间日疟原虫中国分离株传播阻断疫苗候选抗原Pvs25的基因多态性分析

【作者】 郑丽；

【导师】 曹雅明；

【作者基本信息】 中国医科大学 ， 免疫学， 2003， 硕士

【摘要】 前言 疟疾是疟原虫通过媒介昆虫蚊传播的人类最严重的寄生原虫感染性疾病。每年，全世界约有5亿人感染疟疾，近200万人因其死亡，其中大部分为儿童及妊娠妇女。近年来，由于耐药性虫株及耐杀虫剂性蚊的普遍出现，使疟疾的流行进一步恶化，防疟、抗疟形势更为严峻。因此，开发有效的疟疾疫苗已成为当今疟疾防治工作迫切需要解决的课题。 疟疾疫苗研制的障碍主要在于疟原虫复杂的生活史和广泛的抗原变异性。因此，有效的特异性疫苗候选抗原的确定已成为疫苗研制开发的关键。传播阻断疫苗是目前控制疟原虫传播的一种重要战略措施。与红前期和红内期疫苗相比，传播阻断疫苗的突出特点在于它具有有限的抗原多态性和较强的免疫原性。Pvs25是间日疟原虫动合子表面表达的一种主要蛋白，对动合子在蚊中肠中的存活与发育起着重要的作用。该分子目前被认为是研制和开发传播阻断疫苗首选的疫苗候选抗原。但在研究过程中也发现Pvs25在基因水平上存在着一定的多态性，不同地理株之间基因多态性存在地理差异性。这种基因多态性的存在是否影响疫苗实际应用的效果，目前尚不十分清楚。而作为间日疟原虫广泛流行的我国来说，Pvs25基因多态性的基础研究目前尚未开展。 基于以上原因，本论文对我国间日疟原虫单纯流行区湖北、浙江两省31例间日疟原虫分离株的Pvs25全长基因进行了多态性分析，同时与来自孟加拉的分离株进行比较，为间日疟原虫传播阻断疫苗的研制和应用提供必需的前提依据。 材料与方法 在疟疾感染流行季节，采集我国间日疟原虫单纯流行区湖北和浙江两省31例疟原虫镜检阳性患者血样，与日本爱媛大学分子寄生虫学教研室坪井敬文教授惠赠的14例孟加拉分离株一同，提取寄生虫基因组DNA。以基因组DNA为模板，选取基因特异性弓物（Pvs25 primer sense：5’－CAC 17’x GCC AAA ATG AAC TC－3’；anti－sense：5’－AAA GGA CAA GCA GGA TGA TA－3’）。在KOD－PISS聚合酶作用下通过聚合酶链式反应付CR）扩增PVS25基因的全长片段，所得PCR产物直接测序，计算机演绎法进行序列分析（软件）。 实 验 结 果 1．本试验获得的PVs25基因全长646BP，编码对 个氨基酸。 2．在核耷酸水平我们共发现3处点突变，并且均为错义突变（G／C289，T／C389，C／A391人在氨基酸水平，共发现三处氨基酸替换门／Q97，I／T130，Q／K131人其中 Q／K131这一替换仅见于孟加拉分离株。 3·核盲酸改变共产生 4种基因型（G’‘’－C”’－C”‘，C‘”－C’明 C”‘c” C’” A”‘C田’C’‘’一 A”‘）其中 G田’一 C’‘’一C”’是一个新发现的基因型，仅见于中国分离株，并且为中国分离株的主导基因型。 4．核耷酸多态性。值显示，45例疟原虫分离株核耷酸多态性、值为 0．001 17株；中国分离株。值为 0．00050；孟加拉分离株。值为0．00 33；在中国流行区内部，浙江分离株、值为0．00024，湖北分离株。值为0．00066。这些结果提示PvS25基因在不同地 ·2·理株间（湖北、浙江、孟加拉三处比较）或同一地域不同分离株之间基因多态性十分有限，各流行区无显著性差异。 讨 论 本研究首次对我国间日疟原虫单纯流行区分离株传播阻断疫苗候选抗原PvS25的基因多态性特点进行报道。实验结果显示，PvS25基因在不同地理株间（湖北、浙江、孟加拉三处比较）或同一地域不同分离株之间基因多态性十分有限，在各流行区无显著性差异，进一步证实了PvS25是间日疟原虫传播阻断疫苗理想的候选抗原。 传播阻断疫苗是目前控制疟原虫感染的一种重要战略措施。它以蚊阶段虫体特异表达的表面蛋白作为抗原免疫人体，使之产生抗蚊阶段虫体表面蛋白的特异性抗体。蚊吸人被免疫的人血后，蚊胃内发育的虫体与人体内产生的抗体发生抗原抗体反应，从而阻断疟原虫在蚊体内的发育。与其它两种疟疾疫苗即红前期和红内期疫苗相比，传播阻断疫苗鲜明的特点在于抗原变异少，这一发现对疟疾疫苗的研制和开发具有重要的意义。众所周知，疟疾疫苗研制的主要障碍在于疟原虫复杂的生活史和广泛的抗原变异性。因此，候选抗原具有有限抗原变异性这一优点的传播阻断疫苗将具有广阔的发展前景。传播阻断疫苗与其他阶段疫苗或抗疟药物联合应用可能会成为防疟、抗疟的主导措施。 PvS25是间日疟原虫蚊发育阶段合子和动合子表面特异性表达的一种主要蛋白。hUbd等于1998年首次对印度等4例间日疟原虫分离株PvS25基因进行了克隆与序列分析，共发现3处点突变。本研究首次将样本扩展至45例，发现3处点突变，与Tsub一份等的研究结果吻合。统计学分析也脱各不同地理株间基因多态性无显著性差异。TSuboi等于2000年进一步通过问日疟原虫 ·3·实验室S＊1株构建的Pv￡5重组蛋白免疫实验动物小鼠，在小鼠体内产生更多还原

