Studies On The Immune-toxicology Of Penicillic Acid From Penicillium Cyclopium And Its Monoclonal Antibody

Penicillic Acid(PA) is one of the main metabolites generated from Penicillium cyclopium.Many feedstuff raw materials,such as sorghum,barley,oats,wheat,corn and rice were suitable for the production of PA with a high output as the main mycotoxin in moldy feedstuff.To some extent,PA has toxic effect on many animals and the interaction of PA and other mycotoxin on animals will contribute to cumulative toxicity and enhance its toxicity.Hence,rapid and effective approaches of testing should be discovered and research on its toxicology to cope with the serious threat of PA posed to animals and humans.In this article,complete antigens of perficillic acid were synthesized by chemical methods and monoclonal antibodies were prepared through animal immunization and cell fusion technology.Toxicity of penicillic acid to immune organs and cells of mice was researched in vivo and vitro. The main research content and results are as follows:1.PA was conjugated to bovine serum album(BSA) and ovalbumin(OVA) by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride(EDC).The artificial antigens PA-BSA and PA-OVA were identified by Ultra-violet spectrometric scanning, SDS-PAGE and immunization.Results showed that the absorption peak of conjugation were different from that of the carrier protein alone and of the PA.The conjugated ratio of PA and BSA was 23.2:1 and that of PA and OVA was 10.4:1. Balb/c mice were immunized by the artificial antigen of PA-BSA,with PA-OVA as coating antigen.It was experimentally found that the average titer of antiserums was more than 12800 by indirect ELISA.It indicated that the complete antigens were obtained.2.Hybridoma cell strains were selected after fusion of mouse SP2/0 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with Penicillic Acid(PA) conjugated to bovine serum albumin(BSA) by ELISA.A monoclonal antibody specific for PA was produced in vivo by the hybridoma cell. Analysis revealed that the monoclonal antibody was of the IgG1 type,with the titer was 1:2×10~5.The relative cross-reactivity of PA with AFB-1,ZON,T-2,FB were less than 0.01%and with an affinity constant for PA of about 1.54×10~8 liters per mol.It offered a foundation for developing immunoassay method for PA. 3.Spleen lymphocytes were separate and exposed to PA at the dosage of 1,3,5 and 10μg/ml for identify the toxicity of PA in vitro.Anti-oxidase content,the rate of the cell proliferation and DNA damage were examined.Results showed that the activity of is lower than the control,but the concentration of MDA and NO increased which was dose-dependent on PA.Penicillic acid can reduce the activity of antioxidant enzymes.MTT was adopted to observe the spleen lymphocyte proliferation,which showed the proliferation of lymphocytes decreased when exposed to PA.PA with higher dose can cause lymphocyte DNA damage by single cell gel electrophoresis,which indicated PA is a potential carcinogen.4.Three groups of mice were exposed to different dose of PA,and then the effect of PA on mice in vivo,apoptosis,histological and ultra-structural changes of the immune organs of the mice were observed.Observation of transmission electron microscope was carried out to study the ultra-structural changes,and Tunnel analysis was used to study the apoptosis of mice spleen.Results showed that no obvious changes were found in the thymus except partial atrophy and lymphocyte reduced in the highest dose group.More serious injures were found in spleen by seen the lymphocyte death and aggravated with the dose of PA increased.Typical apoptotic lymphocytes were found both in middle and high dose group,and also a significant increase of apoptosis rate compared with the control.5.The expression of apoptosis gene Bcl-2,Bax,p53,Fas and FasL mRNA were measured by RT-PCR in spleen and thymus of mice exposed to PA at different dose. Results showed that the expression of Bcl-2 decreased and Bax,Fas,FasL increased in spleen at middle and high dose group,which induced Bcl-2,Bax,Fas/FasL may play an important role in the process of apoptosis in spleen.But it was different in thymus from the spleen.The expression of Bcl-2 and p53 increased,Fas decreased in thymus.We supposed that PA may inhibit apoptosis in thymus.