恶性疟原虫红内期145／102kDa抗原的超微结构定位

用LR White低温包埋法及胶体金标记免疫电镜细胞化学技术对保护性单克隆抗体M26-32识别的体外培养的恶性疟原虫FCC1/HN株红内期145/102 kDa抗原进行超微结构定位研究。发现金颗粒主要定位在该株原虫红内期环状体、滋养体、裂殖体及裂殖子的细胞质中;一些金颗粒则定位于原虫表面的复合膜或散布在感染红细胞的细胞质中。表明145/102kDa抗原是恶性疟原虫FCC1/HN株红内期各无性发育阶段的共同细胞质抗原,其一部分可途经原虫表面的复合膜而被转运到感染红细胞的细胞质。     

用单克隆抗体鉴定恶性疟原虫裂殖子表面抗原

用海南省恶性疟原虫Fcc7801/HN作为抗原,制备的抗恶性疟原虫单克隆抗体C6株具有明显的抑制体外恶性疟原虫生长的作用,并且与恶性疟原虫Fcc-1/HN,Fcc7802/HN,Fcc8703/JS和伯氏疟原虫、食蟹猴疟原虫有较广泛的交叉免疫荧光反应。用免疫电镜观察,此株单克隆抗体能识别恶性疟原虫裂殖子表面抗原,用免疫印迹法测得该抗原分子量为71kDa,此抗原有可能成为疟疾疫苗的候选抗原。     

3种恶性疟原虫红内期抗原基因的测序和序列分析

目的 测定我国恶性疟原虫海南株（FCC1／HN）谷氨酸富集蛋白（GARP），丝氨酸重复抗原（SERA）和裂殖子表面蛋白1（MSA1）基因序列，并进行序列分析。方法：采用PCR技术从恶性疟原虫FCC1／HN株基因组DNA中扩增GARP，SERA和MSA1基因片段，分别插入到测序载体上进行测序。应用DNAstar软件辅助分析3种抗原基因的结构及3种抗原在不同恶性疟原虫株间的分化情况。结果 恶性疟原虫FCC1／HN株GARP基因全长2263bp，编码682个氨基酸残茯，谷氨酸占23．61％，包含5个典型的氨基酸重复序列；SERA基因全长3448bp，编码995个氨基酸残基，丝氨酸含量为10．65％，包含1个连续32个丝氨酸（S）残基的序列；MSA1基因全长5085bp，编码1694个氨基酸残基，MSWA1的氨基酸序列符合MAD20特征。恶性疟原虫FCC1／HN株与3D7，FC27株GWARP的序列差异主要集中于C－末端；FCC1／HN株与FCR3，3D7，FCBR，Hondulas-1株SERA的序列差异主要集中于N－端，FCC1／HN株与MAD2，3D7，HN1，HN2，FC27，RO－71RO－33，CAMP和Palo-alto株MSA1的同源性高，K1和WELLCOME株MSA1的同源性高，各分离株MSA1的序列差异主要处于第2至16分区。结论 了解了恶性疟原虫FCC1／HN株GARP，SERA和MSA1的初级结构及其编码基因结构。恶性疟原虫FCC1／HN株与其它分离株GARP和SERA的氨基酸序列差异集中于特定区段，FCC1／HN株MSA1不同分区的氨基酸序列与其它分离株MSA1的对应序列存在不同程度的差异。     

