本文選題：豬蛔蟲(chóng)　切入點(diǎn)：GST基因　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：豬蛔蟲(chóng)(Ascaris suum Goeze 1782)是豬體內(nèi)最大的腸道寄生蟲(chóng)之一,能感染豬從而造成嚴(yán)重的幼蟲(chóng)移行癥和生長(zhǎng)障礙,甚至死亡,給養(yǎng)豬業(yè)帶來(lái)巨大的經(jīng)濟(jì)損失。豬蛔蟲(chóng)病(Swine ascariasis)在世界范圍內(nèi)普遍流行,無(wú)論是集約化養(yǎng)殖還是農(nóng)戶散養(yǎng)的豬群均易感染豬蛔蟲(chóng)而引起該病的發(fā)生,我國(guó)豬群的感染率亦在10%~80%。另外,研究發(fā)現(xiàn),豬蛔蟲(chóng)還可以感染雞、牛、羊及靈長(zhǎng)類(lèi)等動(dòng)物和人類(lèi),是一種易被忽視的人獸共患寄生蟲(chóng)病,不僅會(huì)給畜禽養(yǎng)殖造成重大損失,還會(huì)對(duì)人類(lèi)健康帶來(lái)嚴(yán)重的威脅。所以,控制豬蛔蟲(chóng)對(duì)畜牧業(yè)健康發(fā)展和公共衛(wèi)生安全都具有重要的意義。為了更有針對(duì)性地防控豬蛔蟲(chóng)的感染和流行,必須尋找更為有效的疫苗候選基因和藥物靶位,而谷胱甘肽S-轉(zhuǎn)移酶(Gultathione S transferases,GSTs)系統(tǒng)作為線蟲(chóng)主要解毒機(jī)制,是一個(gè)理想的研究目標(biāo)。多年來(lái),關(guān)于GST基因的研究很多,但對(duì)豬蛔蟲(chóng)GST基因的研究相對(duì)不足。故而,本研究擬對(duì)豬蛔蟲(chóng)GST基因編碼序列(Coding sequence,CDS)進(jìn)行克隆并對(duì)其結(jié)構(gòu)和抗原表位進(jìn)行預(yù)測(cè),以分析其作為疫苗候選基因的可能性;對(duì)西北地區(qū)豬蛔蟲(chóng)GST基因單核苷酸多態(tài)性(Single nucleotide polymorphism,SNP)位點(diǎn)進(jìn)行篩選和鑒定并統(tǒng)計(jì)各SNP位點(diǎn)等位基因和基因型頻率,以分析該基因的遺傳多態(tài)性;對(duì)不同發(fā)育階段豬蛔蟲(chóng)GST基因表達(dá)進(jìn)行檢測(cè)并分析藥物對(duì)蟲(chóng)卵和成蟲(chóng)GST基因表達(dá)的影響,以分析該基因在豬蛔蟲(chóng)發(fā)育過(guò)程和藥物代謝過(guò)程中的作用。獲得的結(jié)果如下:1.陜西扶風(fēng)分離到的豬蛔蟲(chóng)GST基因與豬蛔蟲(chóng)和人蛔蟲(chóng)參考基因相似性均為99.5%,但該基因CDS區(qū)的第243和248位核苷酸發(fā)生了突變,并導(dǎo)致編碼的第83位氨基酸發(fā)生改變,另外,預(yù)測(cè)顯示,該區(qū)域二級(jí)結(jié)構(gòu)和抗原表位均十分豐富,且優(yōu)勢(shì)抗原表位最可能出現(xiàn)在肽段43~46和201~204區(qū)域內(nèi)或附近。2.西北地區(qū)豬蛔蟲(chóng)GST基因存在3個(gè)SNP位點(diǎn),分別為SNP2、SNP3和SNP4,各SNP位點(diǎn)優(yōu)勢(shì)等位基因分別為A2、C3和A4,優(yōu)勢(shì)基因型分別為A2A2、C3C3和A4G4,共形成8個(gè)單倍型,同時(shí),各SNP在各地豬蛔蟲(chóng)群體中均呈中度多態(tài)(0.25PIC0.50),且采自陜西地區(qū)的豬蛔蟲(chóng)GST基因各SNP位點(diǎn)及甘肅地區(qū)的豬蛔蟲(chóng)GST基因SNP4位點(diǎn)偏離了哈迪溫伯格平衡(P0.05)。3.豬蛔蟲(chóng)蟲(chóng)卵體外發(fā)育以二分裂的方式進(jìn)行,另外,GST基因在豬蛔蟲(chóng)不同發(fā)育階段表達(dá)穩(wěn)定且沒(méi)有差異,但藥物會(huì)引起成蟲(chóng)和蟲(chóng)卵該基因表達(dá)水平的變化,其中在成蟲(chóng)中的表達(dá)水平顯著升高(P0.001)。綜上所述,豬蛔蟲(chóng)GST基因豐富的抗原表位,預(yù)示著該基因有作為疫苗候選基因的潛力;該基因SNP位點(diǎn)偏離哈迪溫伯格平衡,表明選擇壓力的存在;藥物處理后成蟲(chóng)GST基因表達(dá)水平的顯著升高,提示該基因在藥物代謝中的重要作用并可能與抗藥性的產(chǎn)生有關(guān)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.7

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