日本血吸虫信号转导蛋白Sjwnt_4基因的克隆、表达及功能分析

由Wnt基因家族产物与其它相关基因产物构成的Wnt信号通路,是细胞发育和生长调节的一个关键途径,对动物的发育特别是生殖系统的发育起重要的调节作用。在人类和小鼠中,Wnt4蛋白是性腺分化过程中主要调节因子,在胚胎发育中起着关键作用。利用RACE技术从日本血吸虫19d童虫中首次扩增到一个Wnt家族基因,序列分析表明该基因的完整编码框含1311bp,编码436个氨基酸,理论分子量49.6kD。同源性分析结果表明,该基因的氨基酸序列具有典型Wnt家族蛋白特征,与日本三角涡虫、人Wnt4的氨基酸序列相似性分别达43％、37％,推测为血吸虫的Wnt4基因,命名为Sjwnt4（GenBank登陆号DQ643829）。实时定量PCR分析显示该基因在14d童虫、19d童虫、31d虫体、44d雌虫及44d雄虫中均有表达,其中19d童虫中的表达量明显高于其它发育阶段,44d雌虫中的表达量明显高于雄虫。构建了该基因的原核表达载体pGEX_4T_2_Sjwnt4,应用大肠杆菌系统进行了表达,表达蛋白以包涵体形式存在,Western印迹显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别。Sjwnt4基因及其表达产物的获得,为探索Wnt信号通路对血吸虫发育、生殖的调节提供了重要基础。     

日本血吸虫信号转导蛋白Sjwnt-4基因的克隆、表达及功能分析

由Wnt基因家族产物与其它相关基因产物构成的Wnt信号通路,是细胞发育和生长调节的一个关键途径,对动物的发育特别是生殖系统的发育起重要的调节作用。在人类和小鼠中,Wnt4蛋白是性腺分化过程中主要调节因子,在胚胎发育中起着关键作用。利用RACE技术从日本血吸虫19d童虫中首次扩增到一个Wnt家族基因,序列分析表明该基因的完整编码框含1311bp,编码436个氨基酸,理论分子量49.6kD。同源性分析结果表明,该基因的氨基酸序列具有典型Wnt家族蛋白特征,与日本三角涡虫、人Wnt4的氨基酸序列相似性分别达43%、37%,推测为血吸虫的Wnt4基因,命名为Sjwnt4(GenBank登陆号DQ643829)。实时定量PCR分析显示该基因在14d童虫、19d童虫、31d虫体、44d雌虫及44d雄虫中均有表达,其中19d童虫中的表达量明显高于其它发育阶段,44d雌虫中的表达量明显高于雄虫。构建了该基因的原核表达载体pGEX-4T-2-Sjwnt4,应用大肠杆菌系统进行了表达,表达蛋白以包涵体形式存在,Western印迹显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别。Sjwnt4基因及其表达产物的获得,为探索Wnt信号通路对血吸虫发育、生殖的调节提供了重要基础。     

血吸虫新抗原基因SjMF4的克隆、表达及功能分析

根据东方田鼠天然抗日本血吸虫病的现象 ,首次利用东方田鼠健康血清结合羊抗小鼠IgG3抗体免疫筛选日本血吸虫 (中国大陆株 )成虫cDNA表达型文库 ,获得两个阳性克隆 ,采用RACE技术对其中一cDNA片段进行扩增 ,获一含ORF的基因片段。序列分析表明该基因为一日本血吸虫新基因 ,命名为SjMF4 (SchistosomajaponicumMicrotusfortis 4 ,SjMF4 )。利用ExPASy的ScanProsite软件对此基因编码的蛋白质结构和功能域进行了分析。把该基因克隆到原核表达载体pET 2 8a( ) ,Western印迹显示表达产物具有良好的抗原性。又构建了真核表达质粒pcD NA3 SjMF4重组DNA疫苗 ,小鼠实验表明可诱导一定的保护作用。     

