gtfB immunization, so as to testify the antigenity of the two gene vaccines.</p><p><b>METHODS</b>36 28-day-old Wistar rats were divided into 6 groups, among which 3 experimental groups were vaccinated with pcDNA3-pac, pcDNA3-gtfB or pcDNA3-pac combined with pcDNA3-gtfB, respectively, one positive control was vaccinated with inactive whole cell of S. mutans JBP and other two negative controls were injected with the vector pcDNA3 or PBS buffer, respectively. All vaccines and materials were delivered with 100 micrograms by submandibular gland injection for 3 times. Then the restricted bacterial model of rat was constructed. Following that all rats were fed with cariogenic diet Keyes 2000 for 3 months, saliva and serum samples were collected to assay SIgA or IgG levels by ELASA.</p><p><b>RESULTS</b>The salivary S-IgA levels both in pcDNA3-pac combined with pcDNA3-gtfB group and inactive S. mutans cell group were higher than others (P < 0.01). In groups of pcDNA3 and PBS buffer, they were lowest (P < 0.01). The serum IgG levels in the three experimental groups and positive control were higher than that in negative control (P < 0.05). It was important that salivary SIgA in groups of gene vaccine and inactive S. mutans vaccination reached its peak at the 11th week after the first inoculation and kept until the end of the study.</p><p><b>CONCLUSION</b>Both pcDNA3-pac and pcDNA3-gtfB can express immunogenic protein and induce immune responses of mucosal and humoral immune system in gnobobiotic rats. It is also indicated that the joint gene vaccines immunization is an optimal choice for anticaries strategy.</p>

Expression of plasmid pcDNA3-gtfB in mammalian cell / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at investigating the transcription and expression of recombined plasmid pcDNA3-gtfB which encoding multiple glucosyltransferase-B antigenic gene, and the feasibility of the pcDNA3-gtfB used as gene vaccine.</p><p><b>METHODS</b>The pcDNA3-gtfB was transfected into mammalian cell COS-1 with liposome. The total RNA of COS-1 cell transfected by pcDNA3-gtfB was extracted and purified. Using the total RNA as template, the transcription of pcDNA3-gtfB was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of pcDNA3-gtfB was identified with 5% SDS-PAGE, and then assayed using Western-blotting. The expression product of pcDNA3-gtfB was also assayed by using LSAB method, and cell transfected by pcDNA3 as the negative control.</p><p><b>RESULTS</b>Identified by agarose gel electrophoresis, the target gene fragment had the same molecular size (3.6 kb) as it was predicted, and it indicated that pcDNA-3gtfB was correctly transcribed into mammalian cells. Proved by SDS-PAGE, the molecular weight of the expression product (116-212 kD) was also the same as it was supposed to be. It was also indicated by Western-blotting and LSAB assay that the expression product induced immunizing response.</p><p><b>CONCLUSION</b>As gene vaccine, it is importance that the recombined plasmid could be correctly transcribed and expressed in mammalian cells. It was suggested by RT-PCR, LSAB and Western-blotting that recombined plasmid pcDNA3-gtfB could be correctly transcribed and expressed in mammalian cells, and the expression product could induce immunizing response, which support its use as gene vaccine candidates in the development of anticaries vaccines.</p>

A study on screening effective immunization route of anticaries DNA vaccine pcDNA3-gtfB / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Glucosyltransferase-B (GTF-B) of Streptococcus mutans has been implicated as a principal virulent factor in the development of dental caries. The objective was to use recombined plasmid pcDNA-gtfB expressing multiple antigen of glucosyltransferase-B as gene vaccine to immunize rats through different route, and to investigate the immunization effects of immunization routes.</p><p><b>METHODS</b>A total of 18 Wistar rats were divided into 3 groups, including the quadriceps injection group, the intransal irrigation group and the submandibular gland-targeted injection group. The serum IgG and salivary IgA were assayed by using ELISA after pcDNA3-gtfB immunization. The serum IgG and salivary IgA in different groups were compared using statistical one-way ANOVA.</p><p><b>RESULTS</b>Compared these 3 groups, the serum IgG in the quadriceps injection group was much higher than those of other two groups (P < 0.01), while the salivary IgA of the submandibular gland-targeted injection was much higher than those of other two groups (P < 0.01).</p><p><b>CONCLUSION</b>It is indicated pcDNA3-gtfB is good candidate for anticarious gene vaccine, and submandibular gland-targeted injection is an effective immunization route for stimulating salivary IgA.</p>

F-ATPase subunit uncEBF mRNA expression in Streptococcus mutans clinical isolates / 实用口腔医学杂志

Objective:To investigate the gene expression variety of different genotype of F-ATPase subunit uncEBF in Streptococcus mutans (S.mutans) and to evaluate the relationship among uncEBF gene expression levels, genotypes and the acidurance ability of S.mutans. Methods:The relative expression quantity of uncEBF gene against the housekeeping gene recA was determined by the two-step method of semi-quantitative RT-PCR in 18 clinical isolates of S.mutans(7 with genotype A uncEBF and 11 with genotyp B,10 with high acidurance and 8 with low). A gel documentation system and QUANTITY ONES software were used to assay the data. Results:uncEBF mRNA expression level in the isolates with genotype A uncEBF was higher than that in those with genotype B(P

