| Mycoplasma synoviae (MS) is one of the main pathogenic Mycoplasma of chicken, and it main causes chicken and turkey joint swelling, synovial capsule and tendon inflammation and swelling of solid organs. Although mycoplasma infection does not cause immediate death of poultry, but once infection in chickens it is difficult to be eradicated, so the chickens can spread and long-term resulting in stunted growth, elimination rate increased, meat and eggs production dropped significantly. Mycoplasma synovial cross-infection with other pathogens, increased illness, increased mortality caused serious economic losses to the poultry industry, with the development of the poultry industry harm and losses will increase. Currently, prevention and treatment of mycoplasma drugs for rare and synovial research literature is also rare. Therefore, research and development related to the synovial mycoplasma, Mycoplasma bound for the prevention and treatment of synovial fluid drug development to lay a theoretical foundation. Based on this, I got through the inspection and amplified Mycoplasma WVU1853synovial fluid LDH gene and used it to construct a prokaryotic expression vector was expressed, and then completed the determination of LDH activity, cellular localization and other biological functions.1. According to the GenBank chicken synovial Mycoplasma (Mycoplasma synoviae, MS) lactate dehydrogenase sequence MS53strain on the design of a specific primer pair, obtained by PCR amplification MS WVU1853strain of lactate dehydrogenase gene cloned to pGEM-T Easy, sequenced properly and successfully completed the connection point mutation expressing bacteria pET-28a (+), the recombinant plasmid pET-28a-ldh transformed into E.coli BL21(DE3). Purified expression product (His-LDH) LDH immune rabbit antiserum was prepared and analyzed LDH immunogenicity by Western-blot. The results showed that:His-LDH antiserum capable and purified prokaryotic expression product His-LDH and MS specific reaction.2. By measuring the enzymatic activity of the expression production of His-LDH continuous monitoring method, the results show that His-LDH having activity, which catalyzes the enzymatic reaction and the optimum temperature is40℃; pH7.5, Al3+and Ba2+increase the activity; Cr3+can effectively promote the enzymatic reaction; Cu2+under the action of the enzyme almost inactivation, Fe3+, Mn2+, Ni2+, Zn2+enzyme to produce different degrees of inhibition effects; their Michaelis constant (Km) of0.365mmoL/L, the maximum reaction rate (Vm) of0.98μmoL/L. min-1.3. In order to understand the distribution of LDH in MS, I extracted the MS WVU1853hydrophilic membrane protein and protein Western-blot analysis showed that LDH in MS hydrophilic membrane proteins and proteins are distributed.4. For the study of LDH in MS adhesion, invasion of host cells and their role in immune protection, This study determined by ELISA test team His-LDH antigen titration and further LDH subcellular localization by Western-blot; through His-LDH polyclonal antibody in vitro bactericidal test and blocking cell adhesion and adhesion test for His-LDH immune protection were analyzed. ELISA tests showed His-LDH plasminogen can be combined with blood, and a dose dependent manner; Western-blot results showed that His-LDH antiserum with hydrophobic proteins, hydrophilic proteins and specific binding of purified protein of MS, the size is approximately34kDa. Bactericidal test results prove His-LDH complement antiserum has a good bactericidal effect; cell adhesion and blocking tests showed His-LDH antiserum was blocked on DF1cells is22.5%. From the foregoing, His-LDH has higher immunogenicity; LDH is widespread locating in the cell membrane and cytoplasm of MS; LDH in MS adhesion, there are certain protective immune host cell invasion. |
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