Studied on Immunoassay for Nanobody Against Aflatoxin B1 and Construction of Random Mutation Library

Author:Ren Wen Jie

Supervisor:xu yang


Degree Year:2019





Aflatoxin B1 is the metabolite produced by Aspergillus flavus and Asp.paraciticus,which especially contaminates maize and maize products.It not only threatens the health of human beings and animals,but also brings huge economic losses.Enzyme-linked immunosorbent assay(ELISA)and immunochrommmuno-chromatography assay(ICA)are the two most commonly used which have been widely applied in the detection of aflatoxin as well as food safety detection due to their advantages of high specificity,high throughput,simplicity and rapidity.However,with the increasingly stringent requirements on food safety and quality,these two immunological analysis methods have been unable to accurately analyze the trace target analytes due to the limitations of their low detection sensitivity,and it is difficult to meet the needs of some practical applications.Therefore,how to improve the sensitivity of these two immunological detection methods has become the main problem to be solved.As the smallest genetic engineering antibody at present,nanobody has the advantages of high stability,short production cycle,easy to modify genetic information and low cost,and has been widely used in food safety testing and other fields.In order to improve the detection sensitivity of ELISA technology and colloidal gold immunochromatography technology,this study took the camel source nanobody as the main experimental material,and closely focused on the two strategies of improving the sensitivity of detection signal and improving the affinity of immunological elements:1 Preparation and application of biotinylated nano-antibody(Strategy I:improving the sensitivity of detection signals)Three biotinylated nanobodies,namely G8-L-Biotin(long arm),G8-S-Biotin(short arm)and G8-Biotin(enzyme catalyzed),were obtained by labeling nano-antibodies with chemical Biotin method and enzyme catalyzed Biotin method.BAS-ELISA was established based on these three antibodies,and its IC50 was 7.71 ng/mL(G8-L-Biotin),3.85 ng/mL(G8-S-Biotin)and 1.30 ng/mL(G8-Biotin).Thus,the sensitivity of BAS-ELISA based on G8-Biotin was 6 times and 3 times higher than that based on the other two antibodies.The G8-Biotin and mildew avidin Biotin chain system established the detection of AFB1 in grain BAS-FLEIA(Biotin(strept avidin system plus fluorescent enzyme-linked immunosorbent assay),the method of half inhibitory concentration(IC50)of 0.26 ng/mL,detection limit(LOD)is 0.03 ng/mL,The linear range(IC20-IC80)was0.09-0.74 ng/mL,and there was no significant cross-reaction with other four mycotoxins(DON,FB1,OTA,ZEN).The standard recovery was 96-112.8%,and the coefficient of variation was 3.28-9.07%.2 Expression,activity analysis and application of the nanobody/nano-luciferase fusion(Strategy I:improving the sensitivity of detection signals)Nanobody(clone G8)against AFB1 was fused with nano-luciferase and cloned into a pET22b expression vector,then transformed into Escherichia.coli.The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography,yielding a biologically active fusion protein,select CTZ-Native,CTZ-H and CTZ-400a,three CTZ analogues as substrates of nano luciferase,to the G8-Nluc nano luciferase activity is analyzed,the results showed that nano luciferase has good activity,initial luminous intensity(RLUmax)is the order of CTZ-H(2.3 x 106)>CTZ-400a(1.5 x 106)>CTZ-Native(2.5 x 105),compared with other two similar objects,CTZ-H showed the strongest signal and was selected as the best substrate for the nano-luciferase reaction.In order to increase the maximum luminous intensity of the reaction and prolong the half-life of reaction,determine the best buffer(pH=8.0,containing 1%Tergitol PBS NP-10 of 10 mM,0.25 mg/mL BSA and 8.80 mM EDTA.Na2),and purchase business from Promega Glo-lysis and Passive lysis buffer liquid ratio,the buffer solution.the maximum luminous intensity increased 1.5 times and 1.80 times respectively,and the half-life that extended the 2.50 times and 9 times respectively,It can be used for high-throughput immunoassay based on nano-luciferase and its fusion protein.The BLEIA(bioluminescent enzyme immunoassay)was established with G8-Nluc fusion protein as the probe.The IC50 of this method is 0.41 ng/mL,and the linear range is0.10-1.64 ng/mL.Based on the sensitivity of the G8-Nluc fusion protein BLEIA than the classical two steps based on the G8 nano antibody ELISA(IC50=8.14 ng/mL)about20 times higher sensitivity,corn and oats standard addition recovery,respectively(98-113)%and(91-106)%,coefficient of variation,respectively(2.56-9.47)%and(4.00-8.25)%,and other four kinds of fungus toxin(DON,FB1,OTA,ZEN)no cross reaction.Research and application of immunochromatography based on pentavalent nano-antibody monomer3 Preparation of immunochromatographic strips based on G8-CTB fusion proteinThe prokaryotic expression vector pET25b-CTB-G8 was constructed and transformed into E.coli BL21(DE3),the monomer of the G8-CTB pentamers was obtained after expression purification;Blue AuNFs with an average diameter of 75±5 nm were synthetized and employed as a signal amplification probe for detection of AFB1,by optimizing the NC membrane type,the amount of AFB1-BSA,the successful detection of AFB1 immune chromatography method was developed,the naked eye visual detection limit of 1 ng/mL,resistance 30%methanol concentration,no need for professionals and equipment,the detection time is only 10 minutes.In summary,AuNFs are a novel probe that exhibited excellent sensitivity in the ICTS system and could be used for ultrasensitive detection of other analytes in food safety monitoring,and even medical diagnostics4 Constructionb of random mutation library and panning4.1 Random mutation library was established by error-prone PCR,the positive rate of the mutant library was preliminarily determined to be 89%,the effective storage capacity of the mutant library is 5.96×106 cfu,after the rescue of mutant library with the aid of phage M13K07,the titer of library was 2.1×1013 cfu/mL.Sequencing analysis of10 randomly selected positive clones showed that the mutants were all single gene mutations and distributed evenly.4.2.The artificial antigen AFB1-BSA was used as the target molecule for four rounds of solid match-making and screening.After indirect competition phage-elisa identification,the mutant 5-34 activity disappeared,the sensitivity of 5-23 positive clone was improved,the sensitivity of 5-21 and 5-24 was decreased,and the sensitivity of 5-20,5-22 and 5-25remained unchanged.Analysis and sequencing results showed that the mutation of the third position of the glusteine(3Q)in the FR1 region and the 93rd position of the threonine(93T)in the FR3 region at the same time had a great impact on the activity of the mutants;the 45th position of the lysine(45K)in the FR2 region was changed into the arginine(45R),and the 47th position of the glutamate(E)was changed into the amber codon,which could improve the activity of the mutants.