本文選題：日本血吸蟲 + SjTβRII　； 參考：《廣州醫(yī)學(xué)院》2010年碩士論文

【摘要】：日本血吸蟲病(Schistosomiasis)是一種嚴(yán)重危害人類健康的人畜共患寄生蟲病,蟲卵沉積于肝、腸等組織中形成蟲卵肉芽腫及其纖維化是蟲體致病的最主要原因。目前,血吸蟲病疫苗的研制是血吸蟲病防治工作的重點(diǎn)之一。 轉(zhuǎn)化生長因子β(transforming growth factor-β,TGF-β)超家族成員在細(xì)胞的生長、分化、細(xì)胞外基質(zhì)的形成和免疫調(diào)節(jié)等方面都發(fā)揮著重要作用[1-8],且TGF-β1是目前公認(rèn)的最強(qiáng)的肝纖維化促進(jìn)因子之一[9]。TGF-β在日本血吸蟲(Schistosoma japonicum,S.j)自身的生長發(fā)育過程中,以及在蟲體與宿主的相互作用中都發(fā)揮著重要作用[10]。TGF-β超家族包括一系列具有類似的結(jié)構(gòu)和功能的生長因子家族肽類。TβR(TGF-β受體)有多種,其中I型(TβRI)、II型(TβRII)、III型(TβRIII)分布于多類細(xì)胞膜上。TβRI和TβRII在TGF-β信號轉(zhuǎn)導(dǎo)中起主導(dǎo)作用,TβRII具有絲氨酸/蘇氨酸激酶活性,在生理濃度下可單獨(dú)與TGF-β結(jié)合,結(jié)合后TβRII胞內(nèi)段結(jié)構(gòu)發(fā)生改變,再與TβRI結(jié)合形成TGF-β-TβRII-TβRI復(fù)合體,隨之TβRI胞內(nèi)段發(fā)生磷酸化,引起下游smad蛋白家族的一系列變化,從而將信號傳遞于細(xì)胞胞核內(nèi)[11,12],調(diào)控目的基因表達(dá)。由此推測SjTβRII在SjTGF-β的信號轉(zhuǎn)導(dǎo)中亦發(fā)揮著重要的樞紐作用。曼氏血吸蟲(Schistosoma mansoni S.m) TβRII可通過激活TGF-β信號通路誘導(dǎo)抱雌溝蛋白(Gynecophoral canal protein,GCP)表達(dá),表明其在雄蟲促雌蟲發(fā)育成熟方面起著重要作用,提示TβRII可作為疫苗候選分子。 研究目的: 鑒于SjTβRII在蟲體TGF-β的信號轉(zhuǎn)導(dǎo)中可能發(fā)揮著重要的樞紐作用,以及作為疫苗候選分子的潛在性,本文擬用分子生物學(xué)等技術(shù),獲得SjTβRII全長cDNA,并對其編碼序列進(jìn)行生物信息學(xué)分析;克隆和原核表達(dá)胞外段(SjTβRIIout),且對表達(dá)產(chǎn)物進(jìn)行免疫活性檢測;將重組蛋白免疫動物,評價其免疫保護(hù)性效果;測定SjTβRII在成蟲、蟲卵中的定位和轉(zhuǎn)錄水平。 研究方法: (1)SjTβRII基因全長cDNA序列由上海英濰捷基生物技術(shù)(Invitrogen)有限公司通過RACE方法擴(kuò)增獲得。利用GeneBank,ExPASy等對該基因序列進(jìn)行生物信息學(xué)分析,設(shè)計(jì)特異性引物,提取成蟲總RNA進(jìn)行RT-PCR,對SjTβRII基因全長編碼基因(ORF)進(jìn)行特異性PCR擴(kuò)增、TA克隆。 (2)特異擴(kuò)增SjTβRII基因胞外段(SjTβRIIout),亞克隆至原核表達(dá)載體pET28a(+),構(gòu)建pET28a(+)-SjTβRIIout重組質(zhì)粒,并轉(zhuǎn)化至大腸桿菌BL21(DE3)。經(jīng)IPTG誘導(dǎo)重組蛋白表達(dá),表達(dá)產(chǎn)物行十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析。純化重組蛋白并免疫大鼠制備抗血清,應(yīng)用Western Blotting方法初步鑒定重組蛋白。 (3)用免疫組織化學(xué)法檢測目的基因在蟲體中的定位;采用熒光定量PCR技術(shù),對SjTβRII基因在雌蟲、雄蟲、蟲卵、尾蚴的轉(zhuǎn)錄水平進(jìn)行檢測。 (4)用SjTβRIIout免疫小鼠,評價其動物免疫保護(hù)性效果。動物隨機(jī)分成3組:A組為SjTβRIIout重組蛋白免疫組,B組為SjGST蛋白免疫組(陽性對照組),C組為佐劑對照組。以減蟲率和每克肝內(nèi)蟲卵減少率對目的基因動物免疫保護(hù)性效果進(jìn)行評價。 研究結(jié)果: (1)應(yīng)用RACE方法獲得SjTβRII基因的全長cDNA序列,所測序列長2867 bp,其ORF序列為2283bp,包括110 bp 5’UTR和474 bp 3’UTR(含有AATAAA和poly A等轉(zhuǎn)錄終止修飾點(diǎn))。以成蟲總RNA為模板,應(yīng)用特異性引物經(jīng)RT-PCR擴(kuò)增獲得約2300bp特異條帶,與預(yù)期分子量大小一致。將構(gòu)建的TA克隆重組質(zhì)粒經(jīng)PCR鑒定,出現(xiàn)與目的基因相符的條帶,同時測序結(jié)果表明該序列與Invitrogen公司應(yīng)用RACE方法獲得的SjTβRII基因cDNA序列完全一致。SjTβRII基因cDNA全長2283 bp,編碼760個氨基酸,蛋白的理論分子量為86.488 kDa,等電點(diǎn)為6.47。同源性分析發(fā)現(xiàn)SjTβRII基因氨基酸序列與曼氏血吸蟲、多房棘球絳蟲、紅原雞、黑猩猩、小鼠、斑胸草雀、人、斑馬魚等動物TβRII編碼基因推導(dǎo)的氨基酸序列一致性分別為70.7 %、28.6 %、18.4 %、18.3 %、18.1 %、17.9 %、17.9 %、17.8 %。此全長cDNA序列已獲得NCBI登錄號:FJ753578。 (2)將SjTβRII基因胞外段(SjTβRIIout)克隆入原核表達(dá)載體pET28a(+),通過雙酶切、PCR和測序鑒定,表明重組質(zhì)粒構(gòu)建成功。原核重組表達(dá)質(zhì)粒pET28a-SjTβRIIout在大腸桿菌BL21(DE3)中獲高效表達(dá)。SDS-PAGE分析顯示重組蛋白分子量大小與預(yù)期完全一致。Western-blot結(jié)果表明該蛋白可分別被感染日本血吸蟲和免疫重組蛋白的動物血清所識別。 (3)免疫組織化學(xué)方法定位表明,SjTβRII在成蟲體壁和實(shí)質(zhì)細(xì)胞、蟲卵內(nèi)毛蚴的實(shí)質(zhì)細(xì)胞中均有表達(dá)。Realtime-PCR顯示SjTβRII在日本血吸蟲成蟲、蟲卵、尾蚴各期均特異轉(zhuǎn)錄,且其在蟲卵和尾蚴中的轉(zhuǎn)錄水平高于其在成蟲中的轉(zhuǎn)錄水平。 (4)原核重組蛋白SjTβRIIout免疫BALB/c小鼠后,進(jìn)行日本血吸蟲尾蚴攻擊感染,檢測其免疫保護(hù)效果:與佐劑對照組(FCA)比較減蟲率和減卵率分別為:20.62%、38.23%,SjGST免疫組的減蟲率和減卵率分別為30.18%、45.70%,二者具有顯著性差異(p0.05)。 結(jié)論: (1)應(yīng)用RACE方法首次獲得SjTβRII基因的全長cDNA序列(2283 bp),其推導(dǎo)的氨基酸序列與曼氏血吸蟲TβRII氨基酸序列高度同源,與宿主人和小鼠TβRII氨基酸序列同源性低。 (2)成功構(gòu)建SjTβRIIout原核重組表達(dá)質(zhì)粒,在大腸桿菌中獲得高效表達(dá),純化的重組蛋白具有抗原性和免疫原性。 (3)SjTβRII分布在成蟲體壁和實(shí)質(zhì)細(xì)胞、蟲卵內(nèi)毛蚴的實(shí)質(zhì)細(xì)胞中,在蟲卵、尾蚴中的轉(zhuǎn)錄水平高于成蟲期轉(zhuǎn)錄水平。 (4)原核重組蛋白SjTβRIIout誘導(dǎo)小鼠產(chǎn)生抗血吸蟲感染的保護(hù)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 .
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.11

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本文編號：1923698