Construction And Expression Of Gene Of ScFv Antibody Detecting Eimeria Acervulina Surface Antigens

E.acervulina sporozoites were prepared by incubating sporulated oocysts in trypsin and taurodeoxycholic acid in PBS in incubator. Sporozoites were harvested by centrifugation and purified from cellular debris on diethylaminoethyl cellulose column (DE52).Pelleted sporozoites in phosphate-buffered saline were disrupted by 3 freeze-thaw cycles,warmed to room temperature, sonicated on ice in PBS. The positive hybridoma was choiced by antigens.Total RNA was extracted from chicken hybridoma cell secreting MAbs against E.acervulina.The frist chain of cDNA which derived from the total RNA with Oligo(dT)18Primer by RT was used as template,VH and VL genes was amplified by PCR using gene specific primers. PCR pruduct be examined by agarose gel electrophoresis and purified by agarose gel DNA purificatin kit ,the result shows the genes of VH and VL amplified were about 381bp and 312bp respectively.The purified pruduct was cloned pMD18-T vector.The cloned plasmids were transformed into JM109.Colonies were selected on ampicillin-containing agar plates and plasmids were isolated from several independent clones.the specific recombinant plasmid was identified with RV-M primer and M13-47 primer by colony PCR. The positive cloned were sequenced by Sanger's dideoxy sequencing method. Nucleotide sequence of the VH and VL genes sequenced were compared with the published VH1-JH and Vλ1-Jλsequence from the CB strain chicken.As expected, sequence difference between VH/VL and corresponding germline genes were identified, predominantly localized in complementary determining regions.in particular, the VL gene contained a nucleotide deletion in CDR3.same quality of purified VH and VLgenes were jointed together with a linker DNA by splicing overlap extension PCR and a desired fragment(scfv) was generated.After scfv and expression vector were digested with the corresponding restriction enzymes Nco I and Hindlll and purified on an agarose gel,the scfv gene was ligated into expression vector pET-22b(+)and transformed into competent E.coli JM109 cells.the recombinants was grown on the ampr plate. The aimed was identified with T7 promoter primer and T7 terminator primer by colony PCR.the result indicated that the resultant construct contained the gene of scfv fragment at right orientation.Fermentation of the positive coined was...