The Construction And Effect Analysis Of Pyogenic Streptococcus PcDNA3.1 /Spy0747 Gene Vaccine

Object: Group A streptococcus(GAS), also called s. pyogenes, is a human specific pathogen. It is transmitted by droplets and skin contact and is widely distributed around the world. GAS can cause some mild infections as sore throat, impetigo, also can lead to rheumatic heart disease, arthritis,glomerulonephritis, sequela. More seriously, it also can cause some severe diseases such as sepsis, streptococcal toxic shock syndrome. Usually, the GAS parasitic on the skin and upper respiratory tract; more than 20% of children were asymptomatic carrier; every year, there are about 500000 people died of streptococcus infection. Although, GAS is still sensitive to antibiotics, but reports of drug resistance are also growing. Therefore, in recent one hundred years, people have been looking for vaccine to effectively prevent the pyogenic streptococcus. But, unfortunately, until now, there is no safe and effective vaccine used for clinical purposes. In recent years, using complementary genome technology, people screened some bacterial surface antigens on the surface GAS,Some of which have been validated by animal experiments and have certain immune protective effect. Spy0747 is a DNA enzyme on the surface of the Pyogenic streptococcus,because it is homologous to the SsnA protein, a cell-wall-located DNase expressed in Streptococcus suis serotype2,so it was also designated as SpnA. It requires the participation of Ca2+ and Mg2+ to work. SpnA is widely distributed in the currently known serotypes of Pyogenic streptococcus. It can induce corresponding antibodies and in nuclease assay and these antibodies can inhibit the activity of SpnA. The virulence of spnA knockout mutant strain is markedly reduced.This shows that SpnA has the potential for vaccine development. The gene of SpnA protein was amplified,and then it is connected to eukaryotic expression plasmid to make the nucleic acid vaccine. We verify the effectiveness of the vaccine through animalexperiment.Methods:(1) The corresponding primers was designed by primer5.0. The DNA fragment of Spy0747 was amplified by PCR.(2) The PCR product was linked to PGEM-T simple vector and were transformed to E.coli DH5α,inserted the recombinant clone were selected. The plasmid was identified by BamHⅠand XhoⅠcutting and sequencing.(3)The inserted segment was isolated, linked to pcDNA3 cut by the same enzymes to construct a eukaryotic expressing plasmid of pcDNA3/ Spy0747.After transformed, the recombinant plasmid was identified by enzyme cutting and sequencing.(4)The recombinant plasmid of pcDNA3/ Spy0747 was expressed in E.coli. Then a mass of recombinant plasmid DNA was extracted by alkaline lysis method.(5)C57 mice were randomly individed into 2 groups, every group is 20,and immunized with PBS,Spy0747 。 The same dose(100ul,1ug/ul) GAS was intramuscularly injected to the left hind limb of the mouse at an interval of14 days, and boosted three times.(6)Identifying immune indices: 14 days after every immunization,three of mices in each group were Killed。 The spleen of the mice were cut out.Total RNA was extracted from spleen cells which were used to detect mRNA of cytokines IL-4, IL-12 by Real-time PCR.(7) Mice were challenged with lethal dose(200ul, 1.5*107)GAS to evaluate the protective rates on the mice after the last immunization.The mice survival was recorded and made the survival curve.(8) 3 days after challenge, one of mices in each group were Killed and the heart,kidney, lung and spleen of the mice were cut out. After making pathological section, HE staining, we observed the immune protection on tissue.Results:(1) The gene fragments of Spy0747 was amplified by PCR. The length of the fragments was same as expected.(2) The prokaryotic clone vectors PGEM-T /Spy0747 was su ccessfu lly constructed, DNA sequ encing showed that the sequ ences is identical to the sequence recorded in Genbank.(3) After the gene fragments were inserted to the pcDNA3, the recombined plas mid was identified by enzyme cutting and sequencing,the results were identical asexpected and suggested that the eukaryotic expressing plasmid of pcDNA3/Spy0747 had been constructed successfully.(4) Cytokines IL-4 and Cytokines IL-12 had difference among the groups by Real-time PCR(P<0.05). HE staining showed that heart and liver have a slight injury,and lung and spleen were normal after immunition. Heart and spleen were normal, and lung had a slight injury and liver was damaged seriously after bacterial attack. The protection of the vaccine effect seems to be not quite apparent.Conclusion:(1) pcDNA3.l/Spy074 gene vaccine was constructed successful.(2) Compared with control group, in the experimental group, the IL-4, IL- 12 both had difference, which illustrating the cellular immunity and humoral immunity of mice were changes, but immune group shows no significantly protective effect, pcDNA3.1/Spy0747 protective effect was not obvious.