Gene Cloning, Expression And Characterization Of Protein Tyrosine Phosphatase From Metarhizium Anisopliae Strain CQMa102

In this paper, with the total RNA of Metarhizium anisopliae var. acridum strain CQMa102 as a template and the RT,1P5 and 1P3,2P5 and 2P3 as primers, the gene expression sequence of the protein tyrosine phosphatase of this fungus had been successfully cloned by RT-PCR in vitro. The PTPase gene fragment was cloned into the pPIC9K vector and was transformed into the Pichia pastoris KM71, which was induced with 0.5% methanol after that. The results of SDS-PAGE and enzyme characteristics analysis of the crude enzyme solution after induced culture for 144 hours showed that the PTPase was expressed by the recombinant PD in Pichia pastoris KM71. Some studies have shown that the PTPase which in vitro could change the phosphorylation status of proteins associated with signal transduction (Toll-like receptor) in haemolymph of its host, which may further interfere with the immune system of the host. Therefore, this study will provide reference for the researches of how the PTPase of Metarhizium anisopliae affect the humoral immune system of the locust and the possible theory of how disease fungus interfere with the humoral immune system at the protein levels.The main results of the paper were as follows:■The gene cloning of PTPase in Metarhizium anisopliaeAccording to the known sequence of PTPase gene in Metarhizium anisopliae. we designed degenerate primers to amplify a DNA fragment (about1900bp) from the total RNA of Metarhizium anisopliae. The DNA fragment was connected with pMD19-T vector and transformed into E. coli, then the positive clones were screened by PCR. The result of this gene sequence analysis showed that the total cDNA was about 1960 bp, and it contained two EcoRI digestion sites and six HIS ma-rks which were in primers. The expression gene of PTPase was also in the DNA fragment, which co-uld encode 647 aa putative amino acid. The amino acid sequence contains five peptides sequences which were determined by mass spectrometry to be DITNFFGVKEPEVEK, WYEDLFAEDK, GND SALDLPGLK, NIQYGTGVNFPSNWEPR, ALGDLLEELDDTVR, and by the MALDI-TOF-MS database analyzing and denovo sequencing. And these proved the PTPase gene had been success-fully cloned.■The construction of the recombinant and expression plasmidBased on the characteristics and requirements of the Pichia pastoris expression system, we used the pPIC9K plasmid as the expression vector of PTPase gene in Pichia pastoris. The PTPase gene was connected with pPIC9K plasmid and transformed into E. coli, then the positive clones were screened by PCR. Sequencing analysis of positive clones indicated that the target gene had been inserted into the open reading frame of pPIC9K plasmid, and 5 'end of the target gene was connected to the signal end of pPIC9K plasmid. And these results above proved the recombinant expression vector of PTPase had been constructed successfully.■The integration of target gene and the screening of high-level expression strainThe recombinant expression vector of PTPase was electrically transformed into Pichia pastoris KM71,then the positive clones were screened by PCR combined with geneticin resistance. The positive clones with different geneticin resistance were induced by 0.5% methanol. After that the culture medium in different time period was dialyzed and the relative activity (pNPP as Substrate) was determined, which showed the relative activity of the strain 14 from the concentration of 3.0mg/ml geneticin was significantly higher than that of other strains. The relative activity was highest after cultured for 144h, from which we could tell the target protein may be highly expressed by the strain 14.■SDS-PAGE analysis and characteristics analysis for the crude enzymeThe crude enzyme of strain 14 was analyzed by SDS-PAGE and a protein fragment of 75.6KDa which has similar molecular mass with PTPase of Metarhizium anisopliae was obtained. Substrate specificity analysis of crude enzyme solution showed the digestion of the acid phosphatase to tyrosine residues was specific. The hydrolysis of crude enzyme solution to 8mM pNPP was strongly inhibited by Cu2+ and enhanced by Ca2+; and no obvious effect was shown when Mg2+, Ni2+ and Mn2+ were used. Specific inhibitor of serine/threonine protein phosphatase, such as NaF, had no significant influence on the digestion activity of phosphatase to pNPP. neither were specific inhibitor of acid phosphatase and alkaline phosphatase, such as EDTA, and specific inhibitor of acid phosphatase, such as sodium tartrate. But sodium molybdate and sodium tungstate, which are known as specific inhibitor of PTPase, did affect the digestion activity of phosphatase to pNPP. Therefore, the protein expressed by the target gene was protein tyrosine phosphatase. The optimal temperature for the activity of the purified enzyme against was 60℃as 8mM pNPP was Substrate and 55℃as Substrate was 8mM O-phospho-L-tryosine.