日本血吸虫蛋白酶体α2亚基基因的克隆、表达及功能分析

26S蛋白酶体是一种能够降解大多数内源性蛋白的多亚基复合物,它的蛋白降解作用能够影响细胞周期、转录控制和其他一些重要的细胞进程。本实验利用PCR技术从日本血吸虫18d童虫中首次扩增到蛋白酶体α2亚基基因(GenBank Accession No.AY813725),序列分析表明该基因的开放阅读框(ORF)含708bp,编码235个氨基酸,理论分子量25.84kDa。同源性分析结果显示,该基因为日本血吸虫蛋白酶体α2亚基,命名为SjPSMA2。实时定量PCR分析显示该基因在7d、13d、18d、23d、32d和42d虫体中都有表达,7d和23d虫体表达量低于其他几个时期。构建了该基因的原核表达质粒pET28a(+)-SjPSMA2,在大肠杆菌系统中成功获得了表达,重组蛋白以包涵体形式存在,Western blotting显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别,并且能检测到天然状态下该蛋白的存在。应用重组蛋白免疫BALB/c小鼠后,诱导产生了较高的特异性抗体水平及12.33%的减虫率和35.23%的肝脏减卵率。SjPSMA2基因及其表达产物的获得,为探索蛋白酶体在血吸虫生长发育中的作用提供了重要基础。     

日本血吸虫新基因Sj-MA的克隆、表达及保护性免疫

为发现新基因 ,寻找日本血吸虫病新疫苗候选分子 ,采用Sj雄虫免疫血清筛选Sj成虫cDNA文库。经测序发现新基因Sj MA含有一个完整的阅读框 ,推测其由 2 4 9个氨基酸组成 ,编码分子量为 2 8.8kD的可溶性蛋白质 ,并带有多个能被磷酸化激活的位点 ,提示其可能为一重要的信息传递分子。将Sj MA的cDNA亚克隆至原核表达载体pGEX 5X ,获得Sj MA原核表达的重组体rSj MA/GST ,并在E .coli中高效表达为谷胱甘肽S 转移酶 (GST)融合蛋白 ,分子量为 5 4 .8kD ,Western印迹显示融合蛋白质能被抗雄虫和抗GST血清识别。融合蛋白质免疫小鼠可诱导 34.2 9%的减虫率 ,与对照组有显著性差异 (P <0 .0 0 1 )。表明新基因Sj MA表达的蛋白质能诱导小鼠的抗日本血吸虫的保护性免疫 ,提示其作为日本血吸虫疫苗候选分子的潜在价值     

日本血吸虫磷酸甘油酸变位酶SjPGAM基因的克隆、表达及功能分析

磷酸甘油酸变位酶(Phosphoglycerate mutas)是糖酵解过程中的一种重要酶,主要催化3-磷酸甘油酸转化为2-磷酸甘油酸.利用PCR技术从日本血吸虫19 d童虫中首次扩增到一个PGAM家族基因,序列分析表明该基因的完整编码框含753 bp,编码250个氨基酸,理论分子量28.26 kD,理论等电点7.01.同源性分析结果表明,该基因的氨基酸序列具有典型PGAM家族特征,推测为血吸虫的PGAM基因,命名为SjPGAM(GenBank Accession No.EU374631).实时定量PCR分析显示该基因在14d和19d童虫中的表达量明显高于其他发育阶段,42d雄虫中的表达量高于雌虫.构建了该基因的原核重组表达质粒pET-28a( )-PGAM,在大肠杆菌系统中成功获得了表达,重组蛋白以包涵体形式存在,Western blotting显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别.SjPGAM基因及其表达产物的获得,为探索PGAM基因家族在血吸虫能量代谢过程中碳水化合物转运、新陈代谢调节和生长发育提供了重要基础.     

