| T. pyogenes is a gram-positive corynebacterium, can cause infectious diseases in many economic animals, and it leads to a great economic loss of breeding industry. At present, antibiotics such as β-lactam antibiotics are commonly used to treat T. pyogenes infection. However, the drug-resistant phenomenon has been more serious, because of the widely use of antimicrobial agents,. But the unreasonable use or even abuse of antibiotics caused an increasing drug-resistance situation, and the situation may lead to the change of the expression of virulence factors in bacteria. T. pyogenes contains several kinds of virulence factors. One significant virulence factor of T. pyogenes is neuraminidase, which majorly has a physiological effect such as adhering to cells during the infection to animals. This study aimed to quantify the differences of NanH in isolates in different resistance levels and adhesion of Hela cells, analyze of resistance level of T. pyogenes β-lactam antibiotic resistence and its effects on expression of NanH and cell adhesion ability, This study will lay the ground for further study of T. pyogenes.In this study the expression plasmid pEASY-E1-nanH was established to express the soluble protein of NanH in E. coli. And the protein was administrated to New Zealand rabit to preparation the rabbit antibody against T. pyogenes NanH after purified. The results indicated that the reactivity between antibody and the T. pyogenes NanH protein was fulfilled to carry out test, the antibody titer was 1:1280000.The expression quantity of NanH between 12 T. pyogenes isolates and T. pyogenes standard strain by Western-blot was analyzed, it indicates that the expression quantities of 12 isolates shared a differentiation.Meanwhile,2 isolates expressed more quantity of neuraminidase NanH than the standard strain, among the NanH in 2 isolates was significantly higher than the standard strains,7 strains were significantly lower in comparison with the standard strain,2 strains showed no significant differentiation. The RY04-2 strain had the highest expression quantity of NanH, 4.03 times higher than the standard strains. The lowest expression quantity isolate is TEW01, which expressed only 1/12.5 in comparison with standard strain. By analyzing the results and the β-lactam antibiotic resistance level in the 12 isolates made us predict that the NanH expression quantity has an increasing trend with the increased resistance level.The bacterial adhesion test indicates that the 2 isolates had significantly higher expression quantity of NanH, and also more adherent to the Hela cell when compared with the standard strains. The 4 isolates with lower expression quantity has also the lowest adhesion to the Hela cell, which indicated that the adhesion to Hela cell had a similar relationship with the increase of NanH expression. Meanwhile, the isolates that resistant to 3 or 4 kinds of antibiotics had significantly higher adhesion to Hela cell than the standard strains, and were also higher than majority of isolates that are resistant to 2 or less kinds of isolates. That suggested the NanH expression and adhesion of T. pyogenes increased as the increasing of resistance to p-lactam antibiotics.The study successfully produced the rabbit anti-T. pyogenes NanH polyclonal antibody and detected the influence of different anti-T. pyogenes NanH isolates and NanH expression quantity on adhesion to Hela cell, which further indicated the relationship between drug resistance and NanH expression as well as the influence on cell adhesion. This study investigated the pathogenic mechanism of T. pyogenes, laid the foundation for effective prevention and treatment of infectious diseases caused by T. pyogenes. |
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