3种恶性疟原虫红内期抗原基因的测序和序列分析

目的　测定我国恶性疟原虫海南株 (FCC1/ HN)谷氨酸富集蛋白 (GARP)、丝氨酸重复抗原 (SERA)和裂殖子表面蛋白 1(MSA1)基因序列 ,并进行序列分析。　方法　采用 PCR技术从恶性疟原虫 FCC1/ HN株基因组 DNA中扩增 GARP、SERA和 MSA1基因片段 ,分别插入到测序载体上进行测序。应用 DNAstar软件辅助分析 3种抗原基因的结构及 3种抗原在不同恶性疟原虫株间的分化情况。　结果　恶性疟原虫 FCC1/ HN株 GARP基因全长 2 2 6 3bp,编码6 82个氨基酸残基 ,谷氨酸占 2 3.6 1% ,包含 5个典型的氨基酸重复序列 ;SERA基因全长 344 8bp,编码 995个氨基酸残基 ,丝氨酸含量为 10 .6 5 % ,包含 1个连续 32个丝氨酸 (S)残基的序列 ;MSA1基因全长 5 0 85 bp,编码 16 94个氨基酸残基 ,MSA1的氨基酸序列符合 MAD2 0型特征。恶性疟原虫 FCC1/ HN株与 3D7、FC2 7株 GARP的序列差异主要集中于 C-末端 ;FCC1/ HN株与 FCR3、3D7、FCBR、Hondulas- 1株 SERA的序列差异主要集中于 N-端。FCC1/ HN株与MAD2 0、3D7、HN1、HN2、FC2 7、RO- 71、RO- 33、CAMP和 Palo- alto株 MSA1的同源性高 ,K1和 WEL L COME株 MSA1的同源性高 ,各分离株 MSA1的序列差异主要处于第 2至 16分区。　结论　了解了恶性疟原虫 FCC1/ HN株 GARP、SERA和 MSA1的     

恶性疟原虫融合抗原PfCP-2.9的单克隆抗体制备及功能分析

目的 制备恶性疟原虫（FCC1/HN株）融合抗原PfCP-2.9的单克隆抗体，分析其生物学特性及功能。 方法 用PfCP-2.9免疫BALB/c小鼠，取其脾细胞及SP2/0骨髓瘤细胞在聚乙二醇（PEG1500）作用下进行融合，制备单克隆抗体，并分析其特性。 结果 获得1株能分泌抗PfCP-2.9的小鼠杂交瘤细胞株单克隆抗体F12D，经免疫球蛋白类型和亚类鉴定为IgG1。ELISA和蛋白质印迹法（Western blotting）显示单克隆抗体F12D能与PfCP-2.9发生特异性反应，F12D所识别的PfCP-2.9抗原表位不能耐受还原剂巯基乙醇，表明F12D识别的是构象表位。间接免疫荧光试验（IFA）显示F12D可识别培养的FCC1/HN。体外抑制试验结果显示，F12D终浓度为0.3 mg/ml时，对FCC1/HN的抑制率为56%。 结论 单克隆抗体F12D能与PfCP-2.9发生特异性反应，其所识别表位为构象表位，F12D可识别体外培养的FCC1/HN，并对其生长具有抑制作用。     

恶性疟原虫海南株AMA-1、Pfs230基因的序列分析

目的　测定恶性疟原虫海南 (FCC1/HN)株裂殖子顶端膜抗原 1(AMA - 1)基因和Pfs2 30基因序列 ,并分别进行序列分析。方法　根据AMA - 1基因已知序列合成一对引物 ,用PCR技术从恶性疟原虫FCC1/HN株基因组DNA中扩增AMA - 1基因 ,构建真核表达重组质粒 pcDNA3 -AMA - 1。根据Pfs2 30基因已知序列合成七对引物 ,分 7段从FCC1/HN株基因组DNA中扩增Pfs2 30基因 ,并分别将扩增片段插入pMD - 18T测序载体。用双脱氧链末端终止法测定克隆的AMA -1、Pfs2 30基因序列 ,应用DNAstar软件辅助进行序列分析和同源性比较。结果　PCR扩增得到恶性疟原虫FCC1/HN株AMA - 1和Pfs2 30基因片段。恶性疟原虫FCC1/HN株AMA - 1基因全长 186 9bp ,无内含子 ,编码 6 2 2个氨基酸残基 ,不存在氨基酸重复序列 ,相对分子量约 72 0 4 5kDa ;Pfs2 30基因全长 94 35bp ,无内含子 ,编码 314 4个氨基酸残基 ,分子量为36 4 36kDa。恶性疟原虫FCC1/HN株与FC2 7、7G8、CAMP、FCR3、Thai -Tn、3D7、FVO、KF1916、CMP1、HB3、K1和V1株AMA - 1的同源性在 94 9%以上 ,各株间有 5 3个位置相同的氨基酸残基替代位点 ,并且发生替代的氨基酸残基具二态性。FCC1/HN株分别比 3D7、7G8株Pfs2 30抗原多 9、10个氨基酸残基 ,三个分离株有 2 8个氨基酸替换     

