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Cloning and expression of hyperimmunogenic region of surface protein gene of Theileria annulata schizonts
DANG Zhisheng1, LUO
Jianxun1, JIANG Xishi2,
HUANG Xiaobin2, BAI Qi1, YIN
Hong1
(1. Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China; 2. Southwest University for Nationalities,Chengdu 610041,China) |
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Abstract:The total RNA was extracted from cell cultures of Theileria annulata schizonts and single chain cDNA was synthesized by reverse transcription using oligo(dT)18 as the primers. SBxp gene specific primers were designed by DNAstar software. A 393bp specific fragment was amplified by PCR and ligated into pGEMT Easy vector. It was identified by restriction endonucleases, PCR and sequencing that this fragment contained the complete open reading frame of SBxp gene. After pGEMSBxp recombinant vector was digested, SBxp gene was subcloned into the prokaryotic expression vector pGEX4T1, which was also digested with the same enzymes. Positive clones were identified by restriction endonuclease and PCR.The recombinant Escherichia coli was induced by IPTG and the cultures was collected at different times .The expressed products was tested by SDSPAGE and Western blotting. The results showed that SBxp gene was expressed successfully in E.coli and the 43 ku fusion protein can be recognized by the positive serum of T.annulata.
Key words: Theileria annuata; surface protein gene; hyperimmunogenic region; cloning; expression |
