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绵羊NOS基因的克隆及其在布鲁氏菌侵染过程中的作用

发布时间:2018-06-04 13:12

  本文选题:中国美利奴羊 + 内皮型一氧化氮合酶 ; 参考:《石河子大学》2015年硕士论文


【摘要】:目的:本研究通过对中国美利奴羊e NOS基因编码区SNP位点利用SSCP的方法进行检测,并对有多态性位点的外显子进行克隆,找出与布病易感性相关的SNP位点。用布鲁氏菌M5侵染小鼠后对小鼠e NOS基因的浓度、酶活力进行测定并对肝、脾组织中e NOS基因表达量进行检测,探讨了中国美利奴羊e NOS基因与布鲁氏菌病易感性相关性及布鲁氏菌侵染过程中e NOS的作用,为选育绵羊抗病新品种及布病预防寻求新途径提供一定依据和理论支持。方法:1、将人、绵羊e NOS基因m RNA序列(登录号:NM_000603.4、NM_001129901.1)利用BLAST比对分析找出人、绵羊e NOS基因的外显子序列,再通过Gen Bank中的SNP数据库查找人和绵羊的SNP位点。利用DNAMAN6.0软件对人e NOS基因外显子上的SNP位点进行分析,获得多态性较为丰富的人e NOS基因外显子序列,再利用Gene Doc软件比对找出与这些外显子相似性较高的绵羊e NOS基因外显子序列。2、本实验利用布鲁氏菌虎红平板凝集诊断试剂盒对162只中国美利奴羊血液样本进行布鲁氏菌血清检测,然后利用PCR-SSCP方法对e NOS基因外显子的SNP位点进行检测,后对含有SNP位点的外显子进行克隆测序。通过SHEsis在线分析软件对检测出的SNP位点进行布病易感性分析。3、用布鲁氏菌M5侵染小鼠,分别在侵染后第0、3、7、14、28天检测小鼠e NOS的含量和活力,将测得的数据导入EXCEL中,利用SPSS17.0对侵染组和对照组的含量和活力进行统计分析。4、用荧光实时定量PCR技术对侵染第28天小鼠和对照组小鼠e NOS基因进行扩增,通过2-△△Ct法分析和修正数据,最后将所得结果导入EXCEL中,得到肝、脾组织相对表达量,对生成的各个样本的相对表达量采用SPSS17.0软件进行单因素方差分析。结果:1、通过生物信息学分析,找到与人同源性高的中国美利奴羊e NOS基因的10个外显子,这10个外显子分别是exon2、exon7、exon8、exon13、exon14、exon16、exon17、exon18、exon19、exon20而在人的这10个外显子中多态性较为丰富。2、对162只中国美利奴羊进行虎红平板检测,共检测发现阴性101只,阳性个体为61只,并对出现的阳性样本进行复检,布鲁氏菌检出率为37.65%。利用PCR-SSCP的方法对这162只中国美利奴羊e NOS基因10个外显子进行检测,发现中国美利奴羊e NOS基因exon8序列第142位发生了碱基突变,由G变为A,氨基酸由Ala变为Thr,该突变位点也是一个新的突变位点(ss974768653:A142G)。利用SHEsis在线统计分析软件和SPSS17.0统计分析软件,结果表明该多态性位点的基因型及等位基因频率分布均符合Hardy-Weiberg平衡定律(P0.05),具有群体代表性,但该位点的基因型及等位基因频率分别在侵染组和对照组之间的差异无统计学意义(P0.05)。3、用布鲁氏菌M5侵染小鼠后,发现在侵染后第14天出现了肝、脾肿大。在第0、3、7、14、28天分别测定侵染组和对照组e NOS含量和活力,通过统计分析发现侵染组和对照组e NOS含量没有发生变化,而e NOS活力在侵染后的第14天开始升高。4、利用实时荧光定量对侵染第28天和对照组中小鼠肝、脾e NOS基因进行表达分析发现侵染组中肝和对照组中肝的相对表达量差异不显著,P值为0.685(P0.05);在侵染组和对照组中脾的相对表达量差异不显著,P值为0.512(P0.05)。结论:1、经过PCR-SSCP方法对中国美利奴羊e NOS基因的10个外显子进行多态性分析,最终在exon8上发现1个多态性位点,但是该多态性位点与绵羊布病易感性无关。2、对侵染后的小鼠e NOS含量和活力进行检测后发现,e NOS含量在侵染后没有发生明显变化,而e NOS活力在侵染后第14天出现升高,说明布鲁氏菌侵染小鼠后,并没有引起e NOS含量的变化,而在第14天以后引起e NOS活力升高,这一过程也可能导致NO释放量升高。3、利用实时荧光定量PCR对侵染组和对照组小鼠肝、脾进行相对表达量分析后发现侵染组中肝和对照组肝中e NOS的表达量没有发生明显变化,侵染组中脾和对照组脾中e NOS的表达量没有发生明显变化,说明布鲁氏杆菌M5对小鼠肝、脾组织中e NOS的表达没有影响。
[Abstract]:Objective: to detect the use of SSCP in the SNP loci of the e NOS gene coding region of Chinese Merino sheep, and to clone the exons of the polymorphic loci and find the SNP loci related to the susceptibility to the disease. The concentration of E NOS gene, the activity of the enzyme and the liver and spleen tissue of the mice were detected by the infection of Brucella M5. The expression of E NOS gene was detected, and the correlation between the e NOS gene of Chinese Merino and brucellosis and the role of E NOS in the process of Brucella infection were discussed. It provided a certain basis and theoretical support for the breeding of new varieties of sheep disease resistance and the new way of disease prevention. Method: 1, the m RNA sequence of human and sheep e NOS gene ( Record number: NM_000603.4, NM_001129901.1) using BLAST comparison to find out the exons of human and sheep e NOS gene, and then find the SNP loci of human and sheep through SNP database in Gen Bank. Using DNAMAN6.0 software, the polymorphism of human e NOS gene exons is analyzed by DNAMAN6.0 software, and the polymorphism exons are obtained. Sequence, and then using Gene Doc software to find out the exons of E NOS gene of sheep with high similarity with these exons, this experiment uses Brucella tiger red plate agglutination diagnostic kit to detect Brucella sera in 162 Chinese Merino blood samples, and then PCR-SSCP method is used to detect SNP of the exons of E NOS gene. The exons containing SNP loci were cloned and sequenced. The susceptibility analysis of the detected SNP site was analyzed by SHEsis online analysis software and.3 was used to detect the SNP site. The content and vitality of the mice e NOS were detected on 0,3,7,14,28 days after the infection, and the measured data were introduced into EXCEL, and SPSS was used for SPSS. 17 the content and vitality of the infection group and the control group were statistically analyzed.4. The