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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front. Agric. China    2009, Vol. 3 Issue (4) : 457-465     DOI: 10.1007/s11703-009-0067-1
Research articles |
Construction of a variable regions gene library of antibody against Eimeria acervulian merozoite of chicken
Yuelan ZHAO1,Lei ZHANG1,Li GUO2,Yongzhan BAO2,Jianhua QIN2,
1.College of Chinese Traditional Veterinary Science, Agricultural University of Hebei, Dingzhou 073000, China; 2.College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, China;
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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 (VH) and light chain (VL) genes were amplified by PCR with gene-specific primers. The results showed that the genes of amplified VH and VL were about 420 bp and 390 bp, respectively. PCR products were purified by agarose gel DNA purification kit, and then the VH and VL 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 VH and VL genes were compared with the published VH and VL sequences from the BALB/C mice. The sequences were confirmed with mouse antibodies. As expected, the sequence differences between VH/VL 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 VL/VH had a good diversity.
Keywords Eimeria acervulina merozoite      variable region of heavy chain (VH)      variable region of light chain (VL)      gene library      chicken      
Issue Date: 05 December 2009
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