恶性疟原虫FCC-1／HN株EBA-175基因特异序列的克隆及其鉴定

根据恶性疟原虫红细胞结合抗原EBA-175基因编码序列设计并合成一对引物，通过聚合群链反应(PCR)技术对恶性疟原虫FCC-1／HN株的EBA-175基因进行扩增，用HindⅢ和BarnHⅠ同时消化扩增产物，定向克隆PCDN3载体，转化至感受态大肠杆菌TG1，经抗性筛选和质粒大小快速凝胶电泳鉴定初步获得重组克隆，再经PCR鉴定和HindⅢ／BarnHⅠ酶切鉴定，证实所得的重组克隆含有编码恶性疟原虫FCC-1／HN株EBA-175基因部分序列。     

恶性疟原虫已糖转运体编码基因的体外扩增及克隆

目的：体外扩增恶性疟原虫海南分离株的已糖转运体基因(PfHT1)，并将该基因克隆至pEGFF真核表达载体内使其高效表达，为研究DNA疫苗创造条件。方法：特定PCR引物的设计；恶性疟原虫FCC1／HN株的体外培养；提取基因组DNA；PCR扩增和琼脂糖凝胶电泳分析；酶切、连接及PCR分析鉴定。结果：从恶性疟原虫簿南分离株基因组DNA中扩增出特异性的编码FfhTl的基因序列．片段太小为1516bp，克隆鉴定结果表明插人片段大小正确。结论：体外成功扩增出恶性疟原虫PfHTl编码序列，与预期长度相符，并成功构建pEGFF-Htl真核表达载体。为研究其结构、功能和免疫原性奠定基础，     

恶性疟原虫FCC—1／HN株环子孢子蛋白基因分枝杆菌——大肠杆穿梭表达重组质粒的构建及序列测定

本文对恶性疟原虫环子孢子蛋白(circumsporozoiteprotein,CSP)基因片段进行克隆和序列测定。根据恶性疟原虫837株基因编码序列设计合成一对引物,采用PCR技术从恶性疟原虫FCC-1/HN株基因组DNA中特异扩增CSP基因片段的Ⅰ区、中央重复区、重复区后可变区和Ⅱ区;经纯化的扩增产物用BamHⅠ和KpnⅠ双酶切后,定向克隆入大肠杆菌——分枝杆菌穿梭表达质粒,转化感受态大肠杆菌DH5α,重组克隆经抗性筛选和快速凝胶电泳鉴定,再经PCR和酶切鉴定,并对重组子进行序列测定。结果表明从恶性疟原虫FCC-1／HN株基因组DNA中可特异扩增出约1171bp的基因片段,阳性重组质粒经双酶切和PCR鉴定与预期的结果一致,序列测定表明所克隆的基因和编码环子孢子抗原的基因片段相符。     

寄生虫学

0501961 用pfcrt点突变基因检测技术检测恶性疟原虫氯喹抗药性的初步研究；0501962 恶性疟原虫海南株FEN-1基因的克隆及序列分析；0501963 恶性疟原虫海南株（FCC1／HN）EBA-175Ⅱ区F2结构域基因的克隆与表达；0501964 抗恶性疟原虫谷螽酸脱氢酶单克隆抗体的研制与胶体金免疫层析方法的建立；0501965 伯氏疟原虫RC株和N株感染小鼠IFN-γ mRNA的表达及其对致病的影响。     

恶性疟原虫FCCl／HN株MSAl全基因编码区的体外扩增

恶性疟原虫裂殖子表面蛋白1(MSA1)是抗疟疫苗的候选抗原之一。据已知的MSAl基因序列，自行设计合成了四对引物。应用PCR技术，分四段从恶性疟原虫FCC1／HN株基因组DNA中扩增出垒基因编码区序列。扩增产物经1．2％琼脂塘凝腔电泳鉴定．表明获得了正确的MSA1基因扩增片段。     

