【摘要】：旋毛蟲病是一種嚴(yán)重威脅公共衛(wèi)生安全的重要人獸共患病,感染者主要是通過食入生的或者未加工完全的含有感染性幼蟲的肉而感染。旋毛蟲病一直未得到根本上的控制,近些年來感染率又有反彈的趨勢。絲氨酸蛋白酶抑制劑(SPI)是結(jié)構(gòu)上保守的蛋白質(zhì)家族,廣泛存在于多種生物體內(nèi),是主要的調(diào)節(jié)蛋白之一。在多種生理學(xué)和細(xì)胞學(xué)功能上發(fā)揮重要作用,如凝血、炎癥反應(yīng)、細(xì)胞凋亡等。在多種寄生蟲感染過程中,SPI在蟲體與宿主相互作用中發(fā)揮重要作用。本試驗提取旋毛蟲5日齡成蟲總RNA,根據(jù)Gen Bank中登陸的旋毛蟲絲氨酸蛋白酶抑制劑基因(登錄號:EU263307.1)設(shè)計引物。通過RT-PCR方法獲得蟲體c DNA,PCR方法獲得旋毛蟲成蟲絲氨酸蛋白酶抑制劑(Ts Ad SPI)基因。將獲得的目的基因通過T4連接酶與克隆載體p MD18-T連接,進(jìn)行雙酶切、PCR及測序鑒定。將鑒定正確目的片段與表達(dá)載體p ET-28a連接,經(jīng)過雙酶切、PCR及測序鑒定正確后,用IPTG對目的蛋白進(jìn)行誘導(dǎo)表達(dá)。SDS-PAGE結(jié)果顯示,目的蛋白分子量為44k Da,以包涵體形式存在。對誘導(dǎo)的時間、溫度、IPTG濃度優(yōu)化結(jié)果顯示,誘導(dǎo)時間為5h,溫度為37℃,IPTG濃度為1.0mmol/L時Ts Ad SPI重組蛋白表達(dá)量最大。對Ts Ad SPI重組蛋白進(jìn)行純化,用弗氏佐劑乳化目的蛋白之后背部六點分三次免疫新西蘭大白兔,制備多克隆抗體。免疫之后分離免疫血清,Western-blot方法對Ts Ad SPI重組蛋白的免疫原性進(jìn)行分析,結(jié)果顯示制備的多克隆抗體可以有效識別TsAd SPI重組蛋白且條帶單一。采用間接ELISA方法對制備的多克隆抗體效價進(jìn)行檢測,結(jié)果顯示制備的多克隆抗體效價高達(dá)1:51200。以上實驗結(jié)果表明本研究通過原核表達(dá)系統(tǒng)得到的Ts AdSPI重組蛋白具有良好的免疫原性,可以誘導(dǎo)機(jī)體產(chǎn)生高效且特異性較高的免疫應(yīng)答。分離感染旋毛蟲5天的小鼠血清,Western-blot分析顯示Ts Ad SPI重組蛋白可以被分離得到的血清所識別,證明Ts Ad SPI重組蛋白具有良好的反應(yīng)原性。對旋毛蟲5日齡成蟲及肌幼蟲的Ts Ad SPI重組蛋白免疫組織化學(xué)方法定位發(fā)現(xiàn),在兩個時期蟲體的體內(nèi)和體表均發(fā)現(xiàn)有Ts Ad SPI重組蛋白的分布。運用發(fā)色底物法對Ts Ad SPI重組蛋白的抑制活性進(jìn)行分析,結(jié)果顯示Ts Ad SPI重組蛋白可以有效抑制胰蛋白酶、彈性蛋白酶、糜蛋白酶活性,抑制率分別為88%、72%、42%。本試驗研究結(jié)果表明,Ts Ad SPI重組蛋白可以誘導(dǎo)機(jī)體產(chǎn)生良好的免疫反應(yīng),具有良好的免疫原性。感染旋毛蟲的小鼠血清也可有效識別該重組蛋白,證明Ts Ad SPI重組蛋白良好的反應(yīng)原性。通過Ts AdSPI重組蛋白對消化相關(guān)酶活性的抑制作用,闡明了旋毛蟲抵御宿主消化系統(tǒng)從而入侵宿主的可能機(jī)制。以上結(jié)果表明,Ts Ad SPI重組蛋白可以作為旋毛蟲感染早期診斷的候選抗原,為新型疫苗及藥物的研制奠定堅實的基礎(chǔ)。同時,也為旋毛蟲入侵宿主相關(guān)機(jī)制的闡明奠定一定的理論基礎(chǔ)。
[Abstract]:Trichinellosis is a serious threat to public health and safety of zoonosis. Infected people are mainly infected by feeding raw or raw meat containing infected larvae. Trichinellosis has not been fundamentally controlled and the infection rate has rebounded in recent years. Serine protease inhibitor (SPI) is a conserved family of proteins widely distributed in many organisms and is one of the major regulatory proteins. It plays an important role in many physiological and cytological functions, such as coagulation, inflammation, apoptosis and so on. SPI plays an important role in the interaction between parasites and hosts in the process of parasite infection. In this experiment, the total RNA, of 5-day-old adult Trichinella spiralis was extracted and primers were designed according to the serine protease inhibitor gene (accession number: EU263307.1) of Trichinella spiralis landing in Gen Bank. The (Ts Ad SPI) gene of serine protease inhibitor of adult Trichinella spiralis was obtained by RT-PCR method. The obtained target gene was ligated with the cloned vector p MD18-T by T4 ligase, and identified by double enzyme digestion, PCR and sequencing. The identified target fragment was linked to the expression vector p ET-28a. After double enzyme digestion, PCR and sequencing, the target protein was induced to express by IPTG. The result of SDS-PAGE showed that the molecular weight of the target protein was 44 k Da, as inclusion body. The results of optimization of induction time, temperature and IPTG concentration showed that the expression of Ts Ad SPI recombinant protein was the highest when the induction time was 5 h, the temperature was 37 鈩，

本文編號：2285430