体外培养恶性疟原虫的Pf155/RESA和其它可溶性抗原的研究

Pf155/RESA.是一种分子量为155kDa耐热的可溶性抗原。该抗原与裂殖子的微线体和棒状体有关,当裂殖子入侵红细胞时沉集于膜上。体外培养恶性疟原虫,Pf155/RESA则释放到培养基中。用恶性疟原虫的坦桑尼亚株进行体外培养,离心收集培养24h后的上清。经免疫吸附柱吸附后洗脱,得到吸附的蛋白,其中包括Pf155/RESA及其相关抗原。每毫升内1～4μg这种蛋白即可抑制环状体感染红细胞表面的免疫荧光。洗脱的蛋白用SDS-PAGE和免疫印渍法分析。结果表明,在分子量为10～250kDa间至少有20种多肽,在有抗原     

恶性疟原虫红内期抗原差异分析

选用一组抗恶性疟原虫红内期裂殖体抗原gp195的单克隆抗体,分析海南省、江苏省恶性疟原虫分离株gp195的抗原差异。间接荧光抗体试验表明,6个恶性疟原虫分离株的gp195抗原既合共同性抗原决定簇,也含特异性抗原决定簇。根据原虫对该组单抗的反应差异,可划分为两个主要的gp195血清型。恶性疟原虫FCC_(8705)/JS为Ⅰ型;FCC/HN为Ⅱ型,FCC_(7802)/HN、FCC_(8702)/HN为Ⅱ_2型; FCC_(7801)/HN、FCC_(8701)/HN为Ⅰ Ⅱ混合型。免疫印迹法显示,恶性疟原虫FCC_(8705)/JS、FCC_1/HN、FCC_(8702)/HN、FCC_(7801)/HN gp195分子量分别为210kD_8、200kD_a、200kD_a、198kD_a。其中江苏FCC_(8705)/JS的gp195分子量高于其它三个海南分离株。作者对gp195抗原差异的规律及其对疟疾疫苗研究的影响作了讨论。     

用单克隆抗体测定从巴布亚新几内亚分离的恶性疟原虫的抗原多样性

作者以单克隆抗体结合荧光染色方法,对巴布亚新几内亚分离的恶性疟原虫(PNG)的初次分离株的抗原性进行了测定。所用单克隆抗体除一株(7.5)识别滋养体和裂殖体抗原,另一株(5.1)识别环状体后各原虫期抗原外,其它都是针对裂殖体和裂殖子的。单克隆抗体反应结果,如呈现荧光的寄生虫≥50形为阳性(+),<50%为阴性(-)。当比较     

恶性疟原虫FCC1／HN株Pf12基因体外扩增、克隆及序列分析

目的：构建恶性疟原虫FCC1／HN株Pf12基因原核表达质粒pET28α-Pf12,测定Pf12基因序列,并为FCC1／HN株Pf12的体外表达奠定基础。方法：采用PCR技术从恶性疟原虫FCC1／HN株基因组DNA中扩增出Pf12基因,扩增产物经纯化,用BamHI XhoI双酶切,定向克隆入pET28α质粒,转化大肠杆菌DH5α,再用BamHI XhoI酶切及PCR扩增对重组子进行鉴定。用Sanger双脱氧链终止法进行DNA序列测定,并应用PCGENE软件进行同源性比较及预测抗原表位。结果：筛选出编码FCC1／HN株Pf12基因的原核表达质粒pET28α－Pf12重组质粒的构建,为恶性疟原虫FCC1／HN株Pf12基因的体外表达奠定基础。     

Expression of Plasmodium falciparum surface antigens in Escherichia coli.