日本血吸虫新抗原基因Sj—Ts4的克隆、表达及免疫保护性

为探讨旋毛虫感染小鼠后抗血吸虫感染的分子机制 ,并为血吸虫病疫苗的研究提供新的抗原分子 ,用旋毛虫感染鼠血清筛选日本血吸虫成虫cDNA文库。经过 3轮筛选 ,获得 9个阳性克隆 ,其中新基因Sj Ts4有一完整的编码框 ,编码2 10个氨基酸。该蛋白质的理论分子量为 2 3kD ,等电点为 7.72。将Sj Ts4亚克隆于原核表达载体 pGEX 5X 3,以GST融合蛋白的形式在E .coliDH5α中表达。Western印迹分析显示 ,重组Sj Ts4蛋白 (rSj Ts4)能被慢性感染鼠血清识别。rSj Ts4或与弗氏完全佐剂混合后免疫小鼠 ,均可以诱导产生特异性的IgG抗体 ,抗体效价可高达 1∶2 5 6 0 0。两免疫组的减虫率分别为31.36 %和 36 .80 % ,与对照组相比都具有统计学意义     

日本血吸虫凋亡基因Sjcaspase3的克隆、真核表达 及其功能分析

为深入研究日本血吸虫细胞凋亡机制。利用PCR技术扩增得到Sjcaspase3的全长序列,其ORF含900 bp,编码299个氨基酸,理论分子量为33 509.7 Da,理论等电点为6.39。Real-time PCR分析表明该基因在日本血吸虫生长发育的各个时期均有表达,其中21 d表达量最高,42 d雌虫表达量高于42 d雄虫。成功构建了p XJ40-FLAG-Sjcaspase3重组质粒并转染到Hela细胞内,荧光定量PCR和Western blotting分析表明Sjcaspase3成功在Hela细胞中表达。酶活分析提示重组Sjcaspase3具有切割特异性底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸(DEVD)的活性。流式细胞术检测了Sjcaspase3可诱导Hela细胞发生早期细胞凋亡。研究结果为深入探讨Sjcaspase3的生物学功能及日本血吸虫细胞凋亡机制奠定了基础。     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

日本血吸虫中国大陆株抱雌沟蛋白编码基因的克隆和表达

根据曼氏血吸虫抱雌沟蛋白SmGcp序列和日本血吸虫编码抱雌沟蛋白保守区的基因片段jGcp1分别设计三对引物,以日本血吸虫中国大陆株成虫mRNA为模板 ,用RT－PCR法扩增出大小为1949bp的基因片段。经序列分析推断该基因片段含编码日本血吸虫抱雌沟蛋白基因的阅读框,与SmGcp碱基一致性为85％,其理论推测氨基酸组成与曼氏血吸虫抱雌沟蛋白的一致性为83．7％。将上述扩增的基因片段克隆到表达载体pET28c( )中,在大肠杆菌BL21中获得表达,融合表达产物分子量约为80kD。利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western印迹检测,在预测位置出现了明显的识别条带,说明该编码日本血吸虫中国大陆株抱雌沟蛋白基因的表达产物具有抗原性。     

Bacterial Expression and Characterization of Functional Recombinant Triosephosphate Isomerase from Schistosoma japonicum

The dimeric enzyme triosephosphate isomerase (TPI) converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate, a key reaction in glycolysis. Previous studies of the native enzyme in the human bloodflukes belonging to the genus Schistosoma have indicated that TPI is a promising anti-schistosome vaccine antigen. However, a recombinant form of the enzyme is required as an alternative to the impractical option of using biochemically purified TPI obtained from worm tissue for large-scale vaccine use. We previously cloned and sequenced a full-length cDNA encoding the TPI of the Asian (Chinese strain) schistosome Schistosoma japonicum (SjcTPI). We now report very high level bacterial expression of this cDNA and the subsequent purification of the recombinant protein to >98% homogeneity under nondenaturing conditions. The recombinant SjcTPI (re-SjcTPI) was shown to be enzymatically active with a specific activity of 7687 units/mg protein, an activity higher than that of commercially obtained porcine TPI tested concurrently under the same assay conditions. The K_m value for the re-SjcTPI using glyceraldehyde-3-phosphate as substrate was 406.7 μM, which is similar to the K_m values reported for the yeast enzyme and various mammalian TPIs. With the availability of substantial amounts of enzymatically active and readily purified re-SjcTPI made in bacteria we can now test whether the recombinant protein can induce a similar level of protection in vaccination/challenge experiments as the native, biochemically purified enzyme.     

