| Objective: Group a streptococcus,belonging to a family of streptococcus,is a kind of gram positive bacteria and can cause not only pharyngitis but some serious infections,such as streptococcal toxic shock syndrome,sepsis,and some sequelaes after streptococcus infection such as rheumatic heart disease,arthritis,glomerulonephritis.It is estimated that each year,the cases of pharyngitis caused by s.pyogenes are at more than 500 million and the cases of pyoderma caused by s.pyogenes are at more than 100 million.Some serious pyogenic streptococcus diseases,such as rheumatic heart disease and invasive infectionin,lead to at least 500000 deaths a year.While the mortality of invasive disease is between 15-30%,the mortality of streptococcal toxic shock syndrome can be more than 50%.The prevention of streptococcicosis depends on the rapid diagnosis and treatment of penicillin.Although streptococcus is still sensitive to penicillin,reports of antibiotic resistance increase continually.In the United States,about 20% of the antibiotic prescription of acute respiratory disease is used to streptococcal pharyngitis.Therefore,vaccination,as a good option to control GAS infections,can not only significantly reduce invasive and noninvasive diseases,but reduce the use of antibiotics,so as to promote resistance to the group A streptococcus and other important human pathogens.At present,researchers mainly focu on the M protein as GAS vaccine.But the more than 100 serum types of GAS M proteinthe,the relation between the humoral,cellular immune response induced by M proteins and autoimmune streptococcal infection complications hinder the M protein vaccine development.Other candidate vaccines,such as streptococcus fiber connection protein SfbI etc,were not found in most GAS strains and they show the much diversity of the amino acid sequence among different serotypes,so they are not the ideal vaccine candidates.Recently,reverse vaccinology was used to identify new candidate vaccine from GAS and the advantages of these methods are the efficient use of several GAS genome sequences.Some researchers have systemly filtered out some new conservative protective antigens by antigen omics technology.Through these studies,those researchers have found some new antigens that have the potential to be new vaccine antigens and are highly conservative in GAS clinical isolates.The protein encoded by Spy1536 gene is one of the antigens.Gene knockout research has confirmed that this gene plays an important role in regulating the surface expression of the GAS protein and the process of the interaction between streptococcus cells and host proteins.In this experiment we built pcDNA3.1/Spy1536 eukaryotic expression gene vaccine to validate the protective effect of Spy1536 gene vaccine.Methods:1 The corresponding primers was designed using primer5.0.2 The DNA fragment of Spy1536 was amplified by PCR.3 The PCR product was linked to PGEM-T simple vector and then were transformed into E.coli DH5α which was cultured on LB medium containing 100μg/ml Ampicillin.4 Positive clones were picked and used to extract plasmids which then were identified by BamHⅠand XhoⅠcutting and sequencing.5 The inserted segment was isolated,linked to pcDNA3 cut by the same enzymes to construct a eukaryotic expressing plasmid of pcDNA3/ Spy1536.6 After transformed,the recombinant plasmid was identified by enzyme cutting and electrophoresis.7 The recombinant plasmid of pcDNA3/ 1536 was expressed in E.coli.Thena mass of recombinant plasmid DNAwas extracted by alkaline lysis met h-od.As a control,the pc DNA3.1 was also extracted.8 C57 mice were randomly individed into 3 groups,each of which have 20,and immunized with PBS,Spy1536 and pcDNA3 respectively.Mice were immunized with the same dose at an interval of 14 days,and enhanced three times.Two weeks after each immunization,three mice were Killed in each group.The heart,liver,lung and spleen of the mice were harvested and stored in 10% formaldehyde at 4℃.9 To evaluate the protective effects,mice were challenged with leth-al dose of GAS two weeks after the last immunization.On the third da-y after the challenge,one mice of each group was killed and their the heart,kidney,lung and spleen were harvested.During two weeks after t-he challenge,the physiological state,deaths of the mouse was closely observed,and the survival curve was drew.10 The pathological sections of all organs were done to analyze pathological changes.Total RNA extracted from spleen cells were used to detect mRNA of cytokines IL-4,IL-12 by Real-time PCR.Results: 1 The gene fragments of Spy1536 was amplified by PCR.The length of the fragments was the same as expected;2 The prokaryotic clone vectors PGEM-T/Spy1536 was successfully constructed,and DNA sequencing showed that the sequence accords with that recorded in Genbank;3 After the gene fragments were inserted to the pcDNA3,the recombined plasmid was identified by enzyme cutting and electrophoresis,the results were in line with the prediction and suggested that the eukaryotic expressing plasmid of pcDNA3/ Spy1536 had been constructed successfully;4 According to the Real-time PCR results,IL-4 and IL-12 levels of group Spy1536 were generally higher than that of control groups(P<0.05).IL-12 is increased gradually and its level after challenge is significantly higher than before(P<0.5).HE staining showed that all organs of the control groups and all organs before challenge were normal.After bacterial attack,heart and spleen are normal,liver and lung have a slight injury.The vaccine have a certain protective effect on the mice.Conclusion:1 After enzyme cutting identification and gene sequencing identification,we successfully constructed eukaryotic expression plasmid pcDNA3.L/Spy1536.2 The gene vaccine has a certain protective effect on the mice. |
PDF Full Text Request