Construction of a variable regions gene library of antibody against Eimeria acervulian merozoite of chicken

Abstract

Antigens of Eimeria acervulina merozoites were prepared and purified. BALB/C mice were inoculated with the merozoite soluble antigens of E. acervulina. The total RNA was extracted from the spleen cells of the immunized BALB/C mice. The first chains of cDNA were synthesized by reverse transcription with Oligo (dT)₁₅ Primer, using extracted RNA as a template. The variable regions of heavy chain (V_H) and light chain (V_L) genes were amplified by PCR with gene-specific primers. The results showed that the genes of amplified V_H and V_L were about 420 bp and 390 bp, respectively. PCR products were purified by agarose gel DNA purification kit, and then the V_H and V_L gene libraries were constructed, respectively. The purified products were cloned into PGM-T vectors. The cloned plasmids were transformed into Escherichia coli TOP10. The colonies were selected on ampicillin-containing agar plates and the plasmids were isolated from several independent clones. The selected colonies were sequenced using Sanger’s dideoxy sequencing method. Nucleotide sequences of the V_H and V_L genes were compared with the published V_H and V_L sequences from the BALB/C mice. The sequences were confirmed with mouse antibodies. As expected, the sequence differences between V_H/V_L and the corresponding germline genes were identified and predominantly localized in complementary determining regions. The sequence analysis results of partial clones indicated that the constructed gene libraries of V_L/V_H had a good diversity.

Keywords

Eimeria acervulina merozoite / variable region of heavy chain (V_H) / variable region of light chain (V_L) / gene library / chicken