Gene Cloning, Expression And Analysis Of Nematicidal Activity Of Infectious Extracellular Serine Protease Of Arthrobotrys Oligospora

Gastrointestinal nematodes is one of parasitic diseases, which is seriously retarding the sustainable and healthy development of animal raising, causing high economic losses each year in China. At present, the control of nematode parasites in livestock relies almost exclusively on multiple and regular use of anthelmintics, however, the prophylactic use of anthelmintics has led to the global emergence of multiple anthelmintic resistances in nematodes with low efficacy, the entering of chemical residues into the human food chain, in common with pollution of ecological environment. To overcome the problems, the countries have carried out studies of biologically controlling nematode parasites, especially the use of nematode-trapping fungi and their key virulence factors-infectious extracellular proteases has attracted increasing attention for controlling nematode parasites, and shows tremendous potential for effective prevention and treatment of parasitic nematodes, and has been considered as a human-animal-environment friendly method. Therefore, isolation of nematode-trapping fungi and studies of fungal infectious extracellular proteases become the focus and of great significance and application prospects for the scientific prevention and control of gastrointestinal parasitic nematodes.The present study selected nematode-trapping fungus Arthrobotrys oligospora Xinjiang isolate XJ-XAol with high predatory activity as experimental material, cloned DNAs of fungal infectious serine proteases XAoz1, P12and P186. Subsequently the cDNA gene of mature XAoz1was cloned into yeast expression vector and induced for expression in Pichiapastoris system, and then the nematicidal activity of the recombinant protease was detected. The results revealed gene structural features of the key factor XAoz1from A.oligospora XJ-XAol in the process of infecting nematodes, analyzed the enzymatic properties of recombinant protease reXAozl and measured its nematicidal activity, and laid a theoretical foundation for the development of novel protease spraying biological agents in the control of gastrointestinal nematodes in livestock. Research methods and results are as follows:1. Gene cloning and sequence analysis of extracellular serine proteases from A.oligospora:selecting nematode-trapping fungus A.oligospora Xinjiang isolate XJ-XAol as a tested strain, we successfully cloned DNA genes of three serine proteases XAoz1, P12and P186via PCR (GenBank accession number JF747254, JX898768and JX898769, respectively), carried out bioinformatics analysis of genes and homology analysis in common with constructing the phylogenetic tree. The results showed that the genes of XAoz1(1344bp), P12(1429bp) and P186(1468bp) were successfully cloned. XAozl and P12contained an intron, P186had three introns of different sizes, which began with GT and ended with AG; all three proteases contained catalytic triad in subtilisin-XAozl:Asp164, His203, Ser353; P12:Asp193, His230, Ser383; P186:Asp160, His192, Ser345; and two substrate-binding S1pockets-XAozl:Ser259Leu26oGly261, Ala285Ala286Gly287; P12:Ser289Leu290Gly291, Ala3isAla316Gly317; P186:Ser251Leu252Gly253, Ala277Ala278Gly279. Furthermore, they also had potential glycosylation sites. The similarity was97.5%and98.3%between XAozl and PⅢAozl, respectively, which indicated that the relationship was close and XAozl was likely an ortholog of PⅡ and Aozl, but the relationships of P12, P186with PⅡ, Aozl were distant, with the same among XAozl, P12and P186.2. Expression of extracellular serine protease XAozl from A.oligospora in Pichiapastoris:the cDNA sequence of mature peptide XAozl was amplified through RT-PCR, the recombinant plasmid pPIC9K-XAozl was constructed and linearized by enzyme digestion, and then electroporated into Pichia pastoris GS115. The results showed that:recombinant P.pastoris GS115/pPIC9K-XAozl was screened by phenotype and G418resistance and further confirmed by PCR, and positive recombinant transformants were successfully obtained with G418resistance (1.0mg/mL) and the phenotype His+Mut+; then a positive transformant was chosed and cultured for72-h induction by1.5%methanol, the enzyme activity of recombinant protease (reXAozl) reached the maximum (12168U/mg protein); an expected band about50 kDa was shown by SDS-PAGE and Western blot; reXAozl degraded a broad range of substrates including casein, skimmed milk, BSA, gelatin, denatured collagen and nematode cuticle.3. Purification and nematicidal activity analysis of recombinant serine protease:after72h of1.5%-methanol induction, the cultures of recombinant P. pastoris GS115/pPIC9k-XAozlwas centrifuged, and purified via ultrafiltration and His-Bind affinity chromatography. Thus nematicidal activity of purified recombinant protease reXAozl was analyzed. The results showed that:the optimum activity of reXAozlwas at50℃and pH6.5～9.5with an optimal pH at8.5. The purified protease could degrade a broad range of substrates including casein, skimmed milk, BSA, gelatin, denatured collagen and nematode cuticle. Also, reXAozl could effectively immobilize C.elegans and H.contortus, degrade the nematode cuticle, destroy organizational structures of nematodes and then kill nematodes; the mortality of C.elegans was52%,85%and100%, the mortality of the H.contortus was45%,70%and83%, after12,24and36-h incubation, respectively, which exhibited that reXAozl had nematicidal activity.