尼罗罗非鱼Lck多克隆抗体的制备及鉴定

文章编号：2095-1191（2018）11-2304-07

Preparation and identification of Lck polyclonal

antibody in Nile tilapia

ZHANG Yong-de， LIN Yong， FENG Peng-fei， CHEN Zhong， DU Xue-song，

HUANG Yin， PAN Chuan-yan，  LUO Hong-lin*

（Guangxi Academy of Fishery Sciences/Guangxi Key Laboratory of Aquatic Genetic Breeding and

Healthy Aquaculture， Nanning  530021， China）

Abstract：【Objective】Preparation and identification of Nile tilapia lymphocyte-specific protein tyrosine kinase（Lck） polyclonal antibodies were carried out to lay a foundation for further research on the function and immune mechanism of Lck protein in tilapia. 【Method】The recombinant plasmid pET-B2m-Lck was constructed with seamless cloning technology， and then was transferred into Escherichia coli strain B21（DH3） for expression induction. After purification by Ni-NTA chromatography column， Lck polyclonal antibody was prepared by immunizing Japanese big ear rabbits with purified fusion protein， and the specificity and immunity titer of polyclonal antibody were detected by Western blotting and ELISA. 【Result】The average hydrophilicity（GRAVY） of Nile tilapia Lck protein was -0.454， which was a hydrophilic protein. Lck protein had rich and well-distributed potential epitope site with no typical transmembrane region. The fusion protein obtained by the expression induction of the prokaryotic vector had a molecular weight of about 67.0 kD， and mainly existed in the form of inclusion body. After immunizing Japanese big ear rabbits， a Lck polyclonal antibody with a titer of 1∶256000 was obtained， which could specifically recognize the Lck protein. The concentration of the antibody purified by Protein G affinity chromatography column was above 10 mg/mL， the purity was about 95%， and the antibody was well purified. 【Conclusion】The Lck fusion protein of Nile tilapia obtained by prokaryotic expression induction has good immunogenicity， and the polyclonal antibody obtained after immunity to big ear rabbit has high efficiency and specificity， which can be used in the study of the organism immune mechanism after bacterial infection of tilapia.

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