佐剂对两种旋毛虫丝氨酸蛋白酶抑制剂免疫影响的研究

在寄生虫感染过程中,丝氨酸蛋白酶抑制剂在虫体与宿主相互作用间发挥着重要作用。在免疫过程中,不同佐剂的促免疫应答能力和提高免疫保护能力均不同。为了比较不同佐剂对两种丝氨酸蛋白酶抑制剂(TsAdSPI和TsKaSPI)免疫保护性的影响,通过对免疫剂量进行优化,应用间接ELISA的方法分析两种SPIs抗体应答,分别在肌肉虫攻击感染后第1天、第3天、第7天、第10天剖杀小鼠取成虫并计数,计算成虫减虫率。结果显示,用3种不同剂量(20、50μg和100μg)的重组蛋白免疫小鼠,50μg与100μg蛋白剂量免疫组诱导产生类似的抗体水平,且均高于20μg组;免疫组弗氏佐剂、ISA 206佐剂和铝佐剂分别与重组蛋白乳化后免疫,可诱导相似的免疫应答及成虫减虫率,其中佐剂ISA 206和氢氧化铝的保护效果更好。     

家蚕Kazal型丝氨酸蛋白酶抑制剂基因BmSPI3的克隆与表达特征分析

Kazal型丝氨酸蛋白酶抑制剂(SPI)在机体的发育、免疫等生理过程中发挥重要的作用。从家蚕蛹cDNA文库中获得一条全长为728bp的基因序列,对该基因编码的氨基酸序列进行同源性比对,发现其蛋白具有保守的Kazal型丝氨酸蛋白酶抑制剂结构域(CⅠ-X3-CⅡ-X8-CⅢ-X14-CⅣ-X6-CⅤ-X13-CⅥ),且P1活性位点为赖氨酸(K),因此将该基因命名为BmSPI3(Gen-Bank登录号:DN237641)。通过PCR扩增BmSPI3基因,经BamHⅠ和XhoⅠ双酶切后成功构建了重组表达质粒pGEX-4T-1-BmSPI3,并转化到E.coliBL21(DE3)表达,经SDS-PAGE电泳检测在约36kD处有明显的蛋白表达条带。提取各发育时期蚕体和5龄幼虫各个组织的总RNA进行荧光定量PCR,检测结果表明:BmSPI3在5龄幼虫期表达量最高,卵期最低;在5龄幼虫表皮中的表达量最高,在马氏管的表达量最低。推测BmSPI3对胰蛋白酶和类胰蛋白酶具有抑制作用。     

旋毛虫感染性第一期幼虫分泌丝氨酸蛋白酶

寄生性蠕虫通过产生许多种具有不同生物学活性的蛋白质以刺激宿主的应答。这些蛋白质的一个重要组分便是由许多种线虫和吸虫如曼氏分体吸虫和肝片进吸虫产生的蛋白酶,其在虫体侵人和移行进入宿主组织、吸血及其它生物学活动中起作用。旋毛虫在其生活史的各个时期也产生许多种蛋白质,其中,有些被鉴定为过氧化物酶和重要的抗原。但有关旋毛虫的蛋白水解酶尚未见报道。旋毛虫感染性第一期幼虫在宿主消化酶作用下被释放出来后侵入小肠绒毛而转移到小肠。新生第一期幼虫经小肠侵入肠系膜淋巴或血．液,通过组织,最终侵入骨胳肌形成保姆细胞。…     

