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     

Expression, purification, and human antibody response to a 67 kDa vaccine candidate for schistosomiasis japonica

Schistosomiasis remains a leading cause of morbidity and mortality in the developing tropical world, and vaccines to prevent these infections remain a scientific and public health priority. Sj67 is a 67 kDa Schistosoma japonicum surface membrane protein homologous to a family of actin-binding proteins. Sj67 is recognized by a mouse monoclonal antibody (mAb 6) that confers resistance to challenge infection in passive transfer experiments. These data support Sj67 as a potential vaccine candidate for schistosomiasis japonica. In the present study, we report the ligation-independent cloning of a cDNA encoding thioredoxin/elastin-like polypeptide (ELP)/rSj67 into a pET-32 Xa/LIC vector. Soluble recombinant fusion protein (Thio-ELP-rSj67) was expressed and purified using anion-exchange and size exclusion chromatography. rSj67 was cleaved from the Thio-ELP fusion partner by digestion with Factor Xa protease and purified using hydroxyapatite column chromatography. Endotoxin was reduced by absorption to a polymyxin support. Purified rSj67 had a molecular weight of 67 kDa and N-terminal sequencing confirmed that the first five amino acids of the recombinant protein matched the predicted sequence for the Sj67 gene. In Western blot analysis, rSj67 was recognized by the Sj67 specific mAb 6 antibody. IgG antibodies in sera from schistosomiasis infected volunteers living in an endemic area of the Philippines (n = 13) recognized rSj67 with 4.7-fold greater median fluorescence compared to uninfected North American controls (n = 5) (p < 0.009). Together, these data confirm the expression and purification of recombinant Sj67 and its immuno-reactivity with sera from S. japonicum infected humans.     

Expression, immunolocalization and serodiagnostic value of a myophilin-like protein from Schistosoma japonicum

The cDNA of a Schistosoma japonicum myophilin-like protein was cloned, sequenced, and expressed in Escherichia coli as a recombined protein (rSj myophilin-like protein), and the protein was purified by affinity chromatography. The deduced amino acid sequences of the Sj myophilin-like protein showed significant homology to myophilin, calponin, Np22 and Mp20. Northern blot and RT-PCR analyzes revealed expression of the Sj myophilin-like protein mRNA in eggs, sporocysts, cercariae, hepatic schistosomula and adult worms. Confocal fluorescence microscopy localized the native protein to the muscle of the adult worm. In schistosome-infected rabbits, the rSj myophilin-like protein antibody level, assessed by ELISA, was elevated after infection but was reduced after praziquantel treatment. In humans, the myophilin-like protein antibody level was evaluated by ELISA in sera from 33 non-infected humans and 61 schistosomiasis patients; the results showed a highly significant difference between the two groups with a sensitivity of 57.4%. Taken together, the myophilin-like protein may prove useful for monitoring the therapeutic effect of praziquantel rather than in serodiagnosis of schistosomiasis.     

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.     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

Production and properties of a mouse monoclonal IgE antibody to Schistosoma japonicum

A monoclonal IgE antibody was prepared by fusion of NS-1 myeloma cells with spleen cells of C3H/He mice immunized with an extract of adult worms of Schistosoma japonicum (Sj). The antibody was able to elicit passive cutaneous anaphylaxis in the rat skin against Sj with the highest titer of 1/256,000 in an ascitic form but did not cross-react with any of antigens extracted from S. mansoni, Fasciola hepatica, Paragoniumus westermani, or Trichinella spiralis. Western blot analysis indicated that the monoclonal IgE antibody recognized epitopes on molecules of 82 kDa, 97 kDa, 160 kDa, and 200 kDa, at least some of which were recognized by IgG antibodies of patients with chronic schistosomiasis japonica. The IgE antibody also recognized a 97-kDa antigen expressed on the surface of mechanically transformed schistosomula. Passive transfer of the antibody into mice in an early stage of challenge infection resulted in a partial but significant reduction of recovery of adult worms. However, similar treatment was not effective for the protection if the antibody was given in the postlung stage of the infection. Moreover, eosinophil-mediated damage to schistosomula was observed in vitro in the presence of the monoclonal anti-Sj IgE antibody, whereas the damage was not observed in the presence of another monoclonal IgE antibody with dinitrophenyl specificity.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

