Molecular characterization and polyclonal antibody preparation of intestinal stem cell marker LGR6 in Xiangyun crucian carp 2

【Objective】In this study, the intestinal stem cell marker LGR6 of Xiangyun crucian carp 2 was cloned, and the polyclonal antibody was also prepared, which providede technical support for revealing the anti-disease mechanism of Xiangyun crucian carp 2 from the perspective of intestinal stem cells. 【Method】PCR was used to amplify the open reading frame(ORF) sequence of 3nLGR6 gene, and bioinformatics analysis was conducted by InterProScan, TMHMM Server 2.0, ExPASy Proteomics Server, SignalP 5.0, PSORT Ⅱ Prediction, SoftBerry-Psite, SWISS-MODEL and MEGA 11.0. Using the prokaryotic expression system to express furion protein, BALB/c female mice were vaccinated with purified fusion protein to prepare polyclonal antibodies, and the distribution of 3nLGR6 in intestine of Xiangyun crucian carp 2 was analyzed based on immunohistochemical analysis. 【Result】The ORF sequence of 3nLGR6 gene was 2874 bp in length and encoded 957 amino acids residues. The theoretical molecular weight of 3nLGR6 protein was approximately 105.4 kD, with a theoretical isoelectric point(pI) of 5.44. It had a leucine repeat domain at its N-terminus, a seven transmembrane GPCR domain at its C-terminus, and a signal peptide cleavage site between ¹⁸Gly and ¹⁹Ser(Sec signal peptide). 3nLGR6 had high homology with LGR6 sequences of other amino acid teleost, with the identity of 92.28% with the triploid zebrafish. The closely related genes to LGR6 in fish, including LGR4, RNF43, RSPO2, CTNNB1, APC, and CTNNA1, were mainly involved in the Wnt and Adherens junction signaling pathways. The polyclonal antibody against 3nLGR6 was successfully prepared by immunizing BALB/c female mice with purified 3nLGR6 protein, and immunohistochemistry showed that the antibody could specifically recognize 3nLGR6 in the intestine of Xiangyun crucian carp 2.【Conclusion】3nLGR6 is highly homologous to the LGR6 amino acid sequence of other species. The structure and function of 3nLGR6 are relatively conserved. The prepared mouse anti-3nLGR6 antibody can specifically recognize the endogenous 3nLGR6 in the intestine of Xiangyun crucian carp 2. This result provides technical support for further studies on the expression of LGR6 in teleost tissues and the mechanisms underlying the role of LGR6 regulating intestinal mucosal homeostasis.