Sheep NOS Gene Clone And Its Function Of Brucella Infection

Objective: In this study, the SNP loci of the Chinese Merino sheep e NOS gene coding regions were dectected by the PCR-SSCP method, and the e NOS gene exons with SNP loci were cloned and SNP loci being associated with the susceptibility of brucellosis had been discovered. the content and activities of the mice’s endothelial nitric oxide synthase after being infected with Brucella M5-90 were measured, and the amount of e NOS gene expression in the liver and spleen was assayed by the real-time quantitaive PCR method, thus discussing the association between the Chinese Merino sheep e NOS gene and the susceptibility of the brucellosis as well as the role of e NOS in the process of brucellas infection,which provided a certain basis and theoretical support for seeking a new way for breeding the disease-resistant sheep variety and the brucellosis prevention. Methods: 1.The m RNA sequences of the human and sheep e NOS gene（ID:NM₀00603.4 and NM₀01129901.1） were analyzed through BLAST alignment to find the exons sequences of the human’s and the sheep’s e NOS, and then the e NOS gene SNP loci of the human and the sheep were found from the SNP database of Gen Bank. The SNP loci of the human e NOS gene were analyzed through the DNAMAN6.0 software, and gained the human e NOS exons sequence that they have rich SNP loci.The exons sequences of the sheep e NOS gene,having a high similarity with the human’s these exons, were found through Gene Doc software alignment. 2.In this study, the blood serum samples of 162 Chinese Merino sheep were conducted the Brucellas’ detection through Brucella RBPT（Rose Bengal Plate Test） diagnosis kit, and then the exons’ SNP loci of e NOS gene were detected through PCR-SSCP method and these exons containing SNP loci were cloned and sequenced.SNP loci detected were conducted the brucellosis susceptibility analysis through SHEsis online analysis software. 3.After the mice had been infected with the brucella M5, the content and activity of their e NOS were detected on 0,3,7,14,28 days,respectively.The data measured were input into EXCEL, and the content and activity of e NOS in infection group and control group were conducted the statistic and analysis through the SPSS 17.0 software. 4.The mice e NOS gene in infection group and control group,on 28 days of infection,was amplified through real-time quantitative PCR technology, and the data were analyzed and revised by the 2-△△Ct method and the results were input the EXCEL to gain the relative expression of the liver and spleen tissues.The relative expression of each sample was conducted the one-way ANOVA analysis through SPSS17.0 software. Results: 1.The 10 exons of Chinese Merino sheep e NOS gene, having the high similarity with the human’s,were found by bioinformatic analysis, and they were exon2, exon7, exon8, exon13, exon14, exon16, exon17, exon18, exon19, exon20, respectively.The polymorphism of the human’s 10 exons was more abundant. 2.The 162 Chinese Merino sheep were detected through the RBPT method, showing that 101 were the negative and the positive individual were 61, and the positive samples were retested and the rate of brucella detection was 37.65%.The 10 exons of 162 Chinese Merino sheep e NOS gene were detected by the PCR-SSCP method and showed that the exon8’s 142 th base of Chinese Merino sheep occurred the base mutation.The base G mutated into the A, and amino acid was mutated from Ala to Thr and this mutation locus was a new mutation site（ss974768653:A142G）. The frequencies of the polymorphic loci’s genotype and allele were accord with Hardy-Weiberg balance law（P>0.05） through SPSS17.0 statistical analysis software and the SHEsis online analysis software analysis, and they were a representative group, but the difference of the loci’s genotype and allele frequencies distributuon between the infection group and control group was not statistically significant（P>0.05）. 3. After the mice were infected with brucella M5, the mice’s livers and spleens were enlargement on the 14 th days of infection.The content and activity of e NOS in infection group and control group on 0,3,7,14,28 days, respectively.Through statistical analysis found that the content of e NOS in infection group and control group didn’t chang and the activity of e NOS began to increase on the 14 th day of infection. 4. The e NOS gene of the mice’ livers and spleens in infection group and control group were conducted the expression analysis through real-time quantitative PCR on the 28 th of infection, showing that the difference of the mice’ livers relative expression in infection group and control group wasn’t significant and P value was 0.685（P>0.05）, and the difference of the mice’ spleens relative expression in infection group and control group wasn’t also significant and P value was 0.512（P>0.05）. Conclusion: 1.The 10 exons of Chinese Merino sheep e NOS gene were conducted the polymorphism analysis through PCR-SSCP method, and 1 SNP locus was discovered in exon8 and this locus had no relationship with the susceptibility of sheep brucellosis. 2.The content and activity of the mice’ e NOS were measured after infecting with brucella M5,and the results show that the content of e NOS don’t significantly change and the activity of e NOS on 14 th days of infection begins to increase.The results reveal that the content of e NOS don’t change after infecting with brucella M5 while the activity of e NOS begins to increase, which this process may lead to increasing the content of NO. 3.The mice’ livers and spleens in infection group and control group were conducted the relative expression analysis through real-time quantitative PCR, and the expression of the livers’ e NOS between the infection group and control group don’t change and the expression of spleen’s e NOS don’t significantly change either, showing that the brucella M5 has no influence on the expression of e NOS gene in the mice’ livers and spleens tissues.