猪繁殖与呼吸综合征病毒nsp4抗体制备与鉴定

为了获得猪繁殖与呼吸综合征病毒(PRRSV)nsp4的抗体,根据HP-PRRSV TA-12株(Gen Bank Accession No.HQ416720)的nsp4基因序列,设计并合成一对引物。用RT-PCR扩增后克隆到原核表达载体p ET-28a(+)中,构建重组质粒p ET28a-nsp4,转化至Trasseta(DE3),经IPTG诱导重组蛋白获得了高效可溶性表达,大小约为26 k Da。经镍离子亲和柱(Ni+-NTA)纯化获得了高纯度重组蛋白,将纯化的nsp4蛋白免疫新西兰大白兔制备了多克隆抗体。ELISA检测抗体效价可达106,Western blotting和IFA检测结果表明所制备的多克隆抗体具有良好的免疫反应特异性,能够识别PRRSV感染宿主细胞中的nsp4蛋白。本研究成功制备了针对nsp4的多克隆抗体,为进一步研究nsp4的功能及PRRSV致病机制奠定了基础。     

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。     

Characterization of a sulfurtransferase from Arabidopsis thaliana.

A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants.     

日本血吸虫中国大陆株22.6kD膜相关蛋白基因的克隆及其在大肠杆菌中的高效表达

以日本血吸虫ｍＲＮＡ为模板,用ＲＴＰＣＲ法快速克隆到一大小约６００ｂｐ的ＤＮＡ片段,ＤＮＡ序列分析证实,所扩增到的ＤＮＡ片段中含有日本血吸虫２２６膜相关蛋白（Ｓｊ２２６（Ｃｈ））基因,将该基因重组到表达型质粒ｐＧＥＸ４Ｔ中,表达的ＧＳＴ融合蛋白分子量约４８ｋＤ,用谷胱甘肽琼脂糖凝胶亲和层析柱纯化的重组蛋白不仅纯度好,而且得率高,纯化产量可达４０ｍｇ／Ｌ培养物,免疫试验结果表明该重组蛋白具有良好的抗原性,为其在血吸虫病抗感染中的免疫作用研究创造了条件。     

日本血吸虫中国大陆株26kD GST基因在大肠杆菌中的高效表达

用大肠杆菌高效表达日本血吸虫中国大陆株 2 6k D GST基因 (Sj2 6)并观察表达产物诱导的免疫保护效果 .将 Sj2 6基因亚克隆至 p ET2 8b(+)中构建重组表达质粒 Sj2 6/ p ET,转化大肠杆菌 BL 2 1 (DE3) .Sj2 6在诱导条件下及未诱导条件下均获得高效表达 .表达产物 (r Sj2 6GST)以包含体形式表达 ,分子量为 2 8k D左右 ,能用 6× His亲和层析柱纯化 ,纯化产量为 55～ 60 mg/ L大肠杆菌培养物 .免疫印迹及 ELSA实验证实 r Sj2 6GST具有良好抗原性 .用 r Sj2 6GST不加佐剂直接背部皮下免疫昆明系小鼠 ,获得了 1 9.6% (P<0 .0 5)的减虫率及 31 .9% (P<0 .0 5)的成熟虫体减虫率 ;且免疫组检获的虫体中未成熟虫体的比例为 32 % (66/ 2 0 6) ,明显高于对照组的 1 9.6% (56/2 85) (P<0 .0 1 ) ,说明 r Sj2 6GST免疫不仅可诱导抗日本血吸虫的部分攻击感染作用 ,而且能抑制部分虫体的发育 .     

HSP70基因上游调控序列对GST基因在耻垢分枝杆菌中表达效率的 影响

将外源基因———日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）基因克隆到大肠杆菌分枝杆菌穿梭质粒中,构建成四个不同的表达截体,研究它们在耻垢后分枝杆菌（Ｍｙｃｏｂａｃｔｅｒｉｕｍｓｍｅｇｍａｔｉｓ）中的表达效率。首先将含有结核杆菌热休克蛋白７０（ＨｅａｔＳｈｏｃｋＰｒｏｔｅｉｎ,ＨＳＰ７０）的启动子的质粒ｐＭＴ７０用ＮｃｏＩ切,进行两种不同的修饰后,得到不同的ＳＤ序列;将Ｓｊ２６ＧＳＴ基因克隆进去。再将含ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因的片段切下,克隆到分枝杆菌大肠杆菌穿梭质粒ｐＢＣＧ２０００中,筛选出不同ＳＤ序列、不同方向和不同拷贝数的分枝杆菌表达载体四个。所表达的重组天然Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带。通过薄层扫描分析,发现表达质粒中双拷贝启动子外源基因组合,表达效率最高,是单拷贝组合的１６倍,占分枝杆菌菌体总蛋白的２８％。而不同的克隆方向和不同的ＳＤ序列（两者相差３个碱基）对表达效率的影响不明显。     

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.     