日本血吸虫中国株次黄嘌呤鸟嘌呤磷酸核糖转移酶的克隆、表达及其免疫保护性研究

根据基因库中日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HGPRT) EST (BU803192) 以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjHGPRT基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接,获得SjHGPRT全长cDNA (1 270 bp),经序列分析,推断该片段含有编码SjHGPRT基因的完整阅读框,其编码基因与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SmHGPRT) 全长编码基因碱基一致性为82%,其理论推导的氨基酸组成与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶的一致性约为83%. 将其编码基因克隆到表达载体pQE30上,在大肠杆菌M15中获得准确、高效表达,表达产物分子质量约为28 ku. 用日本血吸虫成虫抗原免疫血清对表达产物进行蛋白质印迹检测,在预测位置上出现明显的识别条带. 重组蛋白动物免疫保护性结果显示：在虫荷、每克肝卵、每克粪卵和雌子宫内卵数方面,疫苗组与对照组比较差异均具有显著性 (P < 0.05,P < 0.01). 结果表明,日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SjHGPRT) 全长cDNA成功克隆并在大肠菌中得到表达,表达产物具有良好的抗原性和动物免疫保护效果,是一种潜在的具有部分免疫保护性的抗血吸虫病疫苗候选分子.     

Heterogeneity of eosinophil chemotactic factors produced by splenic T cells of Schistosoma japonicum-infected mice

Spleen cells of Schistosoma japonicum-infected mice produced eosinophil chemotactic factors (ECF-Ls) upon stimulation with soluble egg antigen preparation (SEA) and Con A, while spleen cells from uninfected mice produced ECF-L upon stimulation with Con A but not with SEA. Depletion of CD4⁺ T cells, but not of CD8⁺ T cells, almost completely removed Con A-induced ECF-L production. In contrast, depletion of CD8⁺ T cells completely abolished SEA-induced ECF-L production while depletion of CD4⁺ T cells did not, indicating that CD4⁺ CD8⁻ T cells and CD4⁻CD8⁺ T cells play essential roles for the production of Con A-induced ECF-L and SEA-induced ECF-L, respectively. Con-A-induced ECF-L had a high affinity to Con A-Sepharose but not to Procion Red agarose. In contrast, SEA-induced ECF-L bound to Procion Red agarose, but not to Con A-Sepharose. A gel permission HPLC analysis revealed that the apparent molecular weight of Con A-induced ECF-L and SEA-induced ECF-L was 16 kDa and 35 kDa, respectively. Both Con A-induced ECF-L and SEA-induced ECF-L had a similar isoelectric point (pI 3.5–3.6). These results indicate that selective stimulation of either CD4⁺ or CD8⁺ T cells of S. japonicum-infected mice produces heterogeneous ECF-L.     

Elucidation of Schistosoma japonicum population dynamics in pigs using PCR-based identification of individuals representing distinct cohorts

The study reported here investigated the interactions of successive infections and acquired resistance of pigs to challenge infections of Schistosoma japonicum. Two morphologically indistinguishable geographical isolates from China (from Anhui and Zhejiang provinces) were used for the infections. The worms of the two isolates were distinguishable by PCR-linked restriction fragment length polymorphism analysis of the nicotinamide adenine dinucleotide phosphate dehydrogenase I gene of the mitochondrial genome. Thirty-two pigs divided into seven groups were used in the experiment. Two groups received a single infection by either the Anhui or the Zhejiang isolate. In Challenge Groups 1, 4, 6, 8 and 12, a primary infection of the Zhejiang isolate was followed by a challenge infection with the Anhui isolate at week 1, 4, 6, 8 or 12 after the primary infection. In this way it was possible to determine whether worms recovered by perfusion originated from the primary or the challenge infection. Only the challenge infection at week 1 resulted in a higher worm burden when compared with a single primary infection with the Zhejiang isolate. The results showed that challenge worms were able to establish, and that the proportion of worms originating from challenge infection increased at the later challenge infections, however without an increase in the total number of worms. In addition, mixed pairs of the two isolates were found in all challenge-infected groups. The results indicate that pigs are able to mount a partial resistance against re-infection with S. japonicum by 4 weeks after a primary infection, but that worms of the challenge infections eventually replace the primary infection. The finding of mixed pairs of the two isolates indicates that worms of S. japonicum are either polygamous or able to wait in solitude for up to 12 weeks for a partner.     

