日本血吸虫TβRII基因全长cDNA的克隆及其胞外段免疫保护功能的研究

发布时间：2018-05-23 06:50

本文选题：日本血吸虫 + SjTβRII　；参考：《广州医学院》2010年硕士论文

【摘要】：日本血吸虫病(Schistosomiasis)是一种严重危害人类健康的人畜共患寄生虫病,虫卵沉积于肝、肠等组织中形成虫卵肉芽肿及其纤维化是虫体致病的最主要原因。目前,血吸虫病疫苗的研制是血吸虫病防治工作的重点之一。转化生长因子β(transforming growth factor-β,TGF-β)超家族成员在细胞的生长、分化、细胞外基质的形成和免疫调节等方面都发挥着重要作用[1-8],且TGF-β1是目前公认的最强的肝纤维化促进因子之一[9]。TGF-β在日本血吸虫(Schistosoma japonicum,S.j)自身的生长发育过程中,以及在虫体与宿主的相互作用中都发挥着重要作用[10]。TGF-β超家族包括一系列具有类似的结构和功能的生长因子家族肽类。TβR(TGF-β受体)有多种,其中I型(TβRI)、II型(TβRII)、III型(TβRIII)分布于多类细胞膜上。TβRI和TβRII在TGF-β信号转导中起主导作用,TβRII具有丝氨酸/苏氨酸激酶活性,在生理浓度下可单独与TGF-β结合,结合后TβRII胞内段结构发生改变,再与TβRI结合形成TGF-β-TβRII-TβRI复合体,随之TβRI胞内段发生磷酸化,引起下游smad蛋白家族的一系列变化,从而将信号传递于细胞胞核内[11,12],调控目的基因表达。由此推测SjTβRII在SjTGF-β的信号转导中亦发挥着重要的枢纽作用。曼氏血吸虫(Schistosoma mansoni S.m) TβRII可通过激活TGF-β信号通路诱导抱雌沟蛋白(Gynecophoral canal protein,GCP)表达,表明其在雄虫促雌虫发育成熟方面起着重要作用,提示TβRII可作为疫苗候选分子。研究目的: 鉴于SjTβRII在虫体TGF-β的信号转导中可能发挥着重要的枢纽作用,以及作为疫苗候选分子的潜在性,本文拟用分子生物学等技术,获得SjTβRII全长cDNA,并对其编码序列进行生物信息学分析;克隆和原核表达胞外段(SjTβRIIout),且对表达产物进行免疫活性检测;将重组蛋白免疫动物,评价其免疫保护性效果;测定SjTβRII在成虫、虫卵中的定位和转录水平。研究方法: (1)SjTβRII基因全长cDNA序列由上海英潍捷基生物技术(Invitrogen)有限公司通过RACE方法扩增获得。利用GeneBank,ExPASy等对该基因序列进行生物信息学分析,设计特异性引物,提取成虫总RNA进行RT-PCR,对SjTβRII基因全长编码基因(ORF)进行特异性PCR扩增、TA克隆。 (2)特异扩增SjTβRII基因胞外段(SjTβRIIout),亚克隆至原核表达载体pET28a(+),构建pET28a(+)-SjTβRIIout重组质粒,并转化至大肠杆菌BL21(DE3)。经IPTG诱导重组蛋白表达,表达产物行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。纯化重组蛋白并免疫大鼠制备抗血清,应用Western Blotting方法初步鉴定重组蛋白。 (3)用免疫组织化学法检测目的基因在虫体中的定位;采用荧光定量PCR技术,对SjTβRII基因在雌虫、雄虫、虫卵、尾蚴的转录水平进行检测。 (4)用SjTβRIIout免疫小鼠,评价其动物免疫保护性效果。动物随机分成3组:A组为SjTβRIIout重组蛋白免疫组,B组为SjGST蛋白免疫组(阳性对照组),C组为佐剂对照组。以减虫率和每克肝内虫卵减少率对目的基因动物免疫保护性效果进行评价。研究结果: (1)应用RACE方法获得SjTβRII基因的全长cDNA序列,所测序列长2867 bp,其ORF序列为2283bp,包括110 bp 5’UTR和474 bp 3’UTR(含有AATAAA和poly A等转录终止修饰点)。以成虫总RNA为模板,应用特异性引物经RT-PCR扩增获得约2300bp特异条带,与预期分子量大小一致。将构建的TA克隆重组质粒经PCR鉴定,出现与目的基因相符的条带,同时测序结果表明该序列与Invitrogen公司应用RACE方法获得的SjTβRII基因cDNA序列完全一致。SjTβRII基因cDNA全长2283 bp,编码760个氨基酸,蛋白的理论分子量为86.488 kDa,等电点为6.47。同源性分析发现SjTβRII基因氨基酸序列与曼氏血吸虫、多房棘球绦虫、红原鸡、黑猩猩、小鼠、斑胸草雀、人、斑马鱼等动物TβRII编码基因推导的氨基酸序列一致性分别为70.7 %、28.6 %、18.4 %、18.3 %、18.1 %、17.9 %、17.9 %、17.8 %。此全长cDNA序列已获得NCBI登录号:FJ753578。 (2)将SjTβRII基因胞外段(SjTβRIIout)克隆入原核表达载体pET28a(+),通过双酶切、PCR和测序鉴定,表明重组质粒构建成功。原核重组表达质粒pET28a-SjTβRIIout在大肠杆菌BL21(DE3)中获高效表达。SDS-PAGE分析显示重组蛋白分子量大小与预期完全一致。Western-blot结果表明该蛋白可分别被感染日本血吸虫和免疫重组蛋白的动物血清所识别。 (3)免疫组织化学方法定位表明,SjTβRII在成虫体壁和实质细胞、虫卵内毛蚴的实质细胞中均有表达。Realtime-PCR显示SjTβRII在日本血吸虫成虫、虫卵、尾蚴各期均特异转录,且其在虫卵和尾蚴中的转录水平高于其在成虫中的转录水平。 (4)原核重组蛋白SjTβRIIout免疫BALB/c小鼠后,进行日本血吸虫尾蚴攻击感染,检测其免疫保护效果:与佐剂对照组(FCA)比较减虫率和减卵率分别为:20.62%、38.23%,SjGST免疫组的减虫率和减卵率分别为30.18%、45.70%,二者具有显著性差异(p0.05)。结论: (1)应用RACE方法首次获得SjTβRII基因的全长cDNA序列(2283 bp),其推导的氨基酸序列与曼氏血吸虫TβRII氨基酸序列高度同源,与宿主人和小鼠TβRII氨基酸序列同源性低。 (2)成功构建SjTβRIIout原核重组表达质粒,在大肠杆菌中获得高效表达,纯化的重组蛋白具有抗原性和免疫原性。 (3)SjTβRII分布在成虫体壁和实质细胞、虫卵内毛蚴的实质细胞中,在虫卵、尾蚴中的转录水平高于成虫期转录水平。 (4)原核重组蛋白SjTβRIIout诱导小鼠产生抗血吸虫感染的保护性免疫。
[Abstract]:Schistosoma japonicum is one of the most important causes of parasitic diseases in human and livestock infected with human health . It is the main reason for the formation of eggs granuloma and fibrosis in liver , intestine and other tissues .