恶性疟原虫FCC1/HN株环子孢子蛋白的原核表达、纯化及其对肝细胞靶向作用的观察

目的从恶性疟原虫FCC1／HN株基因组中扩增得到环子孢子蛋白(circumsporozoite protein,CSP)的全长编码基因,克隆至原核表达载体pET-32c中进行表达纯化,观察重组环子孢子蛋白对肝细胞的结合能力,探讨其作为原发性肝癌基因治疗的靶向分子的可行性。方法根据恶性疟原虫3D7株环子孢子蛋白的编码序列设计一对引物,利用聚合酶链反应(PCR)技术从恶性疟原虫FCC1／HN株基因组中扩增出CSP的全长编码基因,将其克隆到载体pGEM-T中,通过基因测序加以证实;进一步亚克隆至原核表达载体pET-32c中,在大肠杆菌BL21中用IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,采用免疫印迹技术(WB)对纯化的融合蛋白进行免疫反应性检测;采用免疫组化(IHC)技术观察重组环子孢子蛋白对不同组织细胞的结合能力。结果从恶性疟原虫FCC1／HN株基因组中成功扩增到1263 bp的全长CSP基因,该基因在原核系统中经诱导表达出一相对分子质量(Mr)约62×103大小的融合蛋白,表达产物以包涵体的形式存在;通过Ni2+亲和柱纯化获得重组CSP融合蛋白;WB表明,重组CSP融合蛋白能被疟原虫阳性血清特异性识别;免疫组化结果显示重组CSP融合蛋白能够与肝癌和正常肝细胞特异性地结合,与其他组织来源的细胞则未见反应。结论CSP是疟原虫子孢子表面主要的蛋白,重组环子孢子蛋白作为原发性肝癌基因治疗的靶向分子具有一定的潜在应用价值。     

寄生虫学

0500640 细胞因子表达质粒对恶性疟原虫顶端膜抗原1DNA免疫的调节作用，0500641 恶性疟原虫FCC1／HN株醛缩酶编码区基因的克隆及表达，0500642 伯氏疟原虫青蓠素抗性相关的消减cDNA文库构建，0500643 弓形虫SAG1基因的表达及其诱导的细胞免疫应答，0500644 弓形虫RH株致密颗粒蛋白GRA4基因的克隆与表达。     

鼠约氏疟原虫yoelii株Pydyn基因的内含子序列分析

本研究测定并分析鼠约氏疟原虫 (Plasmodiumyoeliiyoelii)动力素蛋白基因 (Pydyn) 3′端内含子序列 ,比较约氏疟原虫yoelii虫株的Pydyn基因与约氏疟原虫 17XNL虫株基因组序列内含子间序列的多态性。方法 :应用PCR技术扩增约氏疟原虫动力素蛋白基因 (Pydyn)基因组 3′端序列 ,将其克隆入pGEM TEasy载体。阳性克隆经酶切和PCR鉴定正确后测序 ,并用分子生物学WINSTAR软件进行基因结构和同源性分析。结果 :用PCR方法成功扩增出约 80 0bp的Pydyn基因特定片段 ,阳性克隆经酶切及PCR扩增确定。基因序列分析表明 ,约氏疟原虫yoelii虫株的Pydyn基因含有 3个内含子 ,并且与约氏疟原虫 17XNL虫株基因组序列在内含子间存在着几个变异。结论 :确定了鼠约氏疟原虫动力素蛋白基因 (Pydyn) 3′端内含子序列 ,其内含子具有序列短且AT含量较高的特点 ,两末端的碱基具有一般真核基因内含子共有的剪接位点。多态性分析表明 ,将约氏疟原虫yoelii虫株的Pydyn基因的 3个内含子与人恶性疟原虫动力素基因Pfdyn内含子保守序列相比 ,Pydyn基因内含子上游下游序列具有一定的同源性。另外 ,约氏疟原虫yoelii虫株与约氏疟原虫 17XNL虫株的内含子间存在着多态性 ,存在着几个变异 ,这些变异属于点突变     

弓形虫缓殖子期特异抗原SAG2C基因的克隆、鉴定与生物信息学分析

目的克隆并鉴定弓形虫PRU株表面抗原SAG2C基因序列和cDNA序列，对比不同毒力弓形虫株（PRU、RH、ME49）中SAG2C基因序列。进行生物信息学分析。方法根据SAG2C基因已知序列设计合成一对引物，应用PCR技术从Prugniaud（PRU）株弓形虫基因组DNA和总RNA中扩增SAG2C基因，克隆入pMD19-T载体并进行序列测定。应用DNAMAN软件、NCBI网站（http：／／www。ncbi．nlm．nih.gov／）分析3种虫株之间SAG2C基因的同源性；利用生物信息学网站ExPASy（http：／／us.expasy．org／）对获得的基因及推导出的氨基酸序列进行生物信息学分析。结果PCR扩增得到弓形虫PRU株SAG2C基因及其全长cDNA序列．酶切及PCR鉴定获得了正确的重组质粒。测序结果表明，获得SAG2C基因序列1225bp，全长cDNA序列1095bp，编码364个氨基酸。同源性分析显示。弓形虫Prugniaud株和RH株SAG2C基因同源性为97．14％；Prugniaud株与ME49株cDNA序列同源性为96．89％；编码氨基酸序列同源性为92％。N端为信号肽，C端疏水序列预测它为糖基磷脂酰肌醇固着蛋白．存在13个潜在抗原表位及多个保守功能区域。结论成功克隆了弓形虫缓殖子期特异抗原SAG2C基因序列及其全长cDNA序列。序列分析显示其为糖基磷脂酰肌醇固着蛋白。     

