旋毛虫水孔蛋白基因的克

发布时间：2018-06-30 01:25

本文选题：旋毛虫 + 水孔蛋白　；参考：《中国农业科学院》2016年硕士论文

【摘要】：旋毛虫病(Trichinellosis)是由旋毛虫(Trichinella spp.)感染引起的一种食源性人兽共患寄生虫病,呈世界性分布,可感染包括人类在内的150余种动物,严重时可以导致宿主死亡,并给畜牧业发展造成巨大的经济损失。水孔蛋白是广泛存在于生物体内的水通道蛋白,属于主要内在膜蛋白家族(Major Intrinsic Protein),可作为潜在的抗寄生虫药物靶点和疫苗抗原候选分子。本研究根据GenBank数据库旋毛虫水孔蛋白基因EST序列设计特异性引物,先用通用引物PR1将旋毛虫肌幼虫总RNA反转录成cDNA,然后以cDNA为模板用特异性引物克隆得到旋毛虫水孔蛋白基因(TsAQP)的完整的开放阅读框(ORF)序列。生物信息学分析表明TsAQP基因由6个外显子和5个内含子构成,其开放阅读框为867 bp,可编码288个氨基酸,理论分子质量为31.01 kDa,理论等电点为8.47。TsAQP含有5个环(A-E),B、E环具有保守的NPA(Asn-Pro-Ala)模体结构。TsAQP蛋白二级结构中α-螺旋占34.38%,β-转角占9.38%,其余为伸展链和无规卷曲。TsAQP与人AQP9氨基酸序列一致性为45.0%,系统进化分析显示TsAQP属于水-甘油水孔蛋白亚家族。将TsAQP基因121-151、78-97(B环)、210-238(E环)和262-288序列连接到pGEX4T-1原核表达载体上,转化大肠杆菌BL21后IPTG诱导表达,分别得到大小为29.3、28.1、29.3、29.0 kDa的重组蛋白。人工合成TsAQP基因的非跨膜区序列TsAQPnt,并构建pET-30a-TsAQPnt重组表达质粒,转化大肠杆菌Rosetta(DE3)后IPTG诱导表达。结果显示,表达的重组蛋白大小为25.2 kDa,主要以包涵体形式存在,能够被旋毛虫感染猪血清识别;qRT-PCR结果表明TsAQP基因在旋毛虫新生幼虫、3日龄成虫和肌幼虫中均有表达,但在新生幼虫时期表达水平相对较高。组织定位结果显示,TsAQP蛋白主要分布在旋毛虫肌幼虫虫体表皮。混合免疫组(GST-121/151、B、E、262/288)和TsAQPnt免疫组BALB/c鼠产生了高水平的抗体,攻虫感染后抗体水平略有下降。混合免疫组较对照组减虫率为20.6%,TsAQPnt免疫组较对照组减虫率为14.7%,表明重组抗原能够诱导免疫小鼠对旋毛虫感染产生一定的免疫保护力。本研究为进一步研究TsAQP的生物学功能奠定基础。
[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 伪 -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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.7

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