Transforming growth factor - 尾 ( TGF - 尾 ) superfamily members play an important role in cell growth , differentiation , extracellular matrix formation and immune regulation , and TGF - 尾1 is one of the most commonly recognized promoting factors of liver fibrosis . TGF - 尾 plays an important role in the growth and development of Schistosoma japonicum ( S.j ) and plays an important role in the interaction of worm and host . The TGF - 尾 superfamily consists of a series of family peptides of growth factor family with similar structure and function . T 尾RI , type II ( T尾RII ) and type III ( T尾RIII ) are distributed on the cell membrane . T 尾RI and T尾RII play a leading role in TGF - 尾 signal transduction .

Purpose of study :

SjT尾RII may play an important pivotal role in the signal transduction of TGF - 尾 in the insect body , and it can be used as a candidate for vaccine candidate . In this paper , the full - length cDNA of SjT尾RII is obtained by molecular biology , and the coding sequence of SjT尾RII is bioinformatic . The recombinant protein was cloned and expressed in prokaryotic expression cell ( SjT尾RIIout ) , the immune protective effect of SjT尾RII was evaluated , and the localization and transcription level of SjT尾RII in adult and insect eggs were measured .

Study method :

( 1 ) The full - length cDNA sequence of SjT尾RII gene was amplified by RACE method .

( 2 ) The recombinant plasmid pET28a ( + ) - SjT尾RIIout was subcloned into the prokaryotic expression vector pET28a ( + ) to construct pET28a ( + ) - SjT尾RIIout recombinant plasmid and transformed into BL21 ( DE3 ) .

( 3 ) detecting the location of the target gene in the worm by immunohistochemistry ; adopting a fluorescence quantitative PCR technique to detect the transcription level of the SjT尾RII gene in the female , the male , the eggs and the cercariae .

( 4 ) Mice were immunized with SjT尾RIIout . The animals were randomly divided into three groups : group A was SjT尾RIIout recombinant protein immunization group , group B was SjGST protein immunization group ( positive control group ) , group C was adjuvant control group .

Results of the study :

( 1 ) The full - length cDNA sequence of SjT尾RII gene was obtained by RACE method . The sequence length was 2867 bp , and its ORF sequence was 2283 bp , including 110 bp 5 ' and 474 bp 3 ' untranslated region ( including transcriptional termination modification points including AATAAA and poly A ) . The deduced amino acid sequence of SjT尾RII gene was 70.7 % , 28.6 % , 18.4 % , 18.3 % , 18.1 % , 17.9 % , 17.9 % and 17.8 % respectively . This full - length cDNA sequence has been obtained from GenBank accession number : FJ753578 .

( 2 ) SjT尾RII gene extracellular segment ( SjT尾RIIout ) was cloned into prokaryotic expression vector pET28a ( + ) . The recombinant plasmid pET28a - SjT尾RIIout was expressed in E . coli BL21 ( DE3 ) .

( 3 ) The localization of SjT尾RII in adult worms , eggs and cercariae of Schistosoma japonicum showed that SjT尾RII had specific transcription at various stages of adult worms , eggs and cercariae of Schistosoma japonicum , and its transcription level in eggs and cercariae was higher than that in adult worms .

( 4 ) In BALB / c mice immunized with SjT尾RIIout of prokaryotic recombinant protein , the immune protective effect of SjT尾RIIout was 20.62 % , 38.23 % and 20.62 % , 38.23 % respectively .

Conclusion :

( 1 ) The full - length cDNA sequence ( 2283 bp ) of SjT尾RII gene was obtained for the first time by RACE method . The deduced amino acid sequence was highly homologous to the amino acid sequence of T . beta . RII of Schistosoma mandii , and had a low homology with the host human and mouse T尾RII amino acid sequence .

( 2 ) The recombinant expression plasmid SjT尾RIIout was constructed successfully . The recombinant protein of SjT尾RIIout was highly expressed in E . coli . The purified recombinant protein had antigenicity and immunogenicity .

( 3 ) SjT尾RII is distributed in the body wall and the real cell of adult worm , and the level of transcription of SjT尾RII in the eggs and cercariae is higher than that of adult stage .

( 4 ) The recombinant protein SjT尾RIIout induces protective immunity against Schistosoma japonicum infection .
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11

【参考文献】