【Abstract】 Malaria constitutes a major human health threat caused by infection with protozoan parasites of the genus Plasmodium. P. vivax represents the most widespread malaria parasite in the world. Malaria also remains one of the leading parasite diseases in China, located in 18 provinces / autonomous regions / municipalities with a coverage of 0. 53 billion population. As the relaping behaving of vivax malaria process and parasite strains resistant to anti - malarial drugs as well as mosquito vectors resistant to insecticides have emerged, new approaches for malaria control are badly needed and intensive research efforts have gone to develop malaria vaccines.Although different complementary approaches to vaccine development are being undertaken in several laboratories, the major obstacle confronting the development of an effective malaria vaccine is the extensive antigenic diversity in natural populations of malaria parasites. Transmission - blocking vaccines (TBVs ) are becoming more attractive because of their limited genetic polymorphism. Pys25 is the ooki-nete surface protein, which plays a role in ookinete survival in the mosquito midgut. Some results have indicated that Pvs25 can be the leading vivax malaria TBV candidate.Although these efforts have been undergone for Pvs25, the data is still limited not enough to identify the feasibility for the application of this kind of TBV. Some results have suggested that the polymorphism of target antigen exists among field isolates, even single amino acidsubstitution can change the primary structure of parasite antigen. This may have an adverse impact on the efficacy of vaccines application. Therefore, it is necessary to acquire the evaluation of the pre - existing polymorphism in Pvs25 before applying it to the vivax malaria endemic regions. Otherwise, P. vivax is popular in extensive areas of China, but there is no report to be published about this study. For these purposes, we provided a detailed analysis of the diversity about this malaria vaccine candidate antigen isolates from Hubei and Zhe-jiang province of China and compared it with parasite population from Bangladesh, which would apply a necessary basic data for the development and application of TBVs against malaria.MATERIALS AND METHODSThe parasite DNA used for the genetic polymorphism assay was obtained from dried filter paper blood spots as described by Sakihama and others. The blood samples were from 45 P. vivax infecting patients during malaria transmission season. As a positive control, ge-nomic DNA extracted from P. vivax Sal 1 strain was used. The genes were PCR amplified and the amplified products were purified, cloned, and sequenced. Both strands were sequenced from at least two clones from two independent PCR amplifications. Computer - based algorithms were used to analyze neucleotide and amino acid sequences and the sequence alignments were done with Mac Vector software. Intrap-opulation and interpopulation diversity indexes were calculated by u-sing DnaSP software.RESULTS1. The Pvs25 gene sequence for 646 nucleotides codes 215 amino acids, which include the complete Pvs25 sequence.2. Three non - synonymous mutations ( G/C289, T/C389, C/ A391) and no synonymous mutations were identified, which were the same with precious report.3. Two conserved (E/Q97, Q/K131) and one half - conserved (I/T130) amino acid substitutions were identified. E/Q97 was located in the second EGF - like domain of Pvs25, while I/T130 and Q/ K131 were located in the third one. The mutation (C/A391) produce the amino acid substitution (Q/K131) , which was only found in Bangladesh isolates.4. These polymorphic nucleotides generated a total of 4 haplo-types (G289-C38-C391,C289-C389- C391,G289-C389-A 391,C289-C389 - A391 ) among the 45 sequences. Haplotypes G289 - C389 - C391 from Chinese isolates was not preciously reported and can be considered predominant in China whose frequency was 80. 6% .5. The nucleotide diversity () in the China samples was 0. 00050, compared to 0.更多还原