A Trial to Develop Peptide Vaccine for Strongyloidiasis

由于粪圆形线虫的可用性有限，并且由于抗原的复杂性，最初我们的目标是克隆S. ratti （Sr）的中性粒细胞趋化因子（NCF）。采用DE52离子交换柱层析与SW3000高效液相色谱或Procion Red亲和层析相结合，得到NCF为单个蛋白峰。但SDS-PAGE显示该峰仍为异质峰。此外，由于Sr-L_3的mRNA仅建立了约5000个克隆，因此未能建立cDNA文库。因此，作为一种替代方法，我们使用免疫Dirofilaria immitis (Di) NCF作为实验材料，因为它已经被我们的手纯化，并且通过Western-blot分析，Di-NCF表位在Sr的几个抗原组分中表达，包括Sr-NCF。从Di成虫中分离Poly-A-RNA，并将其插入到lambdagT11的EcoRI位点。利用…More anti-Di-NCF进一步筛选了2 × 10^5个克隆作为cDNA文库。纯化阳性克隆的噬菌体dna，用EcoRI进行片段化，将插入片段为0.7-2.0 Kb的克隆亚克隆到质粒载体Bluescript SK（-）上。用M13引物双脱氧法测定其DNA序列。同时，用自动测序仪测定纯化的Di-NCF的n端45个氨基酸序列。将4个亚克隆DNA序列的预期氨基酸序列与实际n端氨基酸序列进行比较，发现pD4亚克隆与5′端268-402的DNA序列完全同源。开放阅读框为166 ~ 606，编码34个氨基酸残基的leader序列和112个氨基酸残基的Di-NCF序列。通过计算机辅助同源性检索，发现99-101个氨基酸序列（met - ph - lys）与已知的NCF肽具有相似性，因此该序列被认为是Di-NCF活性位点的表位。事实上，合成的met - ph - lys和fmet - ph - lys在低于已知NCF肽的浓度下显示出NCF活性。将克隆体pD4亚克隆到质粒载体pGEM EX中，获得了与基因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。用该融合蛋白免疫小鼠后，小鼠对Brugia pahagi的易感性反而增加，提示其诱导了阻断抗体或抑制系统。需要对免疫方法作进一步研究。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