本文選題：旋毛蟲 + 水孔蛋白��； 參考：《中國農(nóng)業(yè)科學(xué)院》2016年碩士論文

【摘要】：旋毛蟲病(Trichinellosis)是由旋毛蟲(Trichinella spp.)感染引起的一種食源性人獸共患寄生蟲病,呈世界性分布,可感染包括人類在內(nèi)的150余種動物,嚴(yán)重時(shí)可以導(dǎo)致宿主死亡,并給畜牧業(yè)發(fā)展造成巨大的經(jīng)濟(jì)損失。水孔蛋白是廣泛存在于生物體內(nèi)的水通道蛋白,屬于主要內(nèi)在膜蛋白家族(Major Intrinsic Protein),可作為潛在的抗寄生蟲藥物靶點(diǎn)和疫苗抗原候選分子。本研究根據(jù)GenBank數(shù)據(jù)庫旋毛蟲水孔蛋白基因EST序列設(shè)計(jì)特異性引物,先用通用引物PR1將旋毛蟲肌幼蟲總RNA反轉(zhuǎn)錄成cDNA,然后以cDNA為模板用特異性引物克隆得到旋毛蟲水孔蛋白基因(TsAQP)的完整的開放閱讀框(ORF)序列。生物信息學(xué)分析表明TsAQP基因由6個(gè)外顯子和5個(gè)內(nèi)含子構(gòu)成,其開放閱讀框?yàn)?67 bp,可編碼288個(gè)氨基酸,理論分子質(zhì)量為31.01 kDa,理論等電點(diǎn)為8.47。TsAQP含有5個(gè)環(huán)(A-E),B、E環(huán)具有保守的NPA(Asn-Pro-Ala)模體結(jié)構(gòu)。TsAQP蛋白二級結(jié)構(gòu)中α-螺旋占34.38%,β-轉(zhuǎn)角占9.38%,其余為伸展鏈和無規(guī)卷曲。TsAQP與人AQP9氨基酸序列一致性為45.0%,系統(tǒng)進(jìn)化分析顯示TsAQP屬于水-甘油水孔蛋白亞家族。將TsAQP基因121-151、78-97(B環(huán))、210-238(E環(huán))和262-288序列連接到pGEX4T-1原核表達(dá)載體上,轉(zhuǎn)化大腸桿菌BL21后IPTG誘導(dǎo)表達(dá),分別得到大小為29.3、28.1、29.3、29.0 kDa的重組蛋白。人工合成TsAQP基因的非跨膜區(qū)序列TsAQPnt,并構(gòu)建pET-30a-TsAQPnt重組表達(dá)質(zhì)粒,轉(zhuǎn)化大腸桿菌Rosetta(DE3)后IPTG誘導(dǎo)表達(dá)。結(jié)果顯示,表達(dá)的重組蛋白大小為25.2 kDa,主要以包涵體形式存在,能夠被旋毛蟲感染豬血清識別;qRT-PCR結(jié)果表明TsAQP基因在旋毛蟲新生幼蟲、3日齡成蟲和肌幼蟲中均有表達(dá),但在新生幼蟲時(shí)期表達(dá)水平相對較高。組織定位結(jié)果顯示,TsAQP蛋白主要分布在旋毛蟲肌幼蟲蟲體表皮�；旌厦庖呓M(GST-121/151、B、E、262/288)和TsAQPnt免疫組BALB/c鼠產(chǎn)生了高水平的抗體,攻蟲感染后抗體水平略有下降�；旌厦庖呓M較對照組減蟲率為20.6%,TsAQPnt免疫組較對照組減蟲率為14.7%,表明重組抗原能夠誘導(dǎo)免疫小鼠對旋毛蟲感染產(chǎn)生一定的免疫保護(hù)力。本研究為進(jìn)一步研究TsAQP的生物學(xué)功能奠定基礎(chǔ)。
[Abstract]:Trichinellosis is caused by Trichinella spp. A food borne zoonotic parasitic disease caused by infection can infect more than 150 species of animals, including human beings, which can cause host death and cause huge economic losses to the development of animal husbandry. Aquaporins, which are widely present in organisms, belong to the major intrinsic protein family, and can be used as potential antiparasitic drug targets and vaccine antigen candidate molecules. In this study, specific primers were designed based on EST sequence of Trichinella spiralis aquaporin gene in GenBank database. The complete open reading frame (ORF) sequence of Trichinella spiralis aquaporin gene (TsAQP) was obtained by reverse transcription of total RNA of Trichinella spiralis muscle larvae into cDNAusing cDNA as template and specific primers. Bioinformatics analysis showed that the TsAQP gene was composed of 6 exons and 5 introns, and its open reading frame was 867 BP, which could encode 288 amino acids. The theoretical molecular weight is 31.01 kDa. the theoretical isoelectric point is 8.47. TsAQP contains five rings (A-E) Bon E ring with conserved NPA (Asn-Pro-Ala) motif structure. The 偽 -helix is 34.38, the 尾 -turn angle is 9.38, the rest is extensional chain and random coil. TsAQP is the amino acid sequence of human AQP9. The consistency was 45.0 and phylogenetic analysis showed that TsAQP belonged to the water-glycerol aquaporin subfamily. TsAQP gene 121-151 78-97 (B loop) 210-238 (E loop) and 262-288 sequence were ligated to pGEX4T-1 prokaryotic expression vector, and then transformed into E. coli BL21 and induced by IPTG. The non-transmembrane region of TsAQP gene was synthesized and the recombinant expression plasmid pET-30a-TsAQPnt was constructed. The recombinant plasmid was transformed into E. coli Rosetta (DE3) and induced by IPTG. The results showed that the expressed recombinant protein was 25.2 kDa, mainly in the form of inclusion bodies. The results of qRT-PCR showed that TsAQP gene was expressed in adult and muscle larvae of newborn larvae of Trichinella spiralis. But the expression level was relatively high at the stage of newborn larvae. Tissue localization showed that TsAQP protein was mainly distributed in the epidermis of Trichinella spiralis muscle larvae. The mixed immunization group (GST-121 / 151) and TsAQPNT immunized BALB / c mice produced high levels of antibodies, and the antibody levels decreased slightly after infection. Compared with the control group, the worm reduction rate of the mixed immunization group was 20.6and the rate of the TsAQPnt immunized group was 14.70.The results indicated that the recombinant antigen could induce the immune protection of mice against Trichinella spiralis infection. This study lays a foundation for further study of the biological function of TsAQP.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.7

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