| ObjectiveTo study Haemophilus influenzae type B (Hib),which lead to serious invasive diseases of children.Animals as a model to evaluate the dose relationship between immunogenicity and antibody level of Hib capsular polysaccharide ,to assess the value of PCR technology in the detection of Hib in childhood respiratory diseases ,to discuss the potential application and feasibility of genotyping corresponding with capsular dose in reflecting the strain invasion.So as to guide the clinical therapy and vaccination,to provide some experimental basis for the development of new vaccines .Methods1. Use SD rats as a model,they were given different doses of capsular polysaccharide of Hib PRP-T conjugate vaccine.Saline as a control.Using ELISA to evaluate Hib polysaccharide antibody concentrations in serum.Using SAS 8.1 statistical Data processing software to analyze immunogenicity and dose effect of Hib capsular polysaccharide .2.Totally 159 strains of Haemophilus influenzae isolating from deep sputum of children's respiratory infection from 2009-2010 were enrolled at the author' hospital.The primers were specifically synthesized for Hib,and for Hcs A which was involved in the export of the polysaccharide to the surface of the cell. PCR technology was used to detect Hib strains and identify Hib subtypes.The positive samples of PCR products were sequenced and analysed.Results1.After the last immunization,the average concentration of PRP IgG were higher than 0.15μg/ml,which were induced by each experimental group gave with different doses of capsular polysaccharide.After the immunization of 1,2,4 weeks , PRP IgG level of each experimental group gradually increased, there was a significant statistically difference(P <0.01).2.PRP IgG levels increased with the dose of capsular polysaccharide increasing,which were induced by each experimental group gave with different doses of capsular polysaccharide. The comparison of the average antibody levels among the group before immunization,the group after the immunization of 1 week ,the group after the immunization of 2 week and the group after the immunization of 4 week at the same time showed experimental groups before immunization were no significant differences (p> 0.01),the 0.8μg and 2.5μg polysaccharide groups at 1 week after immunization showed no significant differences (P> 0.01), the differences among the rest of the experimental groups were statistically significant (p <0.01).3.In terms to 159 strains of Haemophilus influenzae, 90 strains were isolated from children with pneumonia (56.6%),39 strains were isolated from children with bronchitis (24.5%).4. 4 clinical isolates showed positive results with bex A gene amplification in 159 Hi clinical isolates,of which NO.69 clinical isolate also showed positive results with Hi-b and HiHcsA-I gene amplification.We aligned this two sequences using BLAST with the respectively identities of 98.5% and 99%.5. 4 clinical isolates showed positive results with bex A gene amplification in 159 Hi clinical isolates,they were typeable Haemophilus influenzae,amounting to 2.52% of all Hi,nontypeable Haemophilus influenzae amounted to 97.48% of all Hi.NO.69 clinical isolate showed positive results with Hi-b and HiHcsA-I gene amplification, we designated it as type I.Conclusion1.The capsular polysaccharide of Hib conjugate vaccine can induce good immune response, the antibody concentrations are increasing along with the time and capsular polysaccharide contents,showing a good dose effect.2.Haemophilus influenzae is a main cause of respiratory diseases in the children. With high sensitivity and specificity, PCR technology can be well operated in the detection and typing of Hib strains. Genotyping aiming to HiHcsA gene possibly reflect the invasion of the Hib strains isolated from children. |
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