Cloning and expression of an adhesin antigen of Streptococcus sanguis G9B in Escherichia coli

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a lambda gt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-beta-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to saliva-coated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.     

日本血吸虫中国大陆株21.7kD蛋白编码基因的克隆和表达

根据日本血吸虫菲律宾株编码21.7kD蛋白的基因设计引物,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558bp的基因片段。经序列分析推断该基因片段为编码日本血吸虫中国大陆株21.7kD蛋白基因的完整阅读框,与菲律宾株该基因的碱基序列同源性为98%。将其克隆到表达载体pET28a(+)中,在大肠杆菌BL21中获得表达,融合表达产物分子量约为25.4kD。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,说明该基因的表达产物具有抗原性。     

Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking.     

日本血吸虫真核翻译起始因子5基因的cDNA克隆和免疫保护功能分析

利用抑制削减杂交法筛选日本血吸虫雌雄虫差异基因,从中获得真核翻译起始因子5基因(eIF5)的 表达序列标。对该序列进行生物信息学分析:对其5’端进行电子延伸,获得含完整开放阅读框的cDNA序列 (AY686501)。将其亚克隆到表达载体pET-28c,进行重组表达。免疫保护效果实验表明,重组表达蛋白具有显著 抑制血吸虫卵在寄主肝脏中的沉积效果。     

Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein

A human lung cDNA expression library was screened by using a rabbit antiserum specific for a human Clara cell 10 kDa protein. The cDNA from two positive clones was sequenced by the dideoxy chain termination method. The nucleotide and primary amino-acid sequence deduced therefrom are presented. The N-terminal amino-acid sequence of the Clara cell 10 kDa protein, purified from bronchoalveolar lavage, was also determined. The deduced and experimentally determined sequences were identical where data for both were available. From the amino-acid composition, deduced and experimentally determined amino-acid sequences, it was determined that the 10 kDa protein in bronchoalveolar lavage consists of two identical 70-amino-acid long polypeptide chains joined by two cystine residues. The size of mRNA for the protein was found to be about 0.6 kb and the monomeric nascent protein, obtained by in vitro translation of lung mRNA was about 7.3 kDa in size. The 10 kDa protein recovered from bronchoalveolar lavage has 61% sequence identity with rabbit uteroglobin, the two proteins have common predicted secondary structures with marked surface differences when comparing predicted and actual structure determined by X-ray diffraction. The differences imply similarity of structure but, not identity of function.     

日本血吸虫三价DNA疫苗pVIVO2 SjFABP/Sj26.SjGAPDH的构建及其免疫保护作用评价

目的:构建含有日本血吸虫中国大陆株脂肪酸结合蛋白(SjFABP),3-磷酸甘油醛脱氢酶(SjGAPDH),26kDa谷胱甘肽S转移酶(Sj26)的三价DNA疫苗,并通过药理实验评价其抗血吸虫感染的免疫保护作用。 方法:  以血吸虫成虫RNA为模板制备cDNA第一链,RT-PCR扩增得到抗原基因片段,再通过重组PCR技术,将Sj26和SjGAPDH融合为Sj26.SjGAPDH基因。将SjFABP和Sj26.SjGAPDH分别克隆入pVIVO2的mcs1和mcs2,获得重组质粒pVIVO2-SjFABP/Sj26.SjGAPDH。瞬时转染MCF-7细胞,通过逆转录PCR（RT-PCR）检测抗原基因在mRNA水平的表达,通过间接荧光免疫（IIF）检测抗原基因在小鼠体内抗原水平表达,验证了DNA疫苗的有效性;并免疫小鼠后进行免疫保护性初步试验。结果:  经过酶切、测序及体内外表达验证,该三价DNA疫苗pVIVO2-SjFABP/Sj26.SjGAPDH构建成功;该三价DNA疫苗在小鼠抗血吸虫感染中诱发减虫率和减卵率达到58.6%和59.8%。结论:  该三价DNA疫苗组具有比单价和双价DNA疫苗组更加良好的免疫保护作用,为日本血吸虫DNA疫苗的研制奠定了基础。