The Idendification Of 16S RRNA Of Pathogens Causing Endometritis In Dairy Cows And Prokaryotic Expression Of Coagulase In Staphylococcus Aureus

Endometritis was one of the most common diseases which pathogenic micro-organisms caused, and it led to great economic loses for the dairy industry. So far, there was no an accurate and quick method to detect the major mastitis pathogens; in addition, there were very few data about molecular epidemiology of pathogens in bovine endometritis, and there was no an effective vaccine against endometritis. Based on the backgrounds above, a series of researchs on dairy cows endometritis were carried out. some methods and results were reported as follows:Partâ… : Isolation and identification of pathogens causing endometritis dairy cowUterine samples from dairy cows with endometritis were collected in Jinan and Taian and pathogens were then isolated and identified. First,bacteria in samples were routinely isolated, purified, cultured and classified. Specific primer was used based on the structural property of 16S rRNA and 16S rRNA gene of unknown strain was amplified on PCR,then ligation of amplified product with pMD19-T constructed clonal vector, finally its sequencing was carried out after correcting through PCR and double digestion and its homology were compared with known bacteria in GeneBank. The results indicated that the pathogens causing endometritis in dairy cows included Acinetobacter junii; Streptococcus .faecalis; Staphylococcus aureus; Staphylococcus intermedius ; Staphylococcus haemolyticus ; Acinetobacter Iwoffi; Streptococcus agalactiae; Bacillaceae; Bacillus subtilis; Escherichia coli; Pseudomonas Migula, respectively, and Staphylococcus aureus and Escherichia coli were important pathogens causing endometritis. The results showed that the pathogen strains causing endometritis in dairy cows was identified successfully and accurately through internationally recognized 16S rRNA sequencing identification technique, which played an important part in preventing and treating endometritis in dairy cows.Partâ…¡Prokaryotic expression of coagulase gene in staphylococcus aureus and analysis of its bioactivityCoagulase was one of major toxical factors in S. aureus, and its gene had one ORF and 2106bp ,and coded 701 amino acids. The gene coding coagulase was amplified from strain of s. aureus in dairy cows with endometritis, and its plasmid pET32a+/coa prokarocyte expressed was constructed after clonal screening and sequencing identification .After expression and analysis on SDS-PAGE, the result indicated: coa gene was expressed confluently in E.coli.and the molecular weight of confluent protein was 100KD. Coagulase test showed that the confluent protein had the abilitiy to coagulate plasma and its titer was 0.012mg/mL, showing that the expressed product had bioactivity, which provided the prerequisite for studying the pathogenicity and immune mechanism and vaccine design.