A Trial to Develop Peptide Vaccine for Strongyloidiasis

类圆线虫病肽疫苗的开发试验

基本信息

批准号：
02557022
负责人：
NAWA Yukifumi
金额：
$ 2.82万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

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

由于粪类圆线虫的可用性有限且由于抗原的复杂性，最初我们的基因克隆目标集中于鼠类圆线虫（Sr）的中性粒细胞趋化因子（NCF），通过使用DE52离子交换柱色谱和SW3000 HPLC或相结合。 Procion Red亲和层析，我们得到了NCF作为单一蛋白峰，但是通过SDS-PAGE，该峰仍然是异质的。此外，由于仅从Sr-L_3的mRNA建立了大约5000个克隆，因此尝试制作cDNA文库失败了。因此，作为替代方法，使用恶丝虫(Di)NCF作为起始材料，因为它已经被纯化。通过蛋白质印迹分析，从 Di 成虫中分离出 Poly-A-RNA，并在 Sr 的几种抗原成分（包括 Sr-NCF）中表达了 Di-NCF 表位。插入到 lambdagT11 的 EcoRI 位点，使用抗 Di-NCF 进一步筛选出约 2 x 10^5 个克隆，将阳性克隆的噬菌体 DNA 进行纯化，通过 EcoRI 进行片段化，得到具有 0.7- 的克隆。将 2.0 Kb 插入片段亚克隆到质粒载体 Bluescript SK(-) 中，使用 M13 引物通过双脱氧方法平行测定它们的 DNA 序列。通过自动测序仪测定了纯化的 Di-NCF 的 N 端 45 个氨基酸序列，当将四个亚克隆 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时，名为 pD4 的亚克隆在5'端开放编码框268-402的DNA序列是编码34个氨基酸残基的前导序列和Di-NCF序列的166-606。通过计算机辅助同源性搜索，发现112个氨基酸序列(Met-Phe-Lys)与已知的NCF肽有相似性，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-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pD4亚克隆到质粒载体pGEM EX中，产生具有基因10的融合蛋白，该融合蛋白表现出与纯化的Di-NCF相似的NCF活性和抗原性，当用该融合蛋白免疫小鼠时，它们对布鲁氏布鲁氏菌具有敏感性。相反增加，表明阻断抗体或抑制系统的诱导需要进一步研究免疫方法。