猫源C11orf96基因表达及其在细胞中的定位分析

doi:10.11843/j.issn.0366-6964.2022.07.035

Abstract

Abstract: The aim of this study was to construct of Felis catus C11orf96 (fC11orf96) gene, prepare polyclonal antibody with good reactivity to the protein and in order to analyze the localization of the C11orf96 gene in cells. Firstly, the fC11orf96 gene cloned by using Crandell Reese Feline Kidney cells (CRFK) cDNA as the template, the recombinant plasmid pET-32a (+)-fC11orf96-Fe was successfully constructed by seamless recombination. Then, the recombinant expression vector was transformed into BL21(DE3), and the recombinant protein fC11orf96-Fe was successfully expressed after IPTG induction. The fC11orf96-Fe protein was used to immunize rabbits to prepare polyclonal antibody. Finally, Western blot and indirect immunofluorescence assay (IFA) were used to analyze the validity and subcellular localization of polyclonal antibody. The results showed that the CDS sequence of the fC11orf96 gene was 372 bp, encoding 124 amino acids. The recombinant protein fC11orf96-Fe exists mainly in inclusion bodies, and the protein size was 49 ku. The polyclonal antibody prepared from the recombinant protein could identify endogenous fC11orf96 protein and eukaryotic expressed foreign protein. And we found that fC11orf96 protein was localized in the cytoplasm. In this study, the fC11orf96 gene was successfully cloned and localized in the cytoplasm, which laid a foundation for further research on the biological functions of the C11orf96 protein.

Key words: C11orf96, gene cloning, polyclonal antibody, subcellular localization

CLC Number:

YANG Hongzao, ZHU Jie, GUO Hongyuan, TANG Aoxing, CHEN Shaoyu, LIU Chuncao, ZHANG Da, YUAN Ligang, LIU Guangqing. Expression and Subcellular Localization Analysis of Felis catus C11orf96[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2390-2395.

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Expression and Subcellular Localization Analysis of Felis catus C11orf96

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