The Mannose-binding Lectin Gene In Sheep And Its Association With Mycoplasma Pneumoniae

Mannose-binding lectin (MBL) is an acute phase protein produced and secreted by liver, which belongs to the collectin family, widespread in the liver and blood. It is an important molecule of the immune system playing an important role in defending the organism from infection, given its ability to activate the complement system and induce phagocytosis. MBL function is directly associated with its complete protein structure, which are determined by the interplay between promoter and structural gene mutations that lead to low serum concentrations, weaken the capability of immune defensive and monitoring, so deficiency of MBL increases the generalised susceptibility of an individual to infectious disease.In addition, an epidemic outbreak of Hu sheep Mycoplasma pneumonia (MP) was in XinJiang especially North Xinjiang because of the necessary of intensive sheep farming and introduction, it was difficult to control and wipe out.Based on the relationship of MBL gene polymorphism, MBL serum level and disease in humans, Mycoplasma pneumonia (MP) of sheep was the disease model of research. First of all, cloned the DNA sequence of MBL gene in sheep to understand its gene structure, the association of MBL plasma concentration and genetic polymorphism was analyzed, screened the mutations which could change the MBL plasma concentration, confirmed the relationship of mutations and sheep Mycoplasma pneumonia infection by experimental infection MP to Chinese Merino, screened out the genotype which was associated with MP resistance. The specific experiment contents and results as following:1. Cloning and bioinformatics analysis on MBL gene DNA sequence in sheep:Nine pairs of primers were designed according to human and bovine MBL gene sequence, combined PCR technique to amplify the MBL gene of sheep, cloning, sequencing and splicing, to analyze bioinformatics with the DNAMAN software and online tool. The results showed that sequence of DNA of the sheep MBL gene was 4462 bp, contain promoter, four exons, three introns which encoded a 249 amino acid, submited it to GenBank (Accession No. FJ977629).Its amino acid sequence had an overall similarity with a comparable region of bovine MBL-C gene (96.39%), phylogenetic tree of amino acid analysis indicated that the sheep MBL gene was MBL-C. Expasy were employed to predict specific structure and function of sheep MBL gene,1-19 amino acids were signal peptide region, exon 1 encoded N-terminus cysteine-rich domain and 8 Gly-X-Y repetitive sequences of collagen-like domain, and exon4 encoded carbohydrate recognition domain(CRD) which had common features of C-type lectin.2. Polymorphisms in exons and introns of MBL gene in Hu sheep:PCR-SSCP method and DNA sequencing techniques were used to analyze the genetic polymorphism of MBL gene 4 exons and 3 introns in Hu sheep. The results showed that exon 1,3,4 and 3 introns of MBL gene had polymorphism, excepted for exon2. While a total of 4 genotypes were identified in exon1, which were controlled by 3 alleles; 3 genotypes were identified in exon3 which were controlled by 2 alleles; 3 genotypes were identified in exon4 which were controlled by 2 alleles; 3 genotypes were identified in intronl, which were controlled by 2 alleles, respectively;3 genotypes were identified in intron2, which were controlled by 2 alleles, respectively; 3 genotypes were identified in intron3, which were controlled by 2 alleles, respectively; 13 new SNPs mutation sites were found in exons,8 SNPs (g.27T> C,g105C>T,g.2017C> G,g.2018T>G,g.2049T>A,g3331T>C,g3334C>T,g.3235 C> T) were synonymous,5 SNPs (g41T>A(Val17 Glu),g43G>A (Val18 Met),g83A>G (Glu31 Gly),g.3243 A> G (Glu 181 Gly), g.3257 G>A (Gly 186 Ser)) were non-synonymous. MBL-A gene exon1 sequence was first found, which was shorter than MBL-C for 9 nucleotides sequence.6 new SNPs mutation sites were found in introns, g.288T>A in intronl, g.1091 T>C,g.1096A>C,g.1770G>C in intron 2, g.2297C>T,g.2331G>A in intron3。 3. Polymorphisms of MBL gene exons and introns and their association with MBL protein levels in serum of the Hu sheep:Using ONE-WAY ANOVA to analyze the relationship of different genotypes and MBL serum level detected by ELISA in Hu sheep, the result show that the MBL serum level of BB genotype was lower than CC genotype (P<0.05) in exon1, GG genotype was lower than HH genotype (P<0.05) in exon4, the hypothesis was that genotype BB and GG were related to disease susceptibility, genotype CC and HH were related to disease resistance.The synonymouses in exon3 couldn't lead to the change of MBL serum level; AA genotype was higher than BB genotype (P<0.05) in intron1, CC genotype was higher than DD,GG genotype (P<0.05) in intron4, AA, CC, GG genotypes may be related to disease resistance; BB, DD, HH genotypes were related to disease susceptibility. The point mutation in exon3 and intron3 couldn't lead to the change of MBL serum level.4. Comparison on the polymorphisms in mannose-binding lectin (MBL) gene of exon1,4 and intron 2,3 in different sheep breeds:Using PCR-SSCP method,5 new SNPs in Germen Merino were found for the first time,105C>T was synonymous, coding Pro, g.27T> C (Leu10Phe), g.43 A> G (Val 15 Met), g44T>A (Met 15 Lys), g86A>G (Thr 29 Ser) were non-synonymous; 4 new SNPs in Chinese Merino were found,105 T> C was synonymous, coding Pro, g.28C>T (Leu10Phe), g.43A>G (Met15Val), g.86G>C (Thr29Ser) were non-synonymous; the comparison results demonstrated that there were significantly different in exon1 in three breeds, the difference which was just one mutation g44T>A (Met 15 Lys) between Germen Merino and Chinese Merino, but the mutations were to be in conformity with each other in exon4, intron2,3 of three breeds. MBL-A was detected in exon1 of Germen Merino but no in Chinese Merino.5. The relationship between different MBL genotypes and MBL serum level in Hu sheep:Using T-test to analyze the relationship of exon1 genotypes of MBL gene and MP antibody level detected by ELISA in Hu sheep, the result showed that CC was more than others in healthy group (P<0.01), it was related to disease resistance; AA and BB genotypes were was more than others in disease group (P<0.05), they were related to disease susceptibility.6. The relationship between different genotypes in Chinese Merino sheep for Mycoplasma pneumonia infected by experimental susceptibility and MBL serum level:Experimental infected Mycoplasma pneumonia to Chinese Merino with different genotypes, the results show that MBL serum level of DD (g.86G>C) genotype was significantly lower than others by ELISA detect, its incidence of MP was 78%, comfirming that genotypes DD was related to MP susceptibility. The MBL serum level of CC (g.43A>G,g.105T>C) genotype was significantly higher than others, its incidence of MP was 22%, comfirming that genotypes CC was related to MP resistance.