| Mastitis, caused by physical factors, chemical factors and biological factors, is a serious disease of substantial IFNlammation in breast. Mastitis pathogen is extensive, including bacterial and non-bacterial pathogens, such as: mycoplasma, fungi, yeast and chlamydia et.al. E. coli, a conditional pathogen, is considered as the one of the most mainly pathogens of mastitis. In recent years, it has become more common that serious mastitis caused by E. coli in farm.A large number of virulence factors and resistance genes are harbored in E. coli. Lipopolysaccharide(LPS), including lipid A, core polysaccharidea and specific polysaccharide, is major component of the cell wall of Gram-negative bacteria and is considered as one of the most important virulence in gram-negative bacteria.In this study, the lps gene, related to core antigen polysaccharide, was amplified by polymerase chain reaction(PCR) from E.coli isolated from the cow milk. Then, the lps gene and the pc DNA3.1 were ligated by T4 DNA ligase. The new expression vector of lps was named pc DNA3.1-His-lps. Furthermore, the plasmid was introduced into the Michigan Cancer Foundation-7(MCF-7) of human with liposome 2000. Moreover, quantitative RT–PCR and western blot were used for testing the consequent of tranfection. It was shown that the vaccine and subunit vaccine of E. coli lps was constructed successfully. To test the immune efficiency, BALC/c mice were injected with plasmid and LPS protein by nasal inhalation and intramuscular route. Furthermore, levels of Ig G, s Ig A, IFN-γ, IL-2, IL-4 and IL-6 cytokines in serum and lung slurry were detected by ELISA.Results are as follows:Firstly, the 1282 bp lps gene was obtained by PCR from wild E. coli strain isolated from milk of cow mastitis. Furthermore, we successfully constructed the pc DNA3.1-lps eukaryotic expression vector.Secondly, the plasmid of pc DNA3.1-lps was transfected into MCF-7 cells when 80% of the T25 flakes were filled. G418 was used for detecting the effect of transfection. It appeared a specific band on 1282 bp. The result of Western blot showed specific bands appeared at 42 k Da, which was consistent with the size of the expected protein.Thirdly, enzyme linked immunosorbent assay(ELISA) was used to analyze the levels of Ig G, s Ig A, IL-2, IL-4, IL-6 and IFN-γ. Antibody titers of all the four immunohistochemical were 1:12800, and no difference was found. However, antibody titers of Ig G in plasmid immunohistochemistry were only 1:3200. Antibody titers of s Ig A in immunohistochemical intranasal groups were higher, up to 1:6400. Conversely, antibody titers of s Ig A in plasmid immunohistochemistry were only 1:1600. Differences of IL-2 and IFN-γ were extremely different(P<0.01) compared with plasmid group and PBS group. The difference was extremely difference(P<0.01) compared with PBS control group, and it was significant different compared with plasmid groups. However, levels of IL-6 were found no different among these groups.Lastly, survival rate of protein groups were 60%~70%, and no significant difference was found. However, survival rate of plasmid groups were only 20%~30%, and mice in PBS group were all died.In addition, immune effect of LPS protein was well, which may provide the oretical basis for the prevention of E. coli mastitis in dairy cows. |
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