幽门螺杆菌omp11基因的克隆及序列分析

目的　对幽门螺杆菌 (Helicobacterpylori,Hp)郑州分离株MEL Hp2 7和NCTC116 37株外膜蛋白基因 (omp11)进行克隆和测序 ;确定不同Hp菌株omp11基因序列的变异性 ,并对该基因编码多肽的化学及免疫学特性进行预测 ,为幽门螺杆菌疫苗抗原的筛选提供数据。方法　提取Hp染色体DNA ,用自行设计的PCR引物 ,从染色体DNA上扩增出omp11基因 ,将其克隆到载体pNEB193中 ,用重组质粒转化大肠杆菌 (E .coliJM10 9)。对重组质粒进行酶切鉴定 ,对插入的omp11基因片段进行测序 ,应用生物信息学软件Omiga2 .0、GeneDoc2 .3和GenBank、Swiss port数据库对 4个Hp菌株 (MEL Hp2 7、NCTC116 37、2 6 6 95、J99)的omp11基因序列进行同源性分析 ,并对该基因编码多肽的主要化学特征和抗原结构域进行预测。结果　MEL Hp2 7和NCTC116 37株omp11基因的核苷酸序列长度均为5 6 1bp ,不同菌株间核苷酸序列的同源性为 96 .6 %～ 98.0 % ,与国内的MEL Hp2 7株同源性最高的菌株是NCTC116 37,二者的同源性为 97.9%。 4株Hpomp11基因编码的氨基酸序列长度均为 186aa ,不同菌株间氨基酸序列的同源性为 98.9%～ 10 0 % ,与MEL Hp2 7株同源性最高的菌株是 2 6 6 95 ,二者的同源性为 99.5 %。预测HpMEL Hp2 7omp11基因编码多肽的相对分子质量 (Mr)     

恶性疟原虫海南分离株FCC1/HN有性期特异抗原Pfs48/45基因的克隆与表达

采用基因工程技术,将编码恶性疟原虫有性期特异抗原Ｐｆｓ８／４５的基因克隆到真核表达质粒ｐｃＤ－ＮＡ３,并进行ＤＮＡ序列测定,再通过磷酸钙—ＤＮＡ共沉淀转化法将重组质粒ｐｃＤＮＡ３－ｐＦＳ４８／４５导入ＨｅＬａ细胞,建立稳定分泌Ｐｆｓ４８／４５蛋白的阳性克隆株。结果显示,我国海南ＦＣＣ１／ＨＮ株Ｐｆｓ４８／４５抗原基因序列与ＮＦ５４株者高度同源,提示该基因在不同虫株间高度保守,是研制疟疾疫苗的理想靶抗原;在ＨｅＬａ细胞中表达的Ｐｆｓ４８／４５蛋白分子量约为４６／４３．５ｋＤａ双联体蛋白,其表达量占细胞培养上清蛋白总量的１８．２７％。经ＷｅｓｔｅｒｎＢｌｏｔ分析显示,表在蛋白能被配子体免疫鼠血清特异性识别,提示表达的重组蛋白Ｐｆｓ４８／４５具有免疫活性。真核表达系统ｐｃＤＮＡ３／Ｐｆｓ４８／４５／ＨｅＬａ的建立为进一步研究重组Ｐｆｓ４８／４５抗原的免疫原性和保护性奠定基础。     

Molecular characterization of Menangle virus, a novel paramyxovirus which infects pigs, fruit bats, and humans

Menangle virus (MenV), isolated in August 1997 following an outbreak of reproductive disease in a piggery in New South Wales, is the second previously unclassified member of the family Paramyxoviridae to be identified in Australia since 1994. Similar to Hendra virus (HeV), MenV appears to be a virus of fruit bats (flying foxes) in the genus Pteropus. No serological cross-reactivity was detected between MenV and other known paramyxoviruses and to facilitate virus classification a cDNA subtraction method was used to obtain viral-specific cDNA from MenV-infected cells. Cloning and sequencing of the products enabled the entire sequences of the NP, P/V, M, F, and HN genes to be determined. Comparison of the nucleotide and deduced amino acid sequences for each gene with members of the family Paramyxoviridae, determination of the P gene mRNA editing strategy, and phylogenetic analyses confirmed that MenV is a new member of the genus Rubulavirus. However the deduced protein sequence of MenV HN exhibited only limited sequence homology when compared with attachment proteins of other paramyxoviruses. Key differences within the amino acid residues considered important determinants of neuraminidase activity suggest MenV HN is unlikely to possess the same degree of neuraminidase activity characteristic of other rubulavirus and respirovirus HN proteins.     

