Analysis of the biological characteristics, gene cloning, and protein expression of glycogen phosphorylase in functional domains from Sparganum mansoni
TAN Qin-yue1, WANG Qing-yin4, FU Rui-jia2,3, ZHOU Xiao-jun5, LIU Ya-mei2, WANG Mei-mei2, LIN Yu-jin2, LU Gang2, LIANG Pei2,3
1.Guangyuan Central Hospital, Guangyuan 628000, China; 2.School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571199, China; 3.Key Laboratory of Tropical Translalional Medicine of Ministry of Education and School of Tropical Medicine and Laboratory Medicine, Hainan medical University,Haikou, Hainan 571199, China; 4.General Hospital of Medical Community of Fenghua People’s Hospital, Ningbo 315500, China;; 5.Hainan General Hospital, Haikou 570311, