| Staphylococcus aureus is one species of gram positive coccuses which can infect many animals. The bacteriological investigation showed that Staphylococcus aureus is one of the important pathogens that cause dairy cattle mastitis, which has endangered the cow breeding industry. In order to prevent and control of dairy cattle mastitis effectively, it is necessary to study an efficient vaccine against the infection of Staphylococcus aureus. In this study, the following experiments were carried out.Firstly, protective antigen genes Staphylococcus aureus strain SHZ1 isolated from cow mastitis:were successfully cloned, sequenced and analysed. FnBP gene and C1fA gene were amplified from Staphylococcus aureus SHZ1 genome DNA by polymerase chain reaction (PCR) technique, respectively. The amplified products of PCR were cloned into T vector and then sequenced. The sequencing results showed that the nucleotide sequence of FnBP gene of Staphylococcus aureus strain SHZl was shared 99.86% sequence identity with that of Staphylococcus aureus (EF127872) reported in Genbank, and there are two bases sequence, but both of these two bases were synonymous mutations. And the nucleotide sequence of C1fA gene of Staphylococcus aureus strain SHZlwas shared 96% sequence identity with that of Staphylococcus aureus (EF207779) reported in Genbank, and the amino acid sequence was shared 100%.Secondly, recombinant poxvirus expressing FnBP gene of staphylococcus aureus was successfully constructed, identified and purified. The pMD18-T-FnBP was digested by samⅠand target gene was recovered. Then FnBP gene was subcloned into pSCll vaccinia virus vector to generate recombinant shutter plasmid pSCll-FnBP. After enzyme digestion and sequencing, the positive clone containing recombinant shutter plasmid pSCll-FnBP was selected, and transfected into BHK-21 cells infected vaccinia virus by lipofectin transfection. The transfected cells were harvested after transfection. The cells were frozen thawed at-70℃and 37℃for three times. The lysed cell pellets from the transfections were fully diluted and then inoculated BHK-21 cell by using blue-white screening recombinant viruses. The recombinant viruses were purified by five rounds of plaque purification in BHK-21 cell. The recombinant viruses expressing FnBP gene was identified by PCR, RT-PCR and western-blotting technique, respectively. The result showed that the FnBP gene has been integrated into the genome of vaccinia virus successfully, and the target gene can be transcripted and translated with the growth of vaccinia virus in BHK-21 cells.Thrirdly, the growth characteristics, safety and immunogenicity of Staphylococcus aureus FnBP recombinant vaccinia virus were studied. The growth characteristics were assayed in BHK-21 cells using TCID50 index. The results showed replication level of recombinant vaccinia virus was not lower than non-recombinant vaccinia virus.30 mice were divided into three groups, and each of the three groups were immunized by different antigens to study the immunity. Recombinant poxvirus multi-point injection was asⅠgroup, vaccinia virus as the II group (control group), PBS as blank control group. After 30 days of inoculation, the signs of internal organs and pathological changes of immunized mice were observed. The results showed that mice immunized by recombinant poxvirus pSC11-FnBP did not show any adverse response, which displays a good security. The IFN-y and phagocytosis of neutrophils in the serum of immunized mice were detected respectively. The result showed that the recombinant poxvirus can induce better cellular immunity than that of the vaccinia virus. The challenge tests showed that the recombinant poxvirus may provide some immunogenicity to protect mice against Staphylococcus aureus infection. This study not only lay a certain foundation to new vaccine against Staphylococcus aureus, but also provided a new thought to prevent and control dairy cow mastitis. |
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