The Identification Of The Cloning,Polyclonal Antibody Preparation And Enzymatic Functional Of Lipoate Protein Ligase From Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae(Mhp)is the pathogeny of swine bronchial pneumonia.The mortality rate is not high after Mhp infection,but Mhp could cause mixed infection by colonizing respiratory ciliary tissue and destroying cilia barrier,so bring great harm to pig industry.Mhp which has a simple structure is difficult to culture and isolate,because it is strict with the environment.So it brings great difficulties to Mhp's further research.Research on the metabolism of Mhp helps us to better understand its growth mechanism.Lipoic acid is a high effective antioxidant which widely exists in animals,plants and microorganisms and plays an important role in the energy metabolism of organisms.LA is a coenzyme of alpha-ketoglutarate dehydrogenase,pyruvate dehydrogenase and glycine cleavage system in the process of energy metabolism of organisms.These multi-enzyme complexes play an important role in energy metabolism,fatty acid synthesis and amino acid degradation of organisms.The metabolism of LA in organisms is mainly catalyzed by related enzymes,and covalently linked to a class of protein molecules containing Lipol Domain(LD).This reaction is also called lipoic acid modificatio,which is important for the growth and metabolism of microorganisms.However,only a few microorganisms have been studied thoroughly the modification pathway of lipoic acid.Studies on some species have found that lipoate protein ligase plays an important role in lipoic acid metabolism in organisms.At present,there is no literature about lipoate protein ligase(Lpl)from Mhp.To analyze whether or not there is a natural Lpl in Mhp,a polyclonal antibody against Mhp rLpl was prepared in this study.The Lpl was detected in Mhp at the protein level using the prepared polyclonal antibody against Mhp rLpl.Then the function of Mhp Lpl was studied by in vitro reaction.The Western blot results showed that Mhp Lpl could transfer lipoic acid molecules to the LD on the Mhp GcvH.This study further explores the structural function of Mhp Lpl.Combining the crystal structure of rLpl protein,the large domain and small domain of Mhp Lpl were cloned and expressed.Through in vitro experiments,we confirmed that its large domain can complete the whole transfer process,and the small domain can't do this function.In order to explore the lipoic acid binding sites on the Mhp GcvH under the catalysis of Mhp Lpl,the site-directed mutagenesis of lysine on its GcvH was carried out by site-directed mutagenesis technique,then the recombinant plasmids with successful mutation were expressed and purified,and the lipoic acid binding sites were determined by vitro experiments.Finally,the results showed that the 56 th lysine was a lipoic acid binding site.In summary,we first discovered the Lpl on Mhp,determined that Mhp Lpl can complete LA metabolic response,and proved the function of Mhp Lpl large domain and small domain.Mhp Lpl was also found to transfer lipoic acid to position 56 of lysine of GcvH.This study helps us to better understand the growth characteristics and metabolic mechanisms of Mhp,and lays the foundation for the study of Mhp lipoic acid metabolism-related enzymes.