Beerntsen B T, Severson D W, Christensen B M
Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison 53706.
Exp Parasitol. 1994 Nov;79(3):312-21. doi: 10.1006/expr.1994.1094.
We report on the initial characterization of an 84-kDa polypeptide that is differentially expressed in Aedes aegypti during melanotic encapsulation immune reactions against filarial worms. [35S]Methionine-labeled hemolymph from mosquitoes inoculated with saline, parasites that are melanized, or parasites that are not melanized was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results show that the level of the 84-kDa polypeptide increases considerably in those mosquitoes undergoing encapsulation reactions against parasites but remains down-regulated in those mosquitoes exposed to parasites that are not melanized or are undergoing wound healing responses (saline-inoculated). Experiments involving glycosidase treatment of hemolymph samples indicate that this polypeptide is not heavily glycosylated. Amino acid microsequencing was performed and two internal sequence fragments (15 continuous amino acids and 12 noncontinuous amino acids) were obtained. Analysis of these sequences to known sequences in a protein database did not yield any conclusive information as to the identify of the 84-kDa polypeptide. Therefore, degenerate oligonucleotide primers were designed, based on the sequence of the 15-amino-acid fragment, and used with the polymerase chain reaction (PCR) to amplify from A. aegypti genomic DNA the region between the primers. The PCR product was cloned and sequenced to verify that the nucleic acid sequence matched the known protein sequence. Screening of an A. aegypti cDNA library with this small PCR-generated clone resulted in the selection of an approximately 540-bp clone. Northern analysis with this larger cDNA clone indicates that it hybridizes to an approximately 2.0-kb message that is differentially expressed in mosquitoes undergoing melanotic encapsulation reactions against filarial worms. Furthermore, sequencing of this approximately 540-bp clone showed that it contained the 15-amino-acid sequence that had been used to design the degenerate PCR primers, indicating that an appropriate clone was selected. However, sequence analysis of this clone at the protein and nucleic acid level did not provide any conclusive answers to the identity or function of the 84-kDa polypeptide.
我们报道了一种84 kDa多肽的初步特征，该多肽在埃及伊蚊针对丝虫的黑化包囊免疫反应中差异表达。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析了用盐水、黑化寄生虫或未黑化寄生虫接种的蚊子的[35S]甲硫氨酸标记的血淋巴。结果表明，在针对寄生虫进行包囊反应的蚊子中，84 kDa多肽的水平显著增加，但在暴露于未黑化寄生虫或正在进行伤口愈合反应（接种盐水）的蚊子中，该多肽水平仍下调。涉及对血淋巴样品进行糖苷酶处理的实验表明，该多肽糖基化程度不高。进行了氨基酸微测序，获得了两个内部序列片段（15个连续氨基酸和12个不连续氨基酸）。将这些序列与蛋白质数据库中的已知序列进行分析，未得到关于84 kDa多肽身份的确切信息。因此，根据15个氨基酸片段的序列设计了简并寡核苷酸引物，并用于聚合酶链反应（PCR）从埃及伊蚊基因组DNA中扩增引物之间的区域。对PCR产物进行克隆和测序，以验证核酸序列与已知蛋白质序列匹配。用这个小的PCR产生的克隆筛选埃及伊蚊cDNA文库，得到了一个约540 bp的克隆。用这个较大的cDNA克隆进行Northern分析表明，它与一个约2.0 kb的信使RNA杂交，该信使RNA在针对丝虫的黑化包囊反应的蚊子中差异表达。此外，对这个约540 bp克隆的测序表明，它包含用于设计简并PCR引物的15个氨基酸序列，表明选择了合适的克隆。然而，对该克隆在蛋白质和核酸水平上的序列分析并未为84 kDa多肽的身份或功能提供任何确凿答案。
