Ying CHEN, Xifeng WANG, Wenqiang ZHONG, Shasha GONG, Jun QIAO, Qingling MENG, Yunfu HUANG, Lulu TIAN, Yanbing NIU, Xingxing ZHANG

Arthrobotrys oligospora Rekombinant Çitinaz AO-379’un Enzim Özellikleri ve Nematod İndirgeyici Aktivitesi

Çitinaz, enfeksiyon sürecinde nematod tutucu mantar tarafından üretilen önemli bir virulans faktörüdür ve nematodların yumurta kabuklarından ayrılmalarında önemli rol oynar. Bu çalışmada, Arthrobotrys oligospora çitinaz AO-379 cDNA sekansı RT-PCR ile amplifiye edildi ve Pichia pastoris GS115’de AO-379 ekspresyonunu oluşturmak amacıyla pPIC9K vektörüne yerleştirildi. Rekombinant AO-379 (reAO-379) nikel iyon affinite kromotografi ile saflaştırıldı, enzim özellikleri ve nematod indirgeme aktivitesi araştırıldı. SDS-PAGE ve Western blot analizleri yaklaşık 44 kDa moleküler ağırlığında olan reAO-379’ın başarıyla elde edildiğini gösterdi. reAO-379 pH 5.5 ve 30°C’de güçlü çitinaz aktivitesi gösterdi. reAO-379 ile Strongylus equinus, Caenorhabditis elegans ve Haemonchus contortus 12, 24 ve 36 saat muamele edildiklerinde sırasıyla S. equinus için %42, %89 ve %100, C. elegans için %50, %90 ve %97 ve H. contortus için %53, %62 ve %84 öldürme oranlarına sahip olduğu gözlemlendi. Fasciola hepatica ve Dicrocoelium chinensis yumurtaları reA-379 ile 24, 48 ve 72 saat muamele edildiğinde, indirgenme oranları F. hepatica yumurtalarında sırasıyla %12, %43 ve %65 iken D. chinensis yumurtalarında sırasıyla %15, %33 ve %55 olarak belirlendi. Bu çalışma, reAO-379’un yetiştiricilikte sindirim sistemi nematodlarına karşı potansiyel bir biyolojik kontrol ajan olarak kullanılabileceğini göstermiştir.

Enzymological Properties and Nematode-Degrading Activity of Recombinant Chitinase AO-379 of Arthrobotrys oligospora

Chitinase is an important virulence factor produced by nematode trapping fungi in the process of infection, and plays an important role in the cleavage of nematodes and their eggshells. In this study, the cDNA sequence of Arthrobotrys oligospora chitinase AO-379 was amplified by RT-PCR and inserted into the vector pPIC9K to induce the expression of AO-379 in Pichia pastoris GS115. The recombinant AO-379 (reAO-379) was purified by nickel ion affinity chromatography, and enzymological properties and nematode-degrading activity of reAO-379 was analyzed. SDS-PAGE and Western blot analysis showed that the reAO-379 with molecular weight of about 44 kDa was successfully obtained. The reAO-379 showed strong chitinase activity at pH 5.5 and 30°C. Using reAO-379 to treat Strongylus equinus, Caenorhabditis elegans and Haemonchus contortus for 12, 24, and 36 h , the killing rates of reAO-379 in S. equinus were 42%, 89% and 100%; in C. elegans were 50%, 90% and 97%; in and H. contortus were 53%, 62% and 84%, respectively. Using reA-379 to treat Fasciola hepatica and Dicrocoelium chinensis eggs for 24, 48 and 72 h, the degradation rates of reAO-379 were 12%, 43% and 65% in F. hepatica eggs, and were 15%, 33% and 55% in D. chinensis eggs, respectively. Our study suggests that the reAO-379 is potentially valuable for development of biological control agent against digestive tract nematodes in livestocks.

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