旋毛虫丝氨酸蛋白酶基因的克隆与分析

以旋毛虫感染猪血清为抗体探针,对旋毛虫3日龄成虫cDNA文库进行免疫筛选,将获得的阳性克隆进行测序,用分子生物学软件对所得cDNA序列进行了分析。序列分析结果显示,阳性克隆Zh68cDNA全长为1372bp,含有1个1287bp的完整的开放阅读框架,编码由429个氨基酸组成的多肽,理论分子质量为47．5ku,等电点为8．45。SMART分析表明,1～18位氨基酸为信号肽序列,37～277位氨基酸为典型的丝氨酸蛋白酶胰蛋白酶类结构域,其中His88、Asp142、Ser233为催化中心的3个主要氨基酸残基,78～80位氨基酸为N-糖基化位点（NCS）,另外还有6个保守的Cys形成二硫键。BLASTn同源性分析表明,与其他生物丝氨酸蛋白酶的基因序列无明显的同源性,为1个新的cDNA分子。BLASTp蛋白质同源性分析表明,与丝氨酸蛋白酶的同源性为30％左右。Southern—blot杂交表明,此基因在旋毛虫基因组中属于多基因家族,具有基因多态性。cDNA的PCR结果表明,此基因在旋毛虫肌幼虫、新生幼虫及成虫期均有表达。     

两种旋毛虫丝氨酸蛋白酶抑制剂对巨噬细胞炎性细胞因子mRNA表达的影响

丝氨酸蛋白酶抑制剂在寄生虫入侵宿主的过程中发挥着关键作用,能够参与到入侵宿主、免疫逃避、炎症反应、凝血系统和细胞迁徙等过程。为了探讨两种丝氨酸蛋白酶抑制剂(Ts Ad SPI和Ts Ka SPI)对巨噬细胞所分泌炎性细胞因子的调节作用,应用实时荧光定量PCR技术对两种SPIs分别处理的小鼠巨噬细胞进行TNF-α、IL-1β、IL-6、IL-12、IL-10、TGF-βmRNA表达水平的检测。结果显示,Ts Ad SPI和Ts Ka SPI均可不同程度的抑制LPS活化的小鼠巨噬细胞促炎性细胞因子TNF-α、IL-1β、IL-6和IL-12 mRNA的表达水平。并且两种SPI均可不同程度的促进巨噬细胞抗炎性细胞因子IL-10和TGF-β的表达。表明这两种旋毛虫SPI可通过调节宿主巨噬细胞影响入侵时宿主的免疫应答。     

寄生性蠕虫丝氨酸蛋白酶抑制剂研究进展

丝氨酸蛋白酶抑制剂(Serpin)是一种蛋白水解酶,广泛存在于生物体中。本文论述了丝氨酸蛋白酶抑制剂的分类、结构、功能及在寄生性蠕虫中的研究进展。指出通过对Serpin的研究,有望使其作为寄生性蠕虫病诊断和疫苗的候选分子,为寄生性蠕虫病防治奠定基础。     

旋毛虫成虫丝氨酸蛋白酶抑制剂的原核表达及免疫原性研究

以旋毛虫成虫的总RNA为模板,应用RT-PCR扩增获得旋毛虫成虫丝氨酸蛋白酶抑制剂(TsAdSPI)基因。经克隆鉴定正确后,将TsAdSPI基因连接到pET-28a表达载体,转化至大肠杆菌BL21(DE3)中进行IPTG诱导表达,并将纯化后的目的蛋白免疫新西兰大白兔,制备多克隆抗体。应用Western-blot及ELISA方法对重组蛋白的免疫原性和抗体效价进行分析。SDS-PAGE结果显示,目的蛋白分子质量为44 k Da,以包涵体形式表达。经Western-blot和ELISA方法分析,制备的多克隆抗体效价可高达1∶51200,表明该蛋白具有较好免疫原性。表明TsAdSPI蛋白可作为旋毛虫感染早期血清学诊断方法的候选抗原,为新型的抗旋毛虫及相关寄生虫药物研发打下结实基础。     

顶复器门原虫丝氨酸蛋白酶抑制剂研究进展

寄生虫可以利用蛋白酶抑制剂在宿主中生存,蛋白酶在宿主-寄生虫关系中处于关键地位.而寄生虫源的蛋白酶抑制剂的研究并未引起应有的重视.论文介绍了顶复器门原虫主要的内源的蛋白酶抑制剂丝氨酸蛋白酶抑制剂的研究概况,以期为顶复器门原虫的入侵机制及防治等研究提供新的思路.     