e NOS gene of the infected mice and the control group was amplified by real time fluorescence quantitative PCR technique. The data were analyzed and modified by 2- Delta Delta Ct method. Finally, the results were introduced into EXCEL to obtain the relative expression of the liver and spleen tissue, and the samples were produced. The relative expression was analyzed by SPSS17.0 software. Results: 1, through bioinformatics analysis, we found 10 exons of the e NOS gene of Chinese Merino sheep with high homology. These 10 exons were Exon2, exon7, exon8, exon13, exon14, exon16, EXON17, exon18, Exon19, and 10 exons. The polymorphism of.2 was more abundant, and 162 Chinese Merino sheep were detected by Tiger red plate, 101 were detected negative, 61 positive individuals were detected, and the positive samples were rechecked. The detection rate of Brucella was 37.65%. using the PCR-SSCP method to detect 10 exons of the 162 Chinese Merino sheep NOS gene, and found that the 10 exons of the 162 Chinese Merino sheep were detected. The 142nd bits of the exon8 sequence of the e NOS gene of the Gome were changed from G to A, the amino acid changed from Ala to Thr, and the mutation site was also a new mutation site (ss974768653:A142G). The results showed that the genotype and allele frequency of the polymorphic loci were used in the SHEsis Online statistical analysis software and SPSS17.0 software. The distribution accorded with the Hardy-Weiberg equilibrium law (P0.05), which had group representation, but there was no significant difference between the genotype and allele frequency between the infection group and the control group (P0.05).3 respectively. After the infection of the mice with Brucella M5, the liver and splenomegaly were found fourteenth days after the infection. The results were measured on the day 0,3,7,14,28 respectively. The content and vitality of E NOS in the infection group and the control group were determined. By statistical analysis, the content of E NOS in the infection group and the control group did not change, while the e NOS activity began to increase in the.4 after the fourteenth day of infection. The expression analysis of the liver and the NOS gene of the spleen E in the infected twenty-eighth days and the control group, and the NOS gene of the spleen E were detected by the real-time fluorescence quantitative analysis. The relative expression of liver in the irradiated group was not significant, the P value was 0.685 (P0.05), the relative expression of spleen in the infection group and the control group was not significant, and the P value was 0.512 (P0.05). Conclusion: 1, 10 exons of the Chinese Merino e NOS gene were analyzed by PCR-SSCP method, and 1 polymorphic loci were found on exon8, but the 1 polymorphic loci were found in exon8. The polymorphic loci were not related to the susceptibility to the susceptibility of sheep brucellosis to.2. After detection of the e NOS content and activity after infection, the content of E NOS did not change obviously after the infection, and the activity of E NOS increased at fourteenth days after infection. It showed that after Brucella infected mice, the NOS content of E was not changed, but after fourteenth days. The activity of E NOS increased, and this process may also lead to the increase of.3 in the release of NO. The expression of E NOS in the liver and the control group of the infected group and the control group was not significantly changed after the analysis of the relative expression of the spleen in the infection group and the control group. The expression of E NOS in the spleen and the control group of the infected group was no more than that of the spleen and the control group. Obvious changes showed that Brucella M5 had no effect on the expression of E NOS in liver and spleen tissues of mice.
【学位授予单位】:石河子大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:S858.26

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