Heat shock cognate 70 is a prominent immunogen in Brugian filariasis.
Heat shock cognate 70 is a prominent immunogen in Brugian filariasis.
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DOI：
10.4049/jimmunol.143.1.299
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发表时间：
1989-07
影响因子：
4.4
通讯作者：
M. Selkirk;D. Denham;F. Partono;R. Maizels
中科院分区：
文献类型：
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作者：
M. Selkirk;D. Denham;F. Partono;R. Maizels
利用印度尼西亚丹中槟槟岛人淋巴丝虫病慢性非微丝虫病病例的混合血清筛选成虫期（雌雄混合）的RNA构建cDNA表达文库，马来丝虫在印尼丹中槟槟岛流行。从两个最具活性的克隆中提取纯化的β -半乳糖苷酶融合蛋白的多克隆抗血清在马来芽孢杆菌和pahangi Brugia的所有阶段（微丝虫，L3和成虫）中鉴定出Mr为70,000的蛋白。从这两个重叠的cdna中衍生出氨基酸序列，鉴定出编码的蛋白是热休克蛋白70家族的成员，并且与组成性表达的“热休克同源70”（hsc70）蛋白最相似。在严格的条件下，将hsc70 cdna与pahangi的RNA和DNA杂交，鉴定出一个2.4 kb的主要转录本，并发现了一个相关基因家族的存在。在高温（43℃）下体外培养pahangi芽孢杆菌幼虫期，hsc70的表达增加，并产生典型的热休克反应，其中5种蛋白（mr 18,500, 22,000, 62,000, 70,000和85,000）仅在微丝虫体中合成。Western blotting交叉反应分析表明，马来芽孢杆菌感染后产生的抗体可靶向布鲁氏菌hsc70的丝状特异性决定因子。然而，用恶性疟原虫和曼氏血吸虫hsc70的重组融合蛋白进行ELISA检测显示，一些布鲁氏丝虫病或班氏丝虫病患者确实产生了与疟原虫和血吸虫同源物交叉反应的抗体。因此，并非所有个体都产生丝状特异性抗体反应，表明该分子不适合用于诊断目的。ELISA纯化的pahangi b -半乳糖苷酶融合蛋白在丝虫病的临床谱中显示了对hsc70的抗体反应。对pahangi、P. falciparum、S. mansoni和大鼠hsc70同源基因衍生的氨基酸序列进行比对，并通过Western blotting和ELISA对两个cDNA克隆产物的免疫反应性进行比较，结果表明这些决定因素主要位于蛋白的C端。
A cDNA expression library constructed from RNA derived from adult stage Brugia pahangi (mixed sexes) was screened with pooled sera from chronic, amicrofilaremic cases of human lymphatic filariasis from the Indonesian island of Tanjungpinang, where Brugia malayi is endemic. Polyclonal antisera raised to purified beta-galactosidase fusion proteins from two of the most highly reactive clones identified a protein of Mr 70,000 in all stages examined (microfilariae, L3 and adults) of both B. malayi and Brugia pahangi. Derivation of the amino acid sequence from these two overlapping cDNAs identified the encoded protein as a member of the heat shock protein 70 family, and showed the closest similarity to the constitutively expressed "heat shock cognate 70" (hsc70) protein. Hybridization of hsc70 cDNAs to RNA and DNA from B. pahangi under stringent conditions identified a major transcript of 2.4 kb and revealed the existence of a family of related genes. In vitro culture of larval stages of B. pahangi at elevated temperatures (43 degrees C) resulted in increased expression of hsc70, and a classic heat shock response in which five proteins (mr 18,500, 22,000, 62,000, 70,000, and 85,000) were exclusively synthesized in microfilariae. Analysis of cross-reactivities by Western blotting implied that antibody generated by infection with B. malayi was directed at filarial-specific determinants of Brugia hsc70. However, ELISA with recombinant fusion proteins for both Plasmodium falciparum and Schistosoma mansoni hsc70 indicated that some individuals with Brugian or Bancroftian filariasis did produce antibodies which cross-reacted with plasmodial and schistosomal homologs. Thus filarial-specific antibody responses were not generated in all individuals, indicating that this molecule would not be suitable for diagnostic purposes. ELISA with a purified beta-galactosidase fusion protein from B. pahangi showed antibody responses to hsc70 across the clinical spectrum of filariasis. Alignment of the derived amino acid sequences from B. pahangi, P. falciparum, S. mansoni and rat hsc70 homologs, and comparison of the immunologic reactivity of the products of the two cDNA clones by Western blotting and ELISA suggested that these determinants were located primarily at the C terminus of the protein.