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首页> 外文期刊>Infection and immunity >Molecular cloning of a serine proteinase inhibitor from Brugia malayi.
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Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

机译：来自马来西亚马齿Bru的丝氨酸蛋白酶抑制剂的分子克隆。

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The antigens produced by the infective-stage larvae of filarial parasites are potentially important targets for a protective immune response. A major impediment to studies on the biochemistry and molecular biology of antigens from infective larvae is a lack of parasite material. By employing a reverse transcription PCR-based strategy which exploited the presence of a conserved 22-nucleotide spliced leader sequence present at the 5' end of a proportion of nematode transcripts, spliced leader-containing cDNAs were amplified from the late-vector-stage larvae of the filarial nematode Brugia malayi. A major 1.4-kb PCR product was cloned into pBluescript. One of the PCR cDNA clones (BmY8) contained a 1,287-bp insert that encoded the first member of the serine proteinase inhibitor (serpin) superfamily to be described from nematodes. Reverse transcription PCR analysis of RNA isolated from different developmental stages of the parasite showed that transcription of the B. malayi serpin (Bmserpin) begins between days 8 and 9 of larval development within the insect vector and continues through to the adult and microfilarial stages. In immunoblot analyses of B. malayi somatic extracts, the native protein was estimated to have a molecular weight of 44,000. In immunoblots using excretory-secretory products from infective- and fourth-stage larvae, a single band with an estimated molecular weight of 75,000 was detected. A quantitative analysis of somatic extracts demonstrated that infective-stage larvae contained 10- to 16-fold-more Bmserpin than adults or microfilariae. Bmserpin was immunogenic in gerbils and was recognized strongly by sera from immunized animals. Bmserpin, which has the potential for modifying host defense responses, may play an important role in parasite survival during the early phase of vertebrate-stage development.
机译：由丝虫寄生虫的感染期幼虫产生的抗原可能是保护性免疫反应的重要目标。感染幼虫抗原的生物化学和分子生物学研究的主要障碍是缺乏寄生物。通过采用基于逆转录PCR的策略，该方法利用了在一定比例的线虫转录本的5'端存在的保守的22个核苷酸的剪接的前导序列的存在，从后期载体阶段的幼虫中扩增了含剪接的前导cDNA。丝虫线虫马来亚。将1.4kb的主要PCR产物克隆到pBluescript中。一个PCR cDNA克隆（BmY8）包含一个1,287 bp的插入片段，该插入片段编码了线虫中要描述的丝氨酸蛋白酶抑制剂（serpin）超家族的第一成员。从该寄生虫不同发育阶段分离的RNA的反转录PCR分析表明，马来芽孢杆菌（Bmserpin）的转录在昆虫载体内幼虫发育的第8天至第9天之间开始，一直持续到成虫和微丝虫阶段。在马来芽孢杆菌体细胞提取物的免疫印迹分析中，天然蛋白质的分子量估计为44,000。在使用感染和第四阶段幼虫的分泌分泌产物的免疫印迹中，检测到一条估计分子量为75,000的单条带。体液提取物的定量分析表明，感染期的幼虫比成人或微丝虫病含有10到16倍多的Bmserpin。 Bmserpin在沙鼠中具有免疫原性，并被免疫动物的血清强烈识别。 Bmserpin具有修饰宿主防御反应的潜力，可能在脊椎动物发育早期的寄生虫存活中发挥重要作用。

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