Relationship between Mutans streptococci transmission from mothers to children and initial adherence properties / 实用口腔医学杂志

Objective:To investigate the relationship between Mutans streptococci (MS) transmission from mothers to children and its initial adherence properties.Methods:200 MS isolates were genotypied by AP-PCR to demonstrate transmission between 20 pairs of mother and child aged 3~4 years, and to detect the transmitted strains and non-transmitted strains of mothers. Then the adherence of the strains to salivary coated hydroxyapatite beads (SHA) were determined by 3H- thymidine incorperation assay.A restriction fragment length polymorphism (RFLP) study of the different regions(spap-a,spap-pv)of the spap were undertaken by endonuclease haeⅢ and AluⅠrespectively. Results:The transmitted strains showed weaker adherence properties than the non-transmitted strains (P

Relationship between genetic diversity of surface protein of Streptococcus mutans and dental caries.Ⅱ Sequence of V-region and P-region gene of surface protein of Streptococcus mutans / 实用口腔医学杂志

Objective: To sequence V-region and P-region gene of surface protein of serotype c Streptococcus mutans clinical isolates with different adherent abilities. Methods: The clinical isolates of serotype c S.mutans included two groups with different spaP-pv AluI genotypes, which were derived from previous studies in our lab. The genome DNA was extracted. The spaP-pv (2 060-3 157 bp) was amplified by PCR, then sequenced and analyzed. Results: Seven sites of AluI 5′-AG↓CT- 3′were included in the sequences of spaP-pv of both genotypes strains. The sequences of spaP-pv of ten a-genotype strains were the same except several site mutations, and so were those of b-genotype strains. Two different DNA fragments were revealed between a and b geotype strains. And they were located in the V-region of spaP. Conclusion: The mutation of gene encoding V-region of S.mutans clinical isolates might be related to the differences of adherent abilities .

An experimental study of gene vaccines pcDNA3-pac and pcDNA3-gtfB of Strept ococcus mutans against dental caries in rats:the influence on weight / 实用口腔医学杂志

0.05);by the end of the experiment that was 24.5?1.05,28. 3?1.29,26.6?1.19,10.2?1.81, 26.3?1.54 and 27.3?1.38 respectively (D vs each of the other groups P0.05). Conclusion: Gene vaccines pcDNA3-pac and pcDNA3-gtfB have no unfavo rable influence on weight of gnotobiotic rats,while the inactive whole cell vac cine has.

PAGE-AgNO_3 staining displays AP-PCR fingerprint of mutans streptococci / 实用口腔医学杂志

Objective:To investigate whether the AP-PCR fingerprint of mutans streptococci(MS) can be displayed by PAGE-AgNO 3 staining. Methods: Amplification products of 200 MS clinical isolates by AP-PCR was separated and stained by ?=3.5%PAGE-AgNO 3 and agarose-EB respectively. Results were compared and the agreement value of Kappa between two methods was calculated. Results: ?=3.5%PAGE-AgNO 3 discerned both homogeneity and heterogeneity of MS genotypes, just as agarose-EB,Kappa value for agreement was 1.00 . Moreover, more bands was showed by PAGE-AgNO 3 staining than by agarose-EB, so PAGE-AgNO 3 gave a clearer pattern than agarose-EB. Conclusion: AP-PCR fingerprint of MS can be displayed by ?=3.5%PAGE-AgNO 3 staining.

Direct pulp capping with a self-etching adhesive and calcium hydroxide on human pulp / 实用口腔医学杂志

Objective:To evaluate the human pulp response following direct pulp capping with Clearfil SE BOND (SB). Methods:45 sound human third molars in 24 volunteers were used. Pulp of 41 molars was mechanically exposed and then the teeth were divided into two groups: in group A the pulp was capped with SB, in group B the pulp was capped with calcium hydroxide (CH), another 4 teeth were served as the control. After 7, 30 and 90 days, the teeth were extracted and processed for light microscopic examination. Results:7-30 days after capping slight inflammatory reaction was observed in group A and group B. The reaction in group A was sligter than that in group B(P

Preparation and antibacteria activity of crude mutacin from clinical isolate of Mutans streptococci / 实用口腔医学杂志

Objective:To obtain crude mutacin produced by clinical isolate of Mutans streptococci (Ms) and to test its antibacteria activity. Methods:The mutacin in supernatant of in vitro cultured clinical isolate of Ms was extracted by chloroform, the antibacteria activity and heat stability of the crude extract were tested by bacteria culture technique. Results:Crude extract of protein was obtained fom clinical isolate of Ms. 10 ml of the liquid extract produced a 19 mm inhibitory zone in cultrue dish, indicating its antibacteria activity. The activity could maintain in 80 ℃ for 120 min, indicating its heat stability. Conclusion: The crude extract can represent the antibacteria activity and heat stability of mutacin.