Construction Of Non-immunized Human ScFv T7Select Phage Library And Selection Of Anti-paraquat ScFv Antibody

Paraquat(1,1’-dimethyl-4,4’-bipyridyl dichloride, PQ) is a widely used nonselective herbicide in agriculture around the world. With increasing use of paraquat, there have been many reports of paraquat poisoning, including accidental and intentional ingestion, skin contact, inhalation, and parenteral administration. The death rate of oral poisoning of paraquat is over90%. More and more people are concerning about it. Except for intense invasion, paraquat could always lead to many inconvertable disease but no antidote or effective treatment for paraquat poisoning has been clinically applied, the survival being mainly dependent on the amount ingested and the time elapsed until the patient is submitted to intensive medical procedures. antibodies are of interest, due to their specificity and affinity to the targets. In particular, the use of partially or completely human antibodies, which elicit no or minimal immune response when administered to patients, is yielding an increasing intreset.With phage display technology, we require no immunization but several series of invitro panning from phage displayed library to obtain antibodies which are always human antibodies unavailable from immunization. Another significant aspect of phage display lies in linking the phenotype of a bacteriophage-displayed peptide or protein with the genotype encoding that molecule, packaged within the same virion. This permits the selection and amplification of specific clones of phage representing desired binding sequences from pools of billions of phage clones. All in all, phage display technology is to be the ideal method for the construction of human scFv library.Here we report the generation and characterization of fully human scFv targeting PQ. The experiment include the construction of human scFv library, animal immunization, biopanning and screening, expression and purification of scFv.The three main procedures are:The expression and purification of T7capsid protein10A and the preparation of the polyclonal of10A which facilitates the screening of paraquat from T7phage display.The isolation of lymphocytes from healthy peripheral blood and extraction of totle RNA from lymphocytes by Trizol method. The preparation of cDNA from mRNA. The rapid access to the variable heavy chain(VH) and variable light chain(VL) region gene pools by the polymerase chain reaction(PCR) and the linkage of VH and VL into scFv. A T7phage antibody library is created by cloning these repertoires as fusion proteins with a minor coat protein of the phage.Particular phage antibodies that specifically bind to PQ can be separated from nonbinding phage antibodies by using two biopanning methods alternatively. The solid phase biopanning is based on coating PQ-OVA/BSA on the immunotubes or immunoplates. The liquid phase biopanning needs to conjugate the carboxylated paraquat derivate with the amino magnetic beads. With4cycles of alternative biopanning, specific phages were enriched. The affinities and specificities of seldom selected phage plaques were identified by phage ELISA and indirect competition ELISA. After the expression, one scFv that possessed high specificity and affinity to paraquat was obtained.We obtained polyclonal antibody of T7phage capsid protein10A which was applied in the screening of scFv library. Being succeed in the construction of a large T7Select phage library with large diversity that could be used in the screening of many different kinds of antibodies.1specific antibody with apparent inhibition ratio was obtained. Because of the immensity of the library and the non-specific binding of phage to the BSA or OVA, the difficulty of the panning and the obtaining of non-specific antibodies was irreversible. Better experimental methods need to be explored.