A Trial to Develop Peptide Vaccine for Strongyloidiasis

由于粪类圆线虫的可利用性有限以及抗原的复杂性，最初我们的基因克隆目标集中在嗜中性粒细胞趋化因子（NCF）。ratti（Sr）.采用DE 52离子交换柱层析和SW 3000高效液相色谱或Procion Red亲和层析相结合的方法，得到了单一蛋白峰的NCF。然而，通过SDS-PAGE，该峰仍然是异质的。此外，Sr-L_3的cDNA文库构建也失败了，仅构建了约5000个克隆。因此，作为一种替代方法，使用犬恶丝虫（Di）NCF作为起始材料，因为它已经由我们的手纯化，并且通过Western印迹分析，Di-NCF表位在Sr的几种抗原组分中表达，包括Sr-NCF。从Di蠕虫中提取Poly-A-RNA，并将cDNA插入到AgdagT 11的EcoRI位点。通过使用PCR进一步选择作为cDNA文库产生的约2 × 10^5个克隆。 ...更多信息 抗Di-NCF。纯化阳性克隆的噬菌体DNA，用EcoRI片段化，将具有0.7- 2.0Kb插入片段的克隆亚克隆到质粒载体BluescriptSK（-）中。用M13引物双脱氧法测定其DNA序列。同时，通过测序仪测定纯化的Di-NCF的N-末端45个氨基酸序列。当将来自四个亚克隆的DNA序列的预期氨基酸序列与实际N-末端氨基酸序列进行比较时，命名为pD 4的亚克隆在5'末端的268-402的DNA序列处显示出完全同源性。开放阅读框为166-606，编码34个氨基酸的前导序列和112个氨基酸的Di-NCF序列。通过计算机同源性搜索，发现99-101个氨基酸序列（Met-Phe-Lys）与已知的NCF肽段具有相似性，因此认为该序列是Di-NCF活性位点的表位。事实上，合成的Met-Phe-Lys以及fMet-Phe-Lys在低于已知NCF肽的浓度下显示出NCF活性。将克隆pD 4亚克隆到质粒载体pGEM EX中，并产生与基因10的融合蛋白。该融合蛋白具有与纯化的Di-NCF相似的NCF活性和抗原性。当用该融合蛋白免疫小鼠时，它们对巴氏丝虫的易感性反而增加，这表明诱导了封闭抗体或抑制系统。需要进一步研究免疫接种的方法。少

Because of limitted availability of Strongyloides stercoralis and because of antigenic complexity, initially our aim of gene cloning was focused on the neutrophil chemotactic factor (NCF) of S. ratti (Sr). By using combination of DE52 ion exchange colum chromatography and SW3000 HPLC or Procion Red affinity chromatograophy, we obtained NCF as a single protein peak. However, this peak was still heterogenous by SDS-PAGE. Furthermore, an attempt to make cDNA library was failed because only about 5000 clones were established from mRNA of Sr-L_3. As an alternative approach, therefore, Dirofilaria immitis (Di) NCF was used as the stating material because it has been already purified by our hand, and Di-NCF epitopes were expressed in several antigenic components of Sr including Sr-NCF by Western-blot analysis. Poly-A-RNA was isolated from Di adult worms and the cDNAs were inserted into EcoRI site of lambdagT11. About 2 x 10^5 clones produced as the cDNA library were further selected by using … More anti-Di-NCF. The phage DNAs of positive clones were purified, fragmented by EcoRI, and clones having 0.7-2.0 Kb inserts were subcloned in to a plasmid vector Bluescript SK(-). Their DNA sequences were determined by dideoxy method using M13 primer. In parallel, N-terminal 45 amino acid sequence of purified Di-NCF was determined by antomatic sequencer. When the expected amino acid sequences from the DNA sequences of four subclones were compared to the actual N-terminal amino acid sequences, subclone named pD4 showed complete homology at the DNA sequence of 268-402 from 5' terminal. Open rcading frame was 166-606 coding the leader sequence of 34 amino acid residues and the Di-NCF sequence of 112 amino acid recidues. By comoputer -aided homology search, 99-101 amino acid sequence (Met-Phe-Lys) had a similarity to known NCF peptide so that this sequence was thougt as the epitope of active site of the Di-NCF. In fact, synthesised Met-Phe-Lys and also fMet-Phe-Lys showed NCF activity at the concentrations lower than of known NCF peptides. The clone pD4 was subcloned into plasmid vector pGEM EX and fusion protein with gene 10 was produced. This fusion protein showed NCF activity and antigenicity similar to those of purified Di-NCF. When mice were immunized with this fusion protein, their susceptibility against Brugia pahagi rather increased, suggesting the induction of blocking antibody or suppressor system. Further study is required as to the methods of immunization. Less