Research On The Preparation And Biological Analysis Of Single-chain Variable Region Fragments Against Enterotoxigenic Escherichia ColiK88

Post Weaning diarrhea(post-weaning diarrhea,PWD)occurred in the first week piglet after weaning or weaning.Clinical symptoms were diarrhea,dehydration,slow growth or stop the growth and caused numerous deaths.It caused huge economic lossesto the world pig industry.Studies have shown that more than 45% of the weaning diarrhea cases was the specific serotype Enterotoxigenic Escherichia coli(ETEC),which accounted for the major part of the K88.ETEC diarrhea caused by the use of antibiotics on the one hand,but drug resistance problem is get worse.On the other hand the use of vaccine therapy for K88 ETEC vaccines are mainly inactivated vaccines,subunit vaccines and genetically engineered vaccine,inactivated vaccine exists large amount of toxins within the residual risks,high cost of production of subunit vaccines,genetically engineered vaccines anti-alone and integrated into strong problem genome.With the development of molecular biology,formulation research and development of novel antibodies against Enterotoxigenic Escherichia coli K88 has received attention.ScFv polypeptide chains of the antibody heavy chain variable region(VH)and light chain variable region(VL)connected together through a short,flexible peptide,the intact antigen-binding site of the smallest functional antibody fragment.ScFv gene engineering antibodies with polyclonal and monoclonal antibodies incomparable advantages: small molecular weight,easy to penetrate;Fc fragment does not contain antibodies,antibody molecules can be avoided nonspecific binding to the receptor;ScFv capable of a variety of systems expression,especially E.coli;also can be genetically engineered into a bi-or multivalent single-chain antibody.Preparation of single chain antibody most commonly used method is a phage-single chain antibody display technology.This study was aimed at ETEC K88 high affinity ScFv,the ScFv its biological function after prokaryotic expression,lay the foundation for the development of antibody preparations original ETEC ETEC K88 FaeG protein expression as an antigen screening.(1)First,primers were designed to amplify the K88 faeG gene,which was ligated into an expression vector pET-28a(+),then using prokaryotic expression and purification by obtained recombinant K88 FaeG protein having activity for the next step single-chain antibody screening foundation.(2)The existing swine single-chain antibody library was used,K88 FaeG was chosen as expressing antigen screening,Screening and enriched by four phage library titer reached 107 CFU / mL,from the fourth round ADS selected randomly picked 48 clones phage antibody prepared using a phage ELISA method for screening to 6 K88 ETEC of swine ScFv.The amino acid sequence of these six ScFv analysiswere confirmed as FR conservative region,CDR3 regions varied greatly,especially VH and VL CDR3 regions.(3)pCANTAB5ehas E-tag label as a carrier,but there is no better method of purification E tag.So we clonedtwo anti-ETEC K88 of ScFv into the prokaryotic expression vector pET-28a(+),thenthe protein was acquired by the prokaryotic expression and purification.Then we study the stability,specificity and affinity of single chain antibodies.It was found that single-chain antibody can be stored at 4 ? one month,the degradation does not occur,-20 and-80 ? in long-term preservation;the affinity may reach 7.21 ± 1.56 and 4.51 ± 0.42 M-1 * 105 Kaff;doesnot react with other pig pathogens such as Enterotoxigenic Escherichia coli K99,Salmonella,Haemophilus parasuis,Pasteurella,epidemic diarrhea virus,transmissible gastroenteritis virus.Bacterial adhesion experiment also shows that our ScFv inhibit enterotoxigenic Escherichia coli K88 on cell adhesion.These preliminary studies of the biological functions ScFv lay the foundation for the development of antibody preparations piglet diarrhea.