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.     

Ultrastructural localization of protective antigens of Plasmodium yoelii merozoites by the use of monoclonal antibodies and ultrathin cryomicrotomy

The production of two hybridoma cell lines secreting monoclonal antibodies (MAb), both of which react specifically with erythrocytic merozoites of Plasmodium yoelii in the indirect immunofluorescence assay, has been reported earlier. MAb 25.77 was reactive with a localized region within each merozoite, while MAb 25.1 appeared to be specific for the plasma membrane of schizonts and merozoites. The parasite antigens recognized by antibodies 25.77 and 25.1 are proteins of 235,000 and 230,000 molecular weight, respectively, both of which induce protective immunity against P. yoelii in mice. In order to establish the precise localization of these protective antigens within erythrocyte merozoites, ultrathin cryomicrotomy was used in conjunction with the MAb and protein A-gold. This technique showed that gold particles were exclusively concentrated over the rhoptries when erythrocytic merozoites were incubated with MAb 25.77. On the other hand, gold particles were distributed uniformly over the merozoite surface when parasites were incubated with MAb 25.1. These results demonstrate, for the first time, that a protective antigen of the erythrocytic stage of P. yoelii is localized within the rhoptries as well as on the merozoite surface.     

Rhoptry secretion of membranous whorls by Plasmodium falciparum merozoites

Multilamellar membranous whorls were localized, by electron microscopy, in elements of the rhoptry-microneme complex from glutaraldehyde-tannic acid (TA)-fixed merozoites of the human malarial parasite, Plasmodium falciparum. These multilaminate structures, which have a dark line-to-dark line periodicity of approximately 5 nm, were also found in the nuclear envelope and closely apposed to the external surface of merozoites. Segmented schizonts, which contain intracellular merozoites, often showed membranous whorls within their parasitophorous vacuoles and closely apposed to the external surface of the parasitophorous vacuole membrane. Whorls were not found in trophozoites, immature schizonts, and uninfected erythrocytes. Most rhoptries in merozoites fixed in glutaraldehyde-TA were electron-lucent whereas rhoptries fixed in glutaraldehyde alone were electron-dense. Some merozoites fixed in glutaraldehyde-TA had both an electron-dense and an electron-lucent rhoptry. These findings suggest that TA induces the premature extrusion of rhoptry materials. Our findings support previous suggestions in the literature that phospholipid materials secreted from merozoite rhoptries are involved in merozoite interaction with host erythrocytes.     

Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease

Compartmentalization of proteins into subcellular organelles in eukaryotic cells is a fundamental mechanism of regulating complex cellular functions. Many proteins of Plasmodium falciparum merozoites involved in invasion are compartmentalized into apical organelles. We have identified a new merozoite organelle that contains P. falciparum rhomboid-1 (PfROM1), a protease that cleaves the transmembrane regions of proteins involved in invasion. By immunoconfocal microscopy, PfROM1 was localized to a single, thread-like structure on one side of the merozoites that appears to be in close proximity to the subpellicular microtubules. PfROM1 was not found associated with micronemes, rhoptries, or dense granules, the three identified secretory organelles of invasion. Release of merozoites from schizonts resulted in the movement of PfROM1 from the lateral asymmetric localization to the merozoite apical pole and the posterior pole. We have named this single thread-like organelle in merozoites, the mononeme.     

