牛偏爱密码子优化后的Beta蛋白多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2015.10.008

Abstract

Abstract:

This study aimed to optimize bet gene of bacteriophage λ so that it can be efficiently expressed in cattle cells. The optimized bet gene was synthesized by genome-wide synthesis method and expressed in prokaryotic expressing vector，the polyclonal antibody was prepared and antibody titer evaluation was preformed.The sequence of bacteriophage λ bet gene was obtained from GenBank(GenBank accession No.:AP005153 ) and optimized with bovine preferred codons. Then synthesized the optimized gene.According to the optimized gene sequence,a pair of primers with the restriction enzyme sites were designed.The bet gene was amplified by PCR and cloned into the pET28a prokaryotic expressing vector. Fusion protein expressed in E.coli BL21 by IPTG induction,then we purified the protein products by affinity chromatography methods and identified by Western blot with His tag antibody. CD1 mice were immunized with the purified fusion protein. Serum with anti-Beta protein polyclonal antibody was obtained, and then the antibody titer was detected by ELISA assay. As a result,confirmed by restriction enzyme digestion and sequencing, pET-bet prokaryotic expressing vector was successfully constructed; The fusion protein was high efficiently expressed, which was confirmed by SDS-PAGE; CD1 mice were immunized with the purified protein, the polyclonal anti-Beta antibody with 1∶41 700 titer was prepared and it could bind His-Beta fusion protein specifically, which was confirmed by ELISA assay and Western blot assay. The successful preparation of anti-Beta protein polyclonal antibody will lay a solid foundation for further studies of the biological functions and applications in bovine and other mammals with bet gene.

CLC Number:

S823.2

QI Yu，XING Yan-ping，ZHOU Huan-min，FANG Jun，QIAN Cheng，WANG Hai-tao. Preparation of Polyclonal Antibody against Beta Protein Optimized with Bovine Preferred Codons[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(10): 1759-1765.

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Preparation of Polyclonal Antibody against Beta Protein Optimized with Bovine Preferred Codons

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