Detection and differentiation of the bovine parainfluenza-3 virus strains studied by amplification and sequencing of the HN gene

A possibility of using the amplification of gene HN fragment in combination with nucleotide cDNA sequencing for the purpose of identification and strain differentiation of bovine parainfluenza-3 virus was demonstrated. A comparative analysis of the primary structure in the studied HN gene fragment revealed 2 genetic groups among the investigated virus' strains and isolates. Group 1 is made up of Northern American viral strains and of Russian isolates, whose primary structure has a high level of homology to the primary SF-4/32 strain structure; group 2 comprises the virus' Russian isolates with a high level of homology to the mentioned strains to Japanese strains' sequences. The biggest differences between the studied strains and the viral isolates amounted to around 8%, when the nucleotide sequences were compared, and to around 4%, when the corresponding amino-acid sequences were compared.     

云南省新分离蚊浓核病毒的分子特征研究

对云南省新分离的14株浓核病毒进行基因序列测定和分析,以了解浓核病毒基因组结构及特性.用浓核病毒基因组扩增引物(DNV3F,DNV3R),RT-PCR扩增片段,PCR产物直接测序,拼接后获得基因序列.通过Clustal X(1.8)、DNAStar、GeneDoc (3.2) 等生物学软件进行核苷酸序列及氨基酸序列分析...     

Primary structure and localization of a conserved immunogenic Plasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle.

A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically dispersed isolates, and a fusion protein containing that sequence was able to stimulate B- and T-cells. Antibodies against GLURP stained erythrocytic stages of the parasite as well as the hepatic stage as detected by electron microscopy.     

Molecular cloning and nucleotide sequence of P, M and F genes of Newcastle disease virus avirulent strain D26

Molecular cloning of most if not all of the genome of an avirulent strain D26 of Newcastle disease virus (NDV) was carried out. cDNA clones were aligned by mutual hybridization and restriction map analysis. The nucleotide sequence of 3672 bases which completed the partial sequence of P gene reported in our previous paper (Ishida, N. et al., 1986, Nucleic Acids Res. 14, 6551-6564), and also covered M and F genes, was determined. Each gene contained one long open reading frame which could code for polypeptides of 395, 364, and 553 amino acid residues, respectively. The deduced amino acid sequences of P and M gene products showed little homology to those of other paramyxoviruses. In contrast, comparison of the amino acid sequence of the F gene product revealed highly conserved regions including the amino terminal sequence of the F1 portion following the putative processing site. There was only one basic amino acid residue at the putative processing site, which would explain the low virulence of this strain.     

The correct sequence of the porcine group C/Cowden rotavirus major inner capsid protein shows close homology with human isolates from Brazil and the U.K.

Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins have revealed a region of extreme sequence divergence. We have been unable to confirm the nucleotide sequence of the Cowden VP6 gene corresponding to this region of divergence. Direct sequencing of a PCR-amplified cDNA pool has revealed a frame shift, and three nucleotide changes, within the published sequence of the porcine (Cowden) VP6 gene. The corrected sequence of the porcine protein revealed a closer homology with VP6 from the Bristol strain and two new human group C rotavirus isolates. Atypical rotaviruses have been detected in the feces of children living in Belém, Brazil, and Preston, U.K. Direct sequencing of PCR-amplified cDNA corresponding to the VP6 gene of one isolate from each location confirmed the presence of a group C rotavirus. The complete nucleotide sequences of the VP6 genes from the group C/Belém and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M(r) 44,669 Da). The Belém VP6 gene demonstrated 97.9% nucleotide homology with the human group C/Bristol VP6 gene and 83.4% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. The Preston VP6 gene demonstrated 99.6% nucleotide homology with the human group C/Bristol VP6 gene and 84.0% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. Remarkably, the deduced amino acid sequence of the Brazilian strain was identical to that of the U.K. isolates.