Gene Cloning, Expression And Biological Activity Of Extracellular Protease Of Arthrobotrys Oligospora

Parasitic nematode is one of parasitic diseases in animal, which is seriously harmful to the development of livestock. For a long time, it is the major measures to control parasite for chemical drugs. With long-term use of chemical substances, however, it not only leads to drug residues and resistance, but also causes serious environmental pollution. Therefore, it is necessary to develop a new approach for the prevention and control of animal. Using nematode biological antagonism-nematophagous fungi to prevent and control animal parasitic nematodes bas received more attention. The molecular mechanisms of nematophagous fungi infestation nematode have highlighted the study. To date, a variety of infection moleculars(including toxin, extracellular enzymes, pathogen hormone, pathogen extracellular polysaccharide, etc.) of nematophagous fungi have been identified, in which the extracellular enzymes(Serine protease, chitinase) was more closely related to the pathogenicity. It plays an important role in degrading components of nematode body wall, inntaking of nutrition and penetrating extracellular polysaccharide.In this study, nematode-trapping fungi were isolated from the collected soil samples of Yili and identified. The nematode-trapping fungus Arthrobotrys oligospora Xinjiang isolate(XJ-A1) with high predatory activity was used as experimental material, from which cDNA of P186 gene was cloned. Then cDNA of P186 gene was cloned into yeast vector and electroporated into Pichia pastoris for expression. Nematicidal activity of the recombinant protease was Determined. Through the above study, molecular characteristics of P186 of A.oligospora(XJ-A1) was preliminary explored, and enzymatic properties and nematicidal activity of recombinant protease reP186 were successfully analyzed, which laid a foundation for the development of novel biological agents to control gastrointestinal nematodes in livestock. Main methods and results are as follows:1. Analysis of molecular characteristics of infectious extracellular protease of Arthrobotrys oligosporaNematode-trapping fungi were isolated with corn meal agar(CMA) from soil samples of Yili, xinjiang province. One isolate was successfully identified as Arthrobotrys oligospora through morphological characteristics and molecular biological techniques. The cDNA of serine protease P12, P186 and P233 of A.oligospora Xinjiang isolate(XJ-A1) were amplified, and chitinase gene AO-801 and AO-483 were also amplified by PCR. The signal peptide, hydrophobicity and hydrophilicity, secondary structure, tertiary structure and functional domains of the encoded proteins were predicted and analyzed. The results showed that these serine proteases have a signal peptide, and all of them contain three active sites of aspartic acid(D), histidine(H) and serine(S), which confirmed that P12, P186 and P233 belong to the extracellular subtilisin-like serine protease families. Moreover, they contain two potential N-terminal glycosylation sites and two substrate-binding S1 pockets. Phylogenetic analysis revealed that P12, P186 and P233 have low sequence identities with that of other different species. Two chitinases have two conserved catalytic domains with the sequence of SXGGW and DGXDXDWE, belonging to the family 18 glycoside hydrolase. They have no signal peptide sequence, indicating those are not secretory proteins. Random coils, alpha-helix and beta-sheet are the major structural elements in secondary structure of chitinase genes AO-801 and AO-483, and with(α/β)8 rounded buckets in tertiary structure. Phylogenetic analysis showed that the relationship of chitinase AO-801 and AO-483 is more close to the insect pathogenic fungi.2. Expression and identification of serine protease P186 gene of Arthrobotrys oligosporaP186 gene was amplified by RT-PCR from total RNA extracted from Arthrobotrys oligospora. The RT-PCR product was cloned into the vector p ET32 a and pPIC9 K to generate recombinant pET32a-P186 and pPIC9K-P186. Then pET32a-P186 plasmid was transformed into E.coli BL21(DE3), while the pPIC9K-P186 plasmid was transformed into Pichia pastoris GS115 for expression.. SDS-PAGE analysis showed that the product had a molecular weight of about 62 kDa. The recombinant Pichia pastoris GS115/pPIC9K-P186 was screened by phenotype and G418 resistance and further confirmed by PCR, and positive transformants were successfully screened by G418 resistance(2.0 mg/m L). The phenotype His+ Mut+ was then cultured for 72 h and inducted by 1.5% methanol. The analysis of SDS-PAGE showed that the product had a molecular weight of about 32 kDa, and the enzyme activity of recombinant protease(reP186) can reach the maximum(13151U/mg protein). Antibody against reP186 was prepared using purified prokaryotic expression product to immunize mice for the detection of reP186 expressed in Pichia pastoris. Analysis of Western blot revealed specific blot band appeared at the appropriate location, indicating that reP186 was successful expressed in Pichia pastoris.3. Purification and biological activity analysis of reP186 serine proteaseThe cultures of recombinant P.pastoris was centrifuged and purified via ultrafiltration and His-Bind affinity chromatography. The substrate specificity test revealed that the scope of the substrate specificity for reP186 was relatively broad. The rate of hydrolyzing casein was relatively high, while the rate of hydrolyzing BSA, gelatin, denatured collage and nematode cortical layer was moderate and it was lowest for collagen. The enzymatic activity of hydrolyzing substrates was gradually increased with the increase in temperature and reached the maximal at 55 oC. The enzymatic activity was relatively stable within the temperature range between 20 and 55 oC but totally loss at 70 oC for 30 min. Purified reP186 displayed relatively high enzymatic activity at the pH range of 6.0-10.0 and displayed the maximal activity at pH 8.0. When reP186 was chelated with the metal ion-chelating agent, ethylenediaminetetraacetic acid(EDTA), it’s enzymatic activity was almost unaffected, whereas PMSF and SSI almost totally inhibited the enzymatic activity of the purified reP186. The killing rates toward C.elegans by reP186 at these time points were 62, 88 and 100%, respectively, and its toward H.contortus were 52, 65 and 84%, respectively. There were significant differences in killing rates between groups treated with reP186 and control groups. After being incubated with this proteinase for 12 h, C.elegans and H.contortus were fixed; the nematode’s stratum corneum began to be degraded. At 24 h after incubation, a large part of the nematode’s body wall was degraded. At 36 h after incubation, the nematode’s body wall and a large part of the organs/tissues inside the body were degraded. No significant changes were observed in the nematode’s body wall in the control groups.