Immunization against Plasmodium falciparum with recombinant polypeptides produced in Escherichia coli

Two proteins produced in recombinant Escherichia coli and containing amino acid sequences from the Plasmodium falciparum precursor to major merozoite surface antigens (PMMSA) have been partially purified. These proteins, together with a preparation of merozoites, have been used to immunize animals. The antibody response and the degree of protection were compared. Animals immunized with merozoites produced antibodies reacting with many P. falciparum proteins, whereas a response specific for PMMSA was detected in those receiving the recombinant material. Incomplete protection was conferred to both groups and there was no apparent correlation between antibody levels and protection.     

P195蛋白及其抗体抑制恶性疟原虫裂殖子入侵人红细胞的研究

目的寻找恶性疟原虫裂殖子表面主要蛋白Ｐ１９５中的红细胞结合位点,为设计疫苗阻断裂殖子入侵红细胞提供实验依据。方法在大肠杆菌中分８段表达Ｐ１９５蛋白。各段蛋白用镍亲和层析柱分离,然后复性。将得到的各段蛋白免疫家兔,制备抗血清。在体外培养疟原虫至成熟裂殖体期,将各段蛋白及其相应的抗血清分别加入到培养基上清中,继续培养２４小时,检查红细胞感染率。通过感染率了解各段蛋白及其抗体对裂殖子入侵红细胞的影响。结果Ｐ１９５蛋白中氨基酸序列为３８３～５９５（Ｍ６）,５９５～８９７（Ｍ７）,１３９７～１６６３（Ｍ１１）的三段蛋白的抗血清具有抑制裂殖子入侵红细胞的作用,而其中Ｍ６蛋白片段也具有抑制裂殖子入侵红细胞的作用。结论Ｐ１９５蛋白中氨基酸序列为３８３～５９５的一段序列,Ｍ６可能含恶性疟原虫识别人红细胞的位点,该位点可以作为疟疾疫苗的候选抗原。     

Stage-specific proteins and glycoproteins of plasmodium falciparum: identification of antigens unique to schizonts and merozoites.

Establishment of highly synchronized cultures of Plasmodium falciparum enabled identification of stage-specific proteins, glycoproteins, and antigens. Comparison of metabolically labeled constituents of rings, trophozoites, mature schizonts, and merozoites indicated the absence of major proteins or glycorproteins unique to rings or trophozoites. A burst of new synthetic activity occurred during schizogony when several schizont- and merozoite-specific proteins and glycoproteins became apparent. In addition to the knob protein, which was previously shown to be associated with protrusions on the host erythrocyte membrane, a major glycoprotein of parasite origin was identified on the surface membrane of schizonts. Analysis of antigens solubilized from different developmental stages indicated that immune sera, which inhibit growth of parasites in vitro, react mainly with merozoite- and schizont-associated antigens.     

A protective merozoite protein of Plasmodium falciparum shares an epitope with surface antigens of Paramecium

A Plasmodium falciparum cDNA expression clone, lambdaPf9, had been identified earlier as a protective epitope, using anti-lambdaPf9 antibodies and combinatorial phagotopes. A segment of the Pf9 gene showed homology with Paramecium immobilization surface antigens such as 51B, 51A and 156G. A synthetic Pf9-peptide was designed from this region, and specific antibodies were raised. Each of these anti-Pf9 antibodies and combinatorial reagents, as well as anti-Paramecium 51B antibodies, recognized the Pf9-peptide on ELISA, and the same protein band in parasite immunoblots. The P. falciparum protein was released from the merozoite membrane fraction on treatment with PI-PLC, indicating the presence of a GPI anchor. Anti-Pf9-peptide antibodies specifically inhibited the growth of P. falciparum in culture. Immunofluorescence assays showed the reactivity of anti-Pf9-peptide sera with P. falciparum merozoites and gametocytes, as well as on the surface of Paramecium tetraurelia. The Pf9-peptide was able to induce proliferation of splenic lymphocytes obtained from mice infected with the rodent malarial parasites Plasmodium berghei and Plasmodium yoelii. These results point towards Plasmodium Pf9 as a conserved novel protective protein, sharing an epitope with Paramecium surface antigens.