兔NLRC3基因克隆及其表达特性研究

doi:10.16431/j.cnki.1671-7236.2021.11.004

Abstract

Abstract: This study was aimed to investigate the sequence, molecular structure, and expression characteristics of Oryctolagus cuniculus NOD-like receptor family CARD domain containing 3 (NLRC3) in vivo and in vitro. The primers were designed according to the predicted sequence published by GenBank (accession No. :XM_017338739.1), NLRC3 gene in rabbit was amplified by PCR and cloned, and its molecular structure was predicted and analyzed by bioinformatics method. The pcDNA3.1-rNLRC3-HA vector was constructed, and the subcellular localization of NLRC3 gene in rabbit was carried out by indirect immunofluorescent assay. The tissue distribution of NLRC3 gene in rabbit was detected by Real-time quantitative PCR. After challenged with enterohemorrhagic Escherichia coli (EHEC), the expression level of NLRC3 gene in rabbit was further analyzed. The results showed that the CDS sequence of NLRC3 gene in rabbit was 3 192 bp. The similarity and phylogenetic tree analysis showed that NLRC3 gene in rabbit had highly similarity with other mammalian species in the same branch. NLRC3 protein in rabbit consisted of N-terminal domain, NACHT domain and LRR domain. Tertiary structural model of NLRC3 protein in rabbit formed a typical horseshoe-like structure in a single curvature, with alpha helix forming the convex surface and beta sheet in the concave faces. Indirect immunofluorescence assay showed that NLRC3 protein in rabbit was located in the intracytoplasm, and did not colocate with mitochondria. NLRC3 protein was detected in all tested tissues of rabbit, with the highest level in spleen, followed by mesenteric lymphnodes and lymphoid follicles. The mRNA expression of NLRC3 gene in rabbit was upregulated in spleen, liver and kidney after EHEC infection. The results showed that NLRC3 gene in rabbit was involved in the immune response after EHEC infection. In summary, NLRC3 gene in rabbit was successfully cloned and analyzed by bioinformatics, it was an intracellular receptor and widely distributed in various tissues, it participated in the immune response after bacterial infection. This study provided basic materials for further exploring the regulatory mechanism of NLRC3 in rabbit in inflammatory response.

Key words: NLRC3 gene; cloning; sequence analysis; subcellular localization; tissue distribution

CLC Number:

GUO Mengjiao, LI Mingtao, TAN Huihui, ZHANG Chengcheng, ZHANG Xiaorong, WU Yantao. Study on Cloning and Expression Characteristic of NLRC3 Gene in Rabbit[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 3940-3949.

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Study on Cloning and Expression Characteristic of NLRC3 Gene in Rabbit

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