旋毛虫成虫丝氨酸蛋白酶抑制剂免疫学及生物活性研究
发布时间：2018-10-21 14:49
【摘要】：旋毛虫病是一种严重威胁公共卫生安全的重要人兽共患病,感染者主要是通过食入生的或者未加工完全的含有感染性幼虫的肉而感染。旋毛虫病一直未得到根本上的控制,近些年来感染率又有反弹的趋势。丝氨酸蛋白酶抑制剂(SPI)是结构上保守的蛋白质家族,广泛存在于多种生物体内,是主要的调节蛋白之一。在多种生理学和细胞学功能上发挥重要作用,如凝血、炎症反应、细胞凋亡等。在多种寄生虫感染过程中,SPI在虫体与宿主相互作用中发挥重要作用。本试验提取旋毛虫5日龄成虫总RNA,根据Gen Bank中登陆的旋毛虫丝氨酸蛋白酶抑制剂基因(登录号:EU263307.1)设计引物。通过RT-PCR方法获得虫体c DNA,PCR方法获得旋毛虫成虫丝氨酸蛋白酶抑制剂(Ts Ad SPI)基因。将获得的目的基因通过T4连接酶与克隆载体p MD18-T连接,进行双酶切、PCR及测序鉴定。将鉴定正确目的片段与表达载体p ET-28a连接,经过双酶切、PCR及测序鉴定正确后,用IPTG对目的蛋白进行诱导表达。SDS-PAGE结果显示,目的蛋白分子量为44k Da,以包涵体形式存在。对诱导的时间、温度、IPTG浓度优化结果显示,诱导时间为5h,温度为37℃,IPTG浓度为1.0mmol/L时Ts Ad SPI重组蛋白表达量最大。对Ts Ad SPI重组蛋白进行纯化,用弗氏佐剂乳化目的蛋白之后背部六点分三次免疫新西兰大白兔,制备多克隆抗体。免疫之后分离免疫血清,Western-blot方法对Ts Ad SPI重组蛋白的免疫原性进行分析,结果显示制备的多克隆抗体可以有效识别TsAd SPI重组蛋白且条带单一。采用间接ELISA方法对制备的多克隆抗体效价进行检测,结果显示制备的多克隆抗体效价高达1:51200。以上实验结果表明本研究通过原核表达系统得到的Ts AdSPI重组蛋白具有良好的免疫原性,可以诱导机体产生高效且特异性较高的免疫应答。分离感染旋毛虫5天的小鼠血清,Western-blot分析显示Ts Ad SPI重组蛋白可以被分离得到的血清所识别,证明Ts Ad SPI重组蛋白具有良好的反应原性。对旋毛虫5日龄成虫及肌幼虫的Ts Ad SPI重组蛋白免疫组织化学方法定位发现,在两个时期虫体的体内和体表均发现有Ts Ad SPI重组蛋白的分布。运用发色底物法对Ts Ad SPI重组蛋白的抑制活性进行分析,结果显示Ts Ad SPI重组蛋白可以有效抑制胰蛋白酶、弹性蛋白酶、糜蛋白酶活性,抑制率分别为88%、72%、42%。本试验研究结果表明,Ts Ad SPI重组蛋白可以诱导机体产生良好的免疫反应,具有良好的免疫原性。感染旋毛虫的小鼠血清也可有效识别该重组蛋白,证明Ts Ad SPI重组蛋白良好的反应原性。通过Ts AdSPI重组蛋白对消化相关酶活性的抑制作用,阐明了旋毛虫抵御宿主消化系统从而入侵宿主的可能机制。以上结果表明,Ts Ad SPI重组蛋白可以作为旋毛虫感染早期诊断的候选抗原,为新型疫苗及药物的研制奠定坚实的基础。同时,也为旋毛虫入侵宿主相关机制的阐明奠定一定的理论基础。
[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，
本文编号：2285430
[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，
本文编号：2285430
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