Jezek Jan, El Ridi Rashika, Salah Mohamed, Wagih Amal, Aziz Haidy W, Tallima Hatem, El Shafie Mohamed H, Khalek Tarek Abdel, Ammou Faten F Abo, Strongylis Constantinos, Moussis Vassilios, Tsikaris Vassilios
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Biopolymers. 2008;90(3):349-57. doi: 10.1002/bip.20788.
Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-Lys-Aib-Gly-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-Lys(Fas14p)-Aib-Gly-OH). Ac-Lys(Fas14p)-Aib-Gly-OH failed to readily discriminate between naïve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.
绵羊肝片吸虫病给畜牧业带来了沉重的负担。我们在此报告肝片吸虫病的免疫诊断，以及用基于巨片形吸虫组织蛋白酶L-半胱氨酸蛋白酶的H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH（Fas14p）序列的肽免疫绵羊后，其对巨片形吸虫攻击感染具有显著的保护作用。该序列以三种不同形式合成：N(α)乙酰化形式（Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH，FasAc14p），在氨基末端带有N(α)乙酰化半胱氨酸（Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH，FasAcCys14p），并通过表位的Val(123)羧基与载体的赖氨酸N(ε)基团之间形成的酰胺键与连续寡肽载体Ac-Lys-Aib-Gly-OH（Ac-SOC(4)）偶联（Ac-Lys(Fas14p)-Aib-Gly-OH）。Ac-Lys(Fas14p)-Aib-Gly-OH未能轻易区分未感染和感染的绵羊。相比之下，游离肽能够可靠地区分未感染寄生虫的绵羊、感染除肝片吸虫以外其他寄生虫的绵羊以及实验性感染肝片吸虫的绵羊。这些数据共同表明，这些肽可能在区分巨片形吸虫感染绵羊的早期和晚期以及低负荷和高负荷方面具有很大用途。选择FasAc14p来确定基于组织蛋白酶L蛋白酶关键酶位点的肽是否能诱导对攻击感染的保护作用。用FasAc14p肽免疫绵羊可诱导白细胞介素-4 mRNA的显著表达以及体液抗体的产生，这些抗体可与新脱囊幼虫完整表面膜上的分子结合，并介导其损耗。免疫反应与虫体回收率显著降低（P < 0.02）23.1%相关，但回收的虫体大小或成熟度没有降低。
