猪蛔虫GST基因的克隆与多态性分析及其在不同发育阶段中的表达

发布时间：2018-03-20 22:00

本文选题：猪蛔虫　切入点：GST基因　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：猪蛔虫(Ascaris suum Goeze 1782)是猪体内最大的肠道寄生虫之一,能感染猪从而造成严重的幼虫移行症和生长障碍,甚至死亡,给养猪业带来巨大的经济损失。猪蛔虫病(Swine ascariasis)在世界范围内普遍流行,无论是集约化养殖还是农户散养的猪群均易感染猪蛔虫而引起该病的发生,我国猪群的感染率亦在10%~80%。另外,研究发现,猪蛔虫还可以感染鸡、牛、羊及灵长类等动物和人类,是一种易被忽视的人兽共患寄生虫病,不仅会给畜禽养殖造成重大损失,还会对人类健康带来严重的威胁。所以,控制猪蛔虫对畜牧业健康发展和公共卫生安全都具有重要的意义。为了更有针对性地防控猪蛔虫的感染和流行,必须寻找更为有效的疫苗候选基因和药物靶位,而谷胱甘肽S-转移酶(Gultathione S transferases,GSTs)系统作为线虫主要解毒机制,是一个理想的研究目标。多年来,关于GST基因的研究很多,但对猪蛔虫GST基因的研究相对不足。故而,本研究拟对猪蛔虫GST基因编码序列(Coding sequence,CDS)进行克隆并对其结构和抗原表位进行预测,以分析其作为疫苗候选基因的可能性;对西北地区猪蛔虫GST基因单核苷酸多态性(Single nucleotide polymorphism,SNP)位点进行筛选和鉴定并统计各SNP位点等位基因和基因型频率,以分析该基因的遗传多态性;对不同发育阶段猪蛔虫GST基因表达进行检测并分析药物对虫卵和成虫GST基因表达的影响,以分析该基因在猪蛔虫发育过程和药物代谢过程中的作用。获得的结果如下:1.陕西扶风分离到的猪蛔虫GST基因与猪蛔虫和人蛔虫参考基因相似性均为99.5%,但该基因CDS区的第243和248位核苷酸发生了突变,并导致编码的第83位氨基酸发生改变,另外,预测显示,该区域二级结构和抗原表位均十分丰富,且优势抗原表位最可能出现在肽段43~46和201~204区域内或附近。2.西北地区猪蛔虫GST基因存在3个SNP位点,分别为SNP2、SNP3和SNP4,各SNP位点优势等位基因分别为A2、C3和A4,优势基因型分别为A2A2、C3C3和A4G4,共形成8个单倍型,同时,各SNP在各地猪蛔虫群体中均呈中度多态(0.25PIC0.50),且采自陕西地区的猪蛔虫GST基因各SNP位点及甘肃地区的猪蛔虫GST基因SNP4位点偏离了哈迪温伯格平衡(P0.05)。3.猪蛔虫虫卵体外发育以二分裂的方式进行,另外,GST基因在猪蛔虫不同发育阶段表达稳定且没有差异,但药物会引起成虫和虫卵该基因表达水平的变化,其中在成虫中的表达水平显著升高(P0.001)。综上所述,猪蛔虫GST基因丰富的抗原表位,预示着该基因有作为疫苗候选基因的潜力;该基因SNP位点偏离哈迪温伯格平衡,表明选择压力的存在;药物处理后成虫GST基因表达水平的显著升高,提示该基因在药物代谢中的重要作用并可能与抗药性的产生有关。
[Abstract]:Ascaris suum Goeze 1782) is one of the largest intestinal parasites in pigs, which can infect pigs and cause severe larval migration, growth disorders and even death. The swine ascariasis is prevalent all over the world. Both intensive farming and farmers' scattered pigs are susceptible to the infection of Ascaris lumbricoides, which is caused by Ascaris lumbricoides. The infection rate of pigs in our country is also at 100.In addition, studies have found that Ascaris suis can also infect chickens, cattle, sheep, primates, and humans. It is an easily neglected zoonotic parasitic disease, which will not only cause great losses to livestock and poultry breeding. It also poses a serious threat to human health. Therefore, controlling Ascaris lumbricoides is of great significance to the healthy development of animal husbandry and public health safety. More effective vaccine candidate genes and drug targets must be sought, and the glutathione S-transferasone GSTs system is an ideal research target as the main detoxification mechanism of nematodes. Many studies have been conducted on the GST gene over the years. However, the studies on GST gene of Ascaris suboides were relatively insufficient. Therefore, the coding sequence of GST gene of Ascaris lumbricoides was cloned and its structure and antigen epitopes were predicted to analyze the possibility of using it as a vaccine candidate gene. The single nucleotide polymorphisms (SNPs) of the GST gene of Ascaris suum in Northwest China were screened and identified, and the alleles and genotype frequencies of each SNP locus were counted to analyze the genetic polymorphism of the gene. The expression of GST gene in Ascaris suum at different developmental stages was detected and the effects of drugs on the expression of GST gene in eggs and adults were analyzed. The results are as follows: 1. The similarity between the GST gene of Ascaris suum isolated from Shaanxi Province and the reference gene of Ascaris suum and human Ascaris lumbricoides is 99.5, but this gene is similar to that of Ascaris lumbricoides. Mutations occurred at the 243rd and 248th nucleotides in the CDS region. In addition, the predicted secondary structure and antigenic epitopes of the region are very abundant. The dominant antigen epitopes were most likely to be found in or near the peptide regions 4346 and 201Y204. There were three SNP loci in the GST gene of Ascaris lumbricoides in Northwest China. The dominant alleles of each SNP locus were A2C3 and A4, and the dominant genotypes were A2A2C3C3 and A4G4, respectively. All SNP were moderately polymorphic in Ascaris lumbricoides populations. The SNP loci of GST gene of Ascaris lumbricoides in Shaanxi and the SNP4 locus of GST gene of Ascaris lumbricoides in Gansu region deviated from the Hardy Weinberg equilibrium (P0.05). 3. The eggs of Ascaris lumbricoides developed in vitro. Education is carried out in a two-split manner, In addition, the expression of GST gene was stable in different stages of Ascaris suum, but the expression level of GST gene was significantly increased in adult worm and egg, and the expression level of GST gene in adult worm was significantly higher than that in adult worm (P 0.001). To sum up, the expression level of GST gene in adult worm was significantly higher than that in adult worm. The abundant antigen epitopes of the GST gene of Ascaris suum indicate the potential of the gene as a vaccine candidate gene, the SNP site of the gene deviates from the Hardy Weinberg equilibrium, indicating the existence of selection pressure. The level of GST gene expression in adult worms increased significantly after drug treatment, suggesting that the gene plays an important role in drug metabolism and may be related to the production of drug resistance.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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