| Staphylococcus aureus (S.aureus) is a Gram-positive bacteria which is widely distributed in nature. S.aureus can produce a variety of exotoxins and enzymes, having a strong pathogenicity and causing a lot of bacterial infections in human and animals, such as skin and soft tissue infections, leading to sepsis, endocarditis, pneumonia, meningitis, etc. It also can cause urinary tract infections, osteomyelitis, arthritis and enteritis. Recently US CDC(Centers for Disease Control) reports, infections caused by Staphylococcus aureus ranked for the second place, after E. coli infections (Wang Fengling,2007). Food poisoning due to the enterotoxin produced by Staphylococcus aureus in the United States, accounting for33%bacterial food poisoning cases, and45%in Canada. Such poisoning incidents issue occurs in China each year, also getting much More. Staphylococcus aureus, one of the major pathogens causing mastitis in dairy cows, can bring tremendous damage to dairy industry worldwide.To successfully sustain an infection, nearly all bacteria, fungi and protozoa require a continuous supply of host iron. Generally there are two ways for bacteria to obtain iron from the environment, one is to obtain exogenous iron-containing host cell proteins, such as hemoglobin, transferrin, ferrichrome; and the second, pathogens can employ low molecular mass (<1kDa) siderophores to obtain iron, which has a high affinity for chelating iron. Staphylococcus aureus is no exception. Except the use of Isd (Iron-regulated surface determinants) system to transport iron from hemoglobin, S. aureus has many ABC transporters to transport siderophores:including transport proteins encoding by sirABC, sstABC, fhuCBG operon respectively. In sirABC operon, sirB and sirC encode protein SirB and SirC located in the transmembrane region through the cell membrane; and SirA is a lipoprotein, located in the external plasma membrane, has the ability to capture iron-siderophores complex.To study protein SirA, the binding protein of iron-siderophores complex,we cloned sirA gene from Staphylococcus aureus genomic DNA by PCR technique. The fragments were recovered and purified, and connected to pColdTM TF frozen expression vector, then transformed into E. coli BL21(DE3) competent cells, expressed recombinant protein SirA-TF by IPTG induction, then recombinant protein SirA-TF were purified by protein purification kit and its purified protein concentration were determined. ICR mice were immunized with SirA-TF recombinant protein for further antiserum preparation, antibody titre of immune serum were measured by indirect ELISA; SirA protein expression of S. aureus in the growth culture with different iron concentration were detected by Western Blot.Mice were immunized with SirA protein, inactivated S. aureus and sterile PBS solution for three times respectively, live S. aureus with107,108,109CFU were prepared for mice nasal colonization after the third immunization for7days. Seven days latter, planting mice were killed to cut the nasal tissue for counting the total number of S.aureus in nasal.The role of sirA in S. aureus pathogenicity was primarily studyed. The experiment was designed to study the effect of anti-SirA polyclonal antibody on growth of S. aureus.Gene sirA was successfully amplified, and cloned into the expression vector pColdTM TF. SDS-PAGE showed that the recombinant protein pCold-sirA successfully expressed in E.coli BL21(DE3) was in a soluble form. ICR mice were immunized with the recombinant protein for antiserum preparation, anti-SirA antibody levels reached at1:64000for ELISA titers two weeks after the boost immune; Western Blot analysis showed that the expression level of SirA recombinant protein is higher when S. aureus grow in low iron medium (TSB with600μM2’2-dipyridyl) than in iron rich condition (TSB with50μM FeCl3).The results of nasal colonization showed that the ability of blocking the adhesive of S. aureus strains in mice immuned with SirA recombinant protein (2283.33±411.86) is higher than the group immuned with the killed S. aureus (4420.00±1408.97)(P<0.05). and which of the former one was significantly higher than the control immuned with PBS (10203.33±1296.93)(P<0.01)7days after the third immune; The S. aureus CFU colonized in the mice immuned with SirA protein (610.00±105.35) is lower than the control immuned with PBS (1683.33±367.46)14days after the third immune.Results of antibody suppression test showed that anti-SirA polyclonal antibody with certain dilution can decrease the growth speed of S. aureus in iron restrict medium significantly.but not in iron rich medium.In summary, the expression of sirA gene in pColdTM TF vector was in a large number of soluble recombinant protein, and the recombinant protein SirA had a good immunogenicity and immune protection, which can be used as a basic-consideration for the control of Staphylococcus aureus infection. |
