The Study On The Relationship Between CpG Island Methylation In The Promoter Of MBL And Mycoplasmal Pneumonia In Sheep

Mannan binding lectin (mannan-binding lectin, MBL), mainly in serum, is secreted by the liver to the mammalian blood is a calcium-dependent C-type serum lectin. MBL human innate immune system as an important first-line anti-infective molecules of the innate immune defense against a variety of pathogenic microorganisms. The MBL gene sequences from the sheep experiment, combining with Mycoplasma pneumoniae infection in sheep, of MBL gene promoter methylation and associated mycoplasma pneumonia of sheep for further diagnosis and treatment of mycoplasma pneumonia in sheep provide important theoretical basis for provide the scientific breeding of fine varieties of reference.1. Cloning and sequence analysis of the promoter region of ovine mannan-binding lectin geneIn order to get the sheep sequence of the promoter region of MBL gene, this study with reference to the laboratory submitted to the NCBI MBL gene in sheep (GenBank: FJ977629.1),3 pairs of specific primers designed to the sheep genomic DNA as template, using TAIL-PCR Get a specific fragment was amplified by the recovery of restructuring to the pMD18-T vector, positive clones were selected and sequenced.The results were as follows:The measurement sequence length 679 bp, and through the authority of biological software and online prediction site analysis and comparison of its structure and found that the intron sequence was obtained DEF, Ets, GAGA-1, TCF/LEF and Otc-3 and other transcription Binding site of the original and control, the test MBL gene from the sheep is a non-TATA-box and DPE and can Ets but normal eukaryotic transcription promoter, the sheep were successfully obtained the promoter region of MBL gene sequence.2. Detection method for methylation in promoters of ovine MBL GeneApparent genetic modification is a class above the level of regulation of gene expression, DNA methylation as an apparent modification of the core, has been one of the hot molecular biology. DNA methylation is the only natural vertebrate DNA chemical modification, such as in the regulation of gene expression play an important role. Gene promoter region and its nearby areas the degree of CpG methylation and transcription inhibition are closely related. In this study, select the suffering of sheep 4 mycoplasma pneumonia, liver extraction of DNA, primer design method based on the principle of BSP primers were designed using methylation detection technology for the BSP gene promoter methylation sequence.The all results were as follows:BSP methylation detection method used successfully to detect the 234 bp amplified fragment, methylation analysis showed that sheep infected with MP in the promoter region of MBL gene 6 CpG sites have 5 sites for the methylation, While the normal sheep in the promoter region of MBL gene 6 with 4 CpG sites methylation, quite different, indicating that this experiment established methylation detection technology platform BSP accurate and reliable.3. Study on the relationship between Mycoplasma pneumoniae and methylation of promoter of MBL gene in sheepMBL human innate immune system as an important first-line anti-inflammatory molecules, has shown with a variety of diseases, such as general immunodeficiency, specific infections, cystic fibrosis (CF), systemic lupus erythematosus (SLE), rheumatoid Yan (RA) and so on. Mycoplasma pneumonia is a sheep in sheep caused by Mycoplasma pneumoniae infection in a specific disease. In this study 43 sheep 3 months old build test groups (drug group 37 attack, the control group 6), with sheep, Mycoplasma pneumoniae (M.ovipneumonia) standard strain Y-98 exchange that intratracheal inoculation of virus groups, the use of BSP Methylation detection technology, the successful detection of the 37 drug groups attack the sheep and six sheep in the control group MBL gene promoter methylation and levels of serum MBL concentration in the situation.The all results were as follows:(1) Before and after virus attack, attack drug group tended to decrease serum levels of MBL, and the difference was very significant (p<0.01), while the control group showed no statistically significant difference;(2) 6 CPC fragment selected CpG sites in the control group,2 to 4 methylation, mainly in CpGl, CpG2, CpG3 and CpG6 the sites were not methylated 4,5; Attack drug group 4 to 6 methylation compared in terms of the control group, CpG3, CpG4 and CpG5 significant methylation;(3) 6 killed in attack drug test detect methylation and sheep serum levels of MBL were significantly lower in the samples, CpG5 methylation rate was significantly higher than the other five CpG sites detected, CpG3 and CpG4 A Glycosylation significantly higher incidence of the remaining three CpG sites, and CpGl and CpG2 methylation sites in the attack rate of drug group and control group were no statistically significant difference, suggesting that the promoter region of MBL gene near Coding sequence of the CpG sites (CpG3, CpG4, CpG5) methylation in the regulation of MBL protein may have more significance.