Cloning, Expression And Immunological Characterization Of Lactate Dehydrogenae Of Mycoplasma Mycoides Subsp Capricolum

Contagious caprine pleuropneumonia(CCPP) was disease that occur specially in goat Mycoplasma. Mycoides subsp. capri. belongs to the so-called"Mycoplasma mycoides cluster",which includes six mycoplasmas. PG3standard strains are Separated by Berieidege and Chu in1951, The main clinical symptoms of Contagious caprine pleuropneumonia(CCPP) are cellulose pneumonia, fever and pleurisy. Contagious caprine pleuropneumonia(CCPP) have high incidence in many areas of our country resulting in a great economic losses and potential threats. Lactate dehydrogenase (LDH) is also known as NAD+oxidase.Lactate dehydrogenase (LDH) is an important catalyst in oxidation-reduction reaction process LDH is one of the key enzymes in the metabolic pathways of glycolysis. LDH is widely present in animal tissues and various microbial cells, the study of Mycoplasma mycoides subspecies lactate dehydrogenase has not been reported. To understand the functions of LDH on the pathogenesis, the immunological properties were investigated in this study. Therefore, this study provided the useful knowledge for finding new drug targets and designing new-type vaccines against Mycoplasma mycoides subsp capricolum.1. Cloning, expression and purification of the LDH proteinThe gene which encodes the LDH have three terminators,so the first step is site-directed mutagenesis The570bp and408bp gene were obtained by polymerase chain reaction (PCR) of the first site-directed mutagenesis The870bp and114bp gene were obtained by polymerase chain reaction (PCR) of the next site-directed mutagenesis The LDH gene were obtained by polymerase chain reaction (PCR) and expressed plasmid pGEX-6P-2-LDH was constructed successfully. The target protein was purified by Glutathione4B,then the purified protein were identified by SDS-PAGE which was identified the target protein fragments were61kDa and were soluble expression.The GST tag of protein was removed by PreScission protease.The SDS-PAGE identification showed that:the target protein fragments were35kDa.2. The immunogenic analysis of LDH protein Cloning, Expressing and Purify LDH protein from Mycoplasma. Mycoides subsp. capri. New Zealand White rabbits (2-month-old) were subcutaneously inoculated with the purified protein emulsified with an equal amount of Freund’s complete adjuvant. PBS-treated animals were taken as negative controls. Antibodies against LDH protein were acquired by immunization of New Zealand White rabbits and the immunoreactivity of the proteins were evaluated through respective antibodies. Western blotting assays proved that purified proteins have perfect immunoreactivity.3. Bioinformatics analysisBio informatics show that, ldh gene encoding318amino acids, and the relative molecular weight (Mr) is82851.1; the total number of atoms is10840; the hydrophilic is0.698, the isoelectric point is5.01. Through the analysis of the signal peptide and the transmembrane region:LDH protein predicted non-secreted protein without signal peptide; LDH transmembrane region may be in the N-terminal sequence of5to25. Protein’s secondary structure shows that:it contains eight helices,13folds,20random coils. Tertiary structure shows that:LDH is a tetramer. Therefore, this study provided the useful knowledge for finding new drug targets and designing new-type vaccines against Brucella.