鸡肝激酶 B1（LKB1）的克隆与原核表达及其在肝脏、心脏和下丘脑的定位。

Cloning and prokaryotic expression of the chicken liver kinase B1 (LKB1) and its localization in liver, heart and hypothalamus.

机构信息

Jiangxi Provincial Key Laboratory for Animal Health, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi, China.

School of Computer and Information Engineering, Jiangxi Agricultural University, Nanchang, Jiangxi, China.

出版信息

Int J Biol Macromol. 2021 Feb 1;169:513-520. doi: 10.1016/j.ijbiomac.2020.12.195. Epub 2020 Dec 29.

DOI:10.1016/j.ijbiomac.2020.12.195

PMID:33385449

Abstract

Liver kinase B1 (LKB1) is a member of the serine/threonine kinase family, which plays an indispensable role in the organism of animals. In the current study, the chicken LKB1 protein gene was amplified by PCR based on the primers and cDNA templates. Then, the cloning vector was constructed and the target gene was cloned. After that, the target gene was inserted into the expression vector to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) host cells and the LKB1 recombinant proteins were successfully expressed by using Isopropyl-β-D-thiogalactopyranoside (IPTG). Finally, purified LKB1 proteins were used as antigen and the rabbit-derived antiserums were collected. The antiserum titer determined by ELISA was not less than 1:128000. The results of Western blot suggested that the polyclonal antibody is highly specific to chicken LKB1 protein. Immunofluorescence indicated that the LKB1 protein is mainly expressed in the cytoplasm of liver, heart and hypothalamus cells of chicken. Our study showed that the LKB1 polyclonal antibodies produced by this method are effective and can be used to further study the role of LKB1 in the pathogenesis of chicken disease.

摘要

肝激酶 B1（LKB1）是丝氨酸/苏氨酸激酶家族的成员，在动物机体中发挥着不可或缺的作用。本研究基于引物和 cDNA 模板，通过 PCR 扩增鸡 LKB1 蛋白基因，构建克隆载体并克隆目的基因，然后将目的基因插入表达载体，构建重组质粒。将重组质粒转化至 BL21（DE3）宿主细胞，异丙基-β-D-硫代半乳糖苷（IPTG）诱导 LKB1 重组蛋白的表达。最后，将纯化的 LKB1 蛋白作为抗原，收集兔源性抗血清。ELISA 法测定的抗血清效价不低于 1∶128000。Western blot 结果表明，该多克隆抗体对鸡 LKB1 蛋白具有高度特异性。免疫荧光结果表明，LKB1 蛋白主要在鸡肝脏、心脏和下丘脑细胞的细胞质中表达。本研究表明，该方法制备的 LKB1 多克隆抗体有效，可用于进一步研究 LKB1 在鸡病发病机制中的作用。