The Research On Construction And Transformation Capability Of High Virulence Streptococcus Mutans With HtrA Deficiency In Children

Objective:The aim of this research is to put recombinant plasmid carrier pUChtrKO which had been built by our experimental group into Streptococcus mutans with HtrA high virulence which had been isolated from high caries susceptible children aged from3to5. Use homologous recombination to construct and identify the high virulence streptococcus mutans with HtrA deficiency, and compare the transformation capability of Streptococcus mutans with HtrA high virulence and deficient virulence.Methods:①onstructing the high virulence Streptococcus mutans with HtrA deficiency:the Streptococcus mutans with HtrA high virulence which had been obtained in preliminary experiment was reanimated and incubated in anaerobic culture, then collected bacteria to prepare competent stimulating peptides cells, put the recombinant plasmid vector which join has been constructed into cells, use homologous recombination technique to construct the high virulence Streptococcus mutans with HtrA deficiency virulence. Observe the morphology of HtrA deficiency strains under microscope and identify the strains by biochemical identification.②Use PCR to identify the high virulence Streptococcus mutans with HtrA deficiency:According to the sequence of HtrA gene, design the primers HtrF/HtrR of HtrA gene and amplify by PCR, then make the amplifying by PCR for the resistant gene primers P1/P2of erythromycin, HtrA gene upstream sequences of primers with resistant gene primers HtrAUF/P1of erythromycin, HtrA gene downstream sequence of primer with resistant gene HtrAUR/P2of erythromycin,and make the identification of PCR for four gene fragments which was amplified by PCR with the genomic DNA of standard Streptococcus mutans containing HtrA gene as a reference.③ompare the transformation capability of Streptococcus mutans with HtrA high virulence and HtrA deficient virulence:The Streptococcus mutans with HtrA high virulence and deficient virulence were reproducted in TPY liquid and cultured in anaerobic condition. Compare the transformation capability of the two strains by counting the number of colonies. Results:The high virulence Streptococcus mutans with HtrA deficiency could be constructed successfully by using homologous recombination technique. Use gel imaging analyzer to analyze the DNA fragments obtained from1%agarose gel electrophoresis. Group1-4were high virulence Streptococcus mutans with HtrA deficiency, and group5-8were Streptococcus mutans with HtrA high virulence; choose DNA with DL2100molecular weight of HtrA standard Streptococcus mutans as Marker. The result shows that there was no specific band in group1at500bp, while there was a bright band in group5;500bp was the DNA molecular weight of HtrA gene fragment. There was a bright band in group2at710bp, while there was no band in group6;710bp was the DNA molecular weight of the erythromycin resistant gene fragment. There were both bright bands in group3and4at1200bp and1100bp. There were no bands in group7and8;1200bp and1100bp were DNA molecular weight of HtrA gene upstream sequences of primers with resistant gene primers HtrAUF/Pl of erythromycin and HtrA gene downstream sequence of primer with resistant gene HtrAUR/P2of erythromycin. The result shows that:At6、9hour, the transformation capabilities of two groups increased obviously; there is significant difference between two groups, P<0.05. At12、24、48hour, the transformation capabilities of two groups increased greatly; there is great significantly difference between two groups, P<0.01.Conclusion:HtrA gene of high virulence streptococcus mutans could be knocked-out by using gene recombination method, and it was consistent with the experimental results by identification of PCR. Transformation capability of Streptococcus mutans with HtrA high virulence was higher than that of HtrA deficient virulence, which could confirm that HtrA was closely related to the virulence of Streptococcus mutans.