Evolution in a multi-host parasite: Chronobiological circadian rhythm and population genetics of Schistosoma japonicum cercariae indicates contrasting definitive host reservoirs by habitat

Schistosomiasis japonica is a disease of profound medical and veterinary importance which has remained endemic in many regions and has re-emerged where previously controlled in China. Although over 40 mammalian species are suspected as reservoirs for Schistosoma japonicum, their relative roles, particularly wildlife, remain to be ascertained. As cercarial emergence is a heritable trait shaped by the definitive hosts’ behaviour, three chronobiological trials of cercarial emergence from field-collected snails from two contrasting ecological regions within China were performed, followed by genetic analyses of the parasites. Two distinct modes were identified, with late afternoon emergence mainly found in the hill region, compatible with a nocturnal rodent reservoir, and early emergence within the marshland consistent with a diurnal cattle reservoir. Furthermore, genetic analyses pointed to a clear separation between cercariae with different biological traits. The phenotypic and genotypic differentiation of the parasites identified here between and within two regions may indicate a strain complex. Such parasite diversity could, in turn, provide an explanation for the different infection scenarios observed between the two regions, and hence have important applied implications in terms of targeted control of key reservoirs.     

棉花GhZIP4基因的克隆及表达分析

根据EST拼接的序列设计引物,利用RT-PCR和PCR方法,从陆地棉‘苏棉18’-cDNA和基因组DNA中分别克隆获得了GhZIP4基因片段.结果表明:(1)GhZIP4基因cDNA序列全长1 487 bp,包含1 269 bp ORF,编码422个氨基酸残基,其氨基酸序列具有典型的ZIP蛋白特征,预测具有8个跨膜结构域,第Ⅲ和第Ⅳ跨膜结构域间存在可变区,在可变区有2个富含His的结构域“HRHSHPHG”和“HSHGHGHD”.(2)氨基酸进化树分析显示,GhZIP4同拟南芥ZIP家族AtZIP4的相似性较高.(3)GhZIP4 DNA序列编码区全长1 778 bp,包含4个外显子和3个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则.(4)半定量分析显示,GhZIP4基因在茎中表达量最高,表明该基因有可能在某些金属离子地上部和根部的动态平衡分布过程中具有重要作用.     

Molecular cloning and expression of a Schistosoma japonicum tegumental membrane-associated antigen from Japanese strain

Diagnosis and vaccine development form the major focus in creating strategies for the control of schistosomiasis. In this study, we established an IgG1 mouse monoclonal antibody (MoAb), SJA111, which strongly reacted with 23–25-kDa Schistosoma japonicum tegumental-associated membrane proteins, but not with eight other parasitic antigens. A λgt 11 cDNA library from the Japanese strain of the Schistosoma japonicum adult worm was screened with SJA111 as a probe. A single positive clone was isolated and the nucleotide sequence of the isolated cDNA was determined. The cDNA clone consisted of 844 bp, and the coding region contained 576 bp which was translated to a 22.6-kDa protein. This region showed 99.0% and 99.3% significant homology with those of the Chinese and Philippine strains of Schistosoma japonicum, respectively. The deduced amino acid sequence of the protein was identical to that of the Philippine strain and only one residue differed from that of the Chinese strain. The recombinant form of the tegumental protein was expressed in Escherichia coli and purified by a combination of ion exchange and affinity chromatography, and the purified protein was found to react with the sera of patients infected with Schistosoma japonicum. This result suggests that this antigen may be useful in the immunodiagnosis of schistosomiasis as well as in the development of an effective vaccine.