Development Of Nanobody Against Aspergillus And Of Antibodies Against Key Enzymes In Aflatoxin Biosynthesis

Aspergillus flavus,a saprophytic pathogenic fungus,is widely found in nature and mainly contaminates peanut,corn and other crops.A.flavus and A.parasiticus produce secondary metabolites,aflatoxin(AFT),which is carcinogenic,teratogenic,immunosuppressive.Aflatoxin was identified as one of the most dangerous mycotoxin in nature.It can seriously endanger human and animals health and cause huge economic losses.So it is important to develop a rapid and sensitive detection method for A.flavus producing aflatoxin.There is theoretical and practical significance for prevention and controlling aflatoxin contamination.The aflR gene is the most important regulatory gene in aflatoxin biosynthesis,which encodes an AFLR protein with a molecular weight of 47 kDa.AFLR is closely related to the transcription of most structural genes such as nor-1,ver-1 and omt-A.AFLR is of transcriptional activator which regulates aflatoxin biosynthesis at transcriptional level.aflD(nor-1),aflM(ver-1)and aflP(omt-A)are three structural genes in aflatoxin biosynthesis.AFLD,AFLM and AFLP proteins,respectively expressed in early,middle and late stage,play a vital catalytic role in aflatoxin biosynthesis.Therefore,the developed antibodies against these key enzymes provides a new direction for exploring the expression and specific functional mechanism of AFLR,AFLD,AFLM and AFLP proteins in A.flavus.At present,the common detection method for A.flavus contamination is immunological analysis based on the specific binding between antigen and antibody(such as enzyme-linked immunoassay,immunochromatography,immunosensor,etc.).Traditional immunological detection methods mainly rely on polyclonal antibodies(pAbs)and monoclonal antibodies(mAbs).However,the development of these antibodies needs animals immunization and hybridoma cell fusion technology,resulting in long production cycle,high cost,instable genetic information and other defects.Using phage display technique,a highly affinity nanobody could be screened out from the antibody library from the immunized alpaca,and the coding gene of nanobody could be obtained simultaneously.Nanobody is very stable,easy for prokaryotic expression for industrial production,and easy to carry out genetic engineering.Compared with traditional antibody,the development of nanobody is simple and has a short cycle in the field of immunological detection.The main contents and innovations of this study are as follows:1.Phage-displayed VHH library was first constructed in this research and a sandwich enzyme immunoassay based on nanobody-pAb was established and applied to the detection of A.flavus in agro-products.Four antigens of A.flavus3.4408 were prepared according to the life cycle of A.flavus,and pAb for A.flavus was purified by immunization with rabbit.At the same time,the immunized alpaca with A.flavus3.4408 was used to construct a nanobody library with capacity of 7×10~8.EA3-VHH against the extracellular of A.flavus and PO6-VHH and PO8-VHH against lysate of A.flavus were successfully obtained.The analysis of these three nanobodies showed that the nucleotide sequences of EA3-VHH and PO8-VHH were identical.This result indicated that the content identified by PO8-VHH could be secreted into cells,which making it possible to detect A.flavus in samples by PO8-VHH.Then they were prokaryotic expressed and purified.The results of Indirect-ELISA and western blotting showed that PO6-VHH had high affinity for A.flavus3.4408,but had little affinity for other A.flavus and A.parasiticus.However,PO8-VHH had high affinity for either A.flavus or A.parasiticus,which indicating that there are differences in antigen recognition properties between different nanobodies.And PO8-VHH is more broad-spectrum to recognize A.flavus.In addition,PO8-VHH can recognize a single component(45 kDa),which is of great significance for exploring the target molecules in A.flavus by using the penetration of nanobody.Sandwich-ELISA based on PO8-VHH and pAb was established.This assay was able to detect A.flavus at concentration as low as 1?g/mL.The detection limit of A.flavus in peanuts and maize was 2?g/mg(mycelial concentration),which was suitable for quantitative detection of samples with A.flavus contamination.This method can be used to detect fungi before they are sufficient to produce high levels of aflatoxin.2.The aflatoxin pathway-specific transcription regulator AFLR was expressed and used to immunize alpaca.Phage antibody library was constructed,and a pair of novel nanobodies against AFLR were screened out for the first time.A sandwich enzyme immunoassay based on nanobody-phage was established and applied to explore the relationship between aflatoxin AFLR content and aflatoxin accumulation.Primers of regulatory gene aflR were designed by using sequence of A.flavus genome.Then aflR was transferred into E.coli for expression and purification.AFLR protein was taken as antigen to immune alpaca to construct nanobody library.A pair of novel nanobodies,R2-VHH and R12-VHH,were obtained.The results of Indirect-ELISA and western blotting showed that two nanobodies were highly sensitive and specific for AFLR and were able to recognize native AFLR protein(~47 kDa)in A.flavus.And the affinity constants were 5.33×10~7 L/mol and 2.66×10~7 L/mol,respectively.The method of quantitative detection for AFLR based on R2-VHH and R12-phage was established.The detection limit of AFLR was 1.25 nmol/L,which was suitable for high sensitivity and quantitative detection of native AFLR.Further studies showed that the accumulation of aflatoxin does not depend on the content of biosynthesis regulator AFLR.The development of identification reagents and quantitative methods paved a way for further reveal the regulatory mechanism of AFLR and its accumulation of aflatoxin in agricultural food.3.AFLD,AFLM and AFLP,which are early,middle and late key enzymes in aflatoxin biosynthesis,were expressed and used to immunize Balb/c mice.Monoclonal antibodies against AFLD,AFLM and AFLP were successfully screened out by immunized Balb/c mice.Indirect enzyme-linked immunosorbent assay based on these monoclonal antibodies provide the core reagent for exploring the mechanism of aflatoxin production of these three key enzymes.AFLD,AFLM and AFLP proteins were obtained by amplifying the corresponding genes from A.flavus3.4408 genome.They were used to immunize Balb/c mice to obtain mAbs by using hybridoma technology.Finally,2A8 1E10 for AFLD,11B2 1D7 and 3G5 4E7 for AFLM,3B4 3B10 for AFLP were successfully screened out.The results of Indirect-ELISA and western blotting showed that mAbs produced by recombinant protein were highly sensitive and specific,and they could recognize corresponding native proteins in A.flavus.In addition,the binding activity of purified mAbs to native protein was determined by Indirect-ELISA.The detection limit of 2A8 1E10 for AFLD protein was 3ng/mL,11B2 1D7 and 3G5 4E7 for AFLM protein were 11 ng/mL and 8 ng/mL,and 3B4 3B10 for AFLP was 1ng/mL.This study laid a foundation for further study of the expression and functional mechanism of AFLD,AFLM and AFLP in A.flavus.