长角血蜱丝氨酸蛋白酶抑制剂基因的原核表达

根据GenBank中的长角血蜱丝氨酸蛋白酶抑制荆(Haemaphysalislongicornis serine proteinase inhibitor-2,HLS2)基因序列设计特异性引物,以长角血蜱饥饿成蜱总RNA为模板,经RT-PCR扩增出1164 bp的HLS2DNA片段.将其与pET-30a载体连接,构建pET-30a-HLS2表达载体,转化JM109大肠杆菌,筛选阳性克隆,经双酶切鉴定及测序分析后转化到E.coli BL21(DE3)表达菌株中,经IPTG诱导后收集菌体进行SDS-PAGE电泳分析.优化表达条件后纯化融合蛋白,用Western blotting鉴定其抗原性.结果表明,获得的HLS2 DNA片段与GenBank中的HLS2(序列号AB162827)序列的同源性为98%.构建的pET-30a-HLS2表达载体在大肠杆菌中表达了约为47 000的HLS2融合蛋白,主要以包涵体形式存在,IPTG终浓度为1.0 mmol/L、28℃诱导7 h后融合蛋白的表达量最高.Western blotting显示该融合蛋白可与兔抗长角血蜱阳性血清反应.     

丝氨酸蛋白酶抑制剂超家族的研究进展

丝氨酸蛋白酶抑制剂(Serpin)是一类分子质量在40~50 k D的蛋白酶抑制剂。已知动物、植物、古细菌、细菌、病毒的基因组中都有serpin基因家族存在,Serpin被认为是参与免疫调节和其他生物学通路的重要因子。迄今为止,Serpin家族已发现了1 500余个成员,分为抑制型和非抑制型两大类。其中,抑制型Serpin采用不可逆的自杀性机制抑制靶蛋白酶,而非抑制型Serpin的作用方式多样。Serpin独特的构象变化使其易于发生突变而导致蛋白质的错误折叠、无活性聚合物形成等,且已证实一些Serpin家族成员因构象改变和聚合导致的疾病。家蚕(Bombyx mori)和家蚕微孢子虫(Nosema bombycis)基因组中都存在多拷贝的serpin基因。本文综述Serpin在不同物种中的分布,Serpin的结构、功能、作用机制、与其相关的疾病等,以及家蚕和家蚕微孢子虫的Serpin的研究进展。     

旋毛虫新生幼虫期特异性T314全长cDNA的克隆及表达

利用PCR将旋毛虫新生幼虫期特异性全长T314cDNA的信号肽祛作后重组到原核表达载体pET-28a。将重组质粒pET28-T314转入克隆菌DH5a和表达菌DE3，分别提取质粒进行酶切，测序鉴定。用KIPTG诱导培养重组表达菌DE3，做菌体裂解物外源表达产物的SDS－PAGE分析。将重组质粒表达的融合蛋白免疫家兔制备抗血清，采用Western blot检测T314cDNA在旋毛虫不同发育时期的表达情况及其天然表达产物的相对分子质量。测序结果显示：外源T314cDNA的PCR产物在重组质粒pET28-T314中阅读框架正确；SDS－PAGE分析结果显示：经IPTG诱导后转化菌DE3的裂解产物与对照菌相比出现了1条相对分子质量约为39000的新条带，大小与外源cDNA融合蛋白的理论推导值38800相符；Western blot检测结果显示：T314cDNA的天然表达产物仅存在旋毛虫新生幼虫，不存在于成虫与肌幼虫，且相对分子质量大小与其在原核重组质粒中表达产物相同。     

Vaccination of rats and pigs against Trichinella spiralis spiralis using the subspecies, T. spiralis nativa.

