Construction Of ScFv Library And Screening Anticlenbuterol Antibody By Ribosome Display

Clenbuterol(Clenbuterol,CL) is a syntheticβ₂-adrenergic receptor agonists.It is often used for prevention and treatment of bronchiectasis as asthma and emphysema and other respiratory diseases caused by bronchial spasms.When its application to the treatment dose of 5～10 times can enhance muscle growth and reduce fat deposition. Because of its unique biological properties,it is often used as feed additives for animal husbandry illegally.As CL has the pharmacological properties of absorb fast in the body, high-fat-soluble,accumulated residue,long half-life,so poisoning incidents continue to occur after the consumption of meat products with residues of CL.So strengthen the detection of CL residues in meat products is necessary.The current physical and chemical detection methods have the shortcomings of high level of equipment,high cost of detection,difficulty in the promotion.Therefore,there is an urgent need to establish some economic,simple and rapid detection methods which are based on the CL-specific antibody.Genetically engineered monoclonal antibody is the reorganization of chemical and structural characteristics of the same monoclonal antibody,a class of immune-specific antibodies of stable property.Of these,single-chain antibody(ScFv) has the properties of low or non-immunogen,small molecular weight,strong tissue penetration,low-cost,mass production and so on.ScFv could be prepared by phage display or ribosome display. Ribosome display(RD) is the first in vitro display technology which totally independent on cells.The capacity is far greater than the phage display technology which is more mature in application.In addition,RD also has advantages of simple library construction and screening,have not selection pressure,through the introduction of mutation and recombination technology to improve the affinity of target protein.In this paper,an naive ScFv library of mouse with effective capacity of 4.24×10¹³ was constructed.The library were identified from the following aspects:expansion and diversity,transcription and anti-retroviral activity,expression activity.Synthesed the CL-BSA use diazotization method.A solid phase screening method used for screening anti-CL ScFv and preliminary determined the sensitivity and specificity of the ScFv.The main results are described as follows.1 Naive ScFv library of mouce constructionExtract the total RNA from spleen cells of Balb/c and C57 mouse without immuning, about 5 weeks old.The first chain cDNA was got through reverse transcription, amplified VH,VL gene fragments,about 350 bp.Amplified the size of 93 bp RD components T(T7 promoter,3'loop,ribosome binding site) and 294 bp interval sequence P(spacer,5'loop) from the carrier PT7PD.The components of T,VH,P,and VL connected by overlap primer extension method(splicing by overlap extension,SOE),and then TH LP ScFv link into full-length clips,about 1 100 bp,to build a naive antibody library of mouse by RD.2 Quality evaluation of ScFv library(1) Amplified purpose fragment of 1 100 bp by forward primer T7B and backward primer PRDR,verify that library can be amplified.(2) Library were connected with PEASY-T3 carrier,sequencing results showed that antibody sequences were different,have no stop codon,with good diversity.(3) Transcribe DNA library in vitro with Promega T7 RiboMAX Express Large Scale RNA Production System,500 bp single-chain mRNA was got,an specific band about 1 100 bp was got by anti-transcribe.Those verify that the library have anti-transcription and transcription activity.(4) The protein expression ability of library was verified by Transcend^TM NonRadioactive Translation Detection Systems.The system enables products of translation biotinylation, ALP labled antibodies linked streptavidin.The results showed that about 40 kD band was obvious.The western blotting result show that the product was anti-CL antibody.3 Screening anti-CL antibody with ScFv libraryScreening anti-CL antibody with artificial antigen CL-BSA as target after transcripttion and translation of the library in vitro by solid phage affinity method.Antibody-ribo-some-mRNA (ARM) triple will be dissociated by changing the Mg²⁺ concentration.Through several rounds of screening,highly specific ScFv enrichment.The antibody sequence could highly expressed in E.coli.The WB verify that the specific protein bands are the anti-CL antibody.4 Analyse the sensitivity and specificity of the CL antibody preliminarilyWorking parameters of competed ELISA technique for detection of CL was optimized, including selection of coating method,optimum dilution ratio of antiserum and medium mediation.An competed ELISA method for the detection of CL was developed.Calibration graphs was formed by the logarithm of the CL concentration against the inhibitory rate,the half inhibitory concentration(IC₅₀) reached 1 ng/mL.Different cross-reactions with the closely related compounds including salbutamol, epinephrine,norepinephrine,isoproterenol,and commonly used antibiotics gentamicin sulfate and vitamin A by poultry and livestock were observed,with cross-reactions rate value all below 0.07%.Results indicated the antibody with nice specific.