Purification And Characteristics Analysis Of Two Activity Proteins From Mushroom

Plant virus disease brings great economical damage. To resolve the problem is always a goal of many researchers. Searching antiviral materials from organ is one of the methods to prevent and control plant virus disease. Two proteins in possession of plant virus resistant activity were purified from mushroom in our research, and the results were the following:An alkaline protein, y3> was purified from the fruiting bodies of mushroom Coprinus contains by means of CM-sepharose FF ion-exchange column chromatography and Superdex?5 High Resolution molecular sieve chromatography. It was confirmed a single subunit by the methods of SDS-PAGE and PAGE.y3 was sacchariferous and the content was estimated to be 30% glucose equivalent when determined by the method of Hydroxybenzene- vitriol. It contained a large proportion of Glx, Arg and Ala with the molecular weight 12.3kDa determined by MALDI-TOF mass spectrometry. The N-terminal 20aa sequence of protein y3 was NRDVAACARFIDDFCDTLTP , which has no homology with other sequences in GenBank, and the accession number was P83477 in Swiss-Prot. Antibody of y3 was prepared and the titer is 2-12-2-13. It could be detected in not only fruiting body but also vegetative mycelium by Western-Blot and ELISA.Activities of y3 had been determined, and the results were the following: the inhibition rate against Tobacco Mosaic Virus infection in Nicotiana glutinosa was 83.0% when the concentration of y3 was 12.5ng.mL-1. y3 was able to agglutinate rabbit and human ervthrocytes at the lowest concentration of 1.562ug.mL-1 and 0.78 lug.mL,-1 but the hemagglutinating activity was inhibited by y-Globulins, Maltose and D-Galactose. Using an assay system based on cancer cell lines MGC80-3, SPC-A1 and SMMC-7721, y3 was studied for its inhibitory ability against cell multiplication, and the IC50 value was 12.5ug.mL-1 to MGC80-3 and SPC-Ai, 30礸-mL-1 to SMMC-7721. Phenomena like apoptosis was observed via microscope when SPC-Ai was treated with y3 and stained by yihongY . Protein y3 could not inhibit the growth of some bacteria and fungi experimented.Another single subunit protein YP46-46 was isolated from the fruiting bodies of mushroom Pleurotus citrinoptteatus with a procedure involving ammoniumsulfate precipitation, ion-exchange chromatography on DEAE-cellulose, and gel filtration on Sephacryl橲-200 High Resolution. The protein has a molecular weight of about 27.4kDa by SDS-PAGE. It was almost not sacchariferous, with a large proportion of acid amino acid. It was novel in respect that possesses a N-terminal sequence of NRDVAACARFIDDFCDTLTP, which has no homology with other sequences in GenBank. and the accession number was P83481 hi Swiss-Prot. Antibody of YP46-46 was prepared and the titer is 2-2.Activities of YP4646 have also been determined. The inhibition rate against Tobacco Mosaic Vims was 50.0% when the concentration of YP46-46 was 0.24g.L. YP4646 could also not inhibit the growth of some bacteria and fungi experimented. Using an assay system based on stomach cancer cell line MGC-803, liver cancer cell line SMMC7721 and lung cancer cell line SPC-A1, YP46-46 was studied for its inhibitory ability against cell multiplication, and the IC value was about 20mg.L.A primary study was done to analyse the mechanism of y3 inhibition to TMV. The result showed that y3 could inactivate TMV in vitro and inhibit its multiplication h N. tabacum var. K326., The inhibition rate against TMV was 50.0% when the concentration of y3 was 2.0g.mL.While RNase was added to the mixture of TMV and y3,the infection rate of TMV was 61.74%, decreased by 38.26% hi contrast with the control. Meanwhile, it was showed that y3 broke TMV when they were mixed and observed through electron microscope. The protein could prevent the aggregation of TMV-CP in vitro and the link with its antibody. It was showed that y3 could alleviate the symptom of plant infected by TMV in a certain extent by means of detecting then chlorophyll.Based on the primer designed according to the N- terminal amino acid sequence of y3, Partial cDNA a...