Rats and pigs were vaccinated against Trichinella spiralis spiralis either by feeding infective larvae of the subspecies, Trichinella spiralis nativa in musculature or by gavage. The number of larvae established in the musculature of vaccinated nonchallenged and vaccinated challenged rats and pigs were negligible and statistically comparable, while highly significant infections were established in the nonvaccinated challenged rats and pigs. High vaccination doses of T. spiralis nativa gave virtually complete protection to challenge with T. spiralis spiralis in pigs. The results of one trial in rats with a lower vaccination dose of larvae suggest that there is a minimal vaccination dose of larvae required to elicit marked resistance to challenge. The low numbers of muscle larvae established due to the high vaccination doses of larvae confirm the low infectivity of the subspecies, T. spiralis nativa in rats and pigs.     

旋毛虫T3223-6 cDNA的表达及鉴定

利用PCR技术将T3223-6 cDNA扩增克隆到原核表达载体pET-28a,将重组质粒转入克隆菌Nova-blue,提取质粒进行酶切和测序鉴定后转入表达菌BL21star(DE3).用1 mM IPTG诱导培养重组表达菌,对菌体裂解物进行SDS-PAGE分析,检测重组蛋白的表达情况.用旋毛虫感染猪血清和正常血清,通过western blotting检测重组蛋白的反应原性.结果表明:经IPTG诱导后重组转化菌的裂解产物出现44 kD左右的表达带,大小与理论值相符;western blotting检测结果显示重组蛋白可以被旋毛虫感染猪血清识别,具有反应原性.     

Detection of Trichinella spiralis nativa antibodies in porcine sera by ELISA using T. spiralis spiralis excretory-secretory antigen

Enzyme-linked immunosorbent assay examination of sera from pigs vaccinated with T. spiralis nativa infective larvae and/or challenged with T. spiralis spiralis larvae using a T. spiralis spiralis excretory-secretory antigen showed a significant cross-reaction between the two species of Trichinella. Eight of 12 pigs vaccinated with a high dose of T. spiralis nativa reacted positively 28 days postvaccination while the remaining four pigs had high but negative ELISA optical density readings. Five of six pigs challenged with the homologous species reacted positively 28 days postchallenge but the sixth pig remained negative despite having a muscle infection of 5.6 larvae/g of musculature.     

Pathogenesis and serodiagnosis of experimental Trichinella spiralis spiralis and Trichinella spiralis nativa infections in cattle.

Trichinella spiralis spiralis infections were established in cattle by gavage and by feeding infected musculature in the ration. Trichinae were present in greatest numbers in masseter, tongue and diaphragm. Trichinella spiralis nativa had a low infectivity to cattle although a light infection was established in one cow by a heavy challenge. Cattle had an aversion to eating musculature unless it was camouflaged with molasses. Clinical signs of reluctance to eat and masticate were observed between 10 and 30 days postinfection. Eosinophil counts started to increase at seven days and peaked at about 30 days postinfection. By day 60 eosinophil counts returned to near preinfection levels but in animals examined greater than 90 days postinfection, the counts were variable. Focal lesions of eosinophilic myositis were observed up to about 90 days postinfection. Little cellular reaction was observed surrounding trichinae after muscle invasion and cyst development was completed except for cysts undergoing disintegration. Seroconversion occurred in all cattle examined between 7 and 14 days postinfection. Seroconversion was associated with IgG1 and IgG2 immunoglobulins. Peak levels of antibody occurred between 30 and 60 days. Cattle examined at 182 and 369 days postinfection showed a gradual decrease in antibody levels over time.     

Role of protease inhibitors and acylation stimulating protein in the adipogenesis in 3T3-L1 cells

Treatment of AIDS (HIV) and hepatitis C virus needs protease inhibitors (PI) to prevent viral replication. Uses of PI in therapy are usually associated with a decrease in body weight and dyslipidemia. Acylation stimulating protein (ASP) is a protein synthesized in adipocytes to increase triglycerides biosynthesis, for that the relation of PI and ASP to adipogenesis is tested in this work. ASP expression was increased during 3T3-L1 differentiation and reached a peak at day 8 with cell maturation. Addition of PI during adipocytes differentiation dose dependently and significantly (p < 0.5) inhibited the degree of triglycerides (TG) accumulation. Moreover, presence of ASP (450 ng/mL) in media significantly (p < 0.5) stimulated the degree of TG accumulation and there was additive stimulation for ASP when added with insulin (10 µg/mL). Finally, when ASP in different doses (Low, 16.7; Medium, 45 and High, 450 ng/mL) incubated with a dose of ×150 PI, ASP partially inhibited the PI-inhibited adipogenesis and TG accumulation. The results in this study show that PI inhibit lipids accumulation and confirm role of ASP in TG biosynthesis and adipogenesis.     

Spatial distribution of Trichinella britovi, T. pseudospiralis and T. spiralis in red foxes (Vulpes vulpes) in Hungary

The red fox (Vulpes vulpes) is considered one of the main reservoir of Trichinella spp. in Europe. As limited information on Trichinella infection in wildlife of Hungary is available, 2116 red foxes, representing more than 3% of the estimated fox population of the country, were screened to detect Trichinella larvae by a digestion method. Trichinella larvae from the 35 positive foxes were identified by a multiplex PCR as Trichinella britovi (30 isolates, 85.7%), Trichinella spiralis (4 isolates, 11.4%), and Trichinella pseudospiralis (1 isolate, 2.9%). The true mean intensity of T. britovi, T. spiralis and T. pseudospiralis larvae in lower forelimb muscles was 23.6, 3.5 and 13.5larvae/g, respectively. T. spiralis was detected only in the southern and eastern regions. The non-encapsulated T. pseudospiralis was recorded for the first time in Hungary. Although the overall true prevalence of Trichinella infection in foxes was only 1.8% (95% confidence interval, CI=1.5-2.1%), the spatial analysis reveals different risk regions. In the north-eastern counties bordering Slovakia and Ukraine (21% of the Hungarian territory), the true prevalence of Trichinella infection is significantly higher than that observed in other regions (6.0%, CI=4.8-7.1%). In the southern counties bordering Croatia, Serbia and Romania (41% of the Hungarian territory), the true prevalence of Trichinella infection is moderate (1.4%, CI=1.0-1.8%). In the north-western and central counties (38% of Hungarian territory), the prevalence of Trichinella infection is significantly lower (0.2%, CI=0.1-0.4%) than that of the other regions. Based on the statistical analysis and the evaluation of epidemiological data, none of the counties can be considered free of Trichinella infection. In the past decade, Trichinella infection has been detected only in few backyard pigs, and only few wild boar-related autochthonous infections in humans were described. Nevertheless, these results highlight the need of the maintenance of a strict monitoring and control programmes on Trichinella infection in farmed and hunted animals of Hungary.     

Complement membrane attack complex formation and infectivity of Trichinella spiralis and T. nativa in rats

Rats readily become infected with Trichinella spiralis but are more resistant to T. nativa. We infected complement factor C6-deficient (C6?) rats and control (C6+) rats with T. spiralis and T. nativa to compare the effects of membrane attack complex on these parasites in vivo. The 2000 larvae infection dose per rat yielded 652 lpg (larvae per gram) in the C6? group and 608 lpg in the C6+ group with T. spiralis, whereas with T. nativa the corresponding figures were only 1.05 and 1.87 lpg. The difference between the Trichinella species was evident, but the infection intensity was unaffected by the C6 deficiency. When newborn larvae were incubated in C6-deficient and control rat sera for 24 h in vitro, no changes in viability were observed. Immunohistochemistry revealed that the musculature of cross-sectioned adults and certain stichocytes bound human complement factors C3, C8 and C9, but not C1q. Interestingly, the outermost layer of the cuticle and the newborn larvae did not show any binding activity. Similar findings were obtained with immunofluorescence microscopy of intact newborn larvae. These results indicate that both T. spiralis and T. nativa have efficient mechanisms to protect themselves against complement attack. The difference in infectivity for rats between the two species, however, is not due to a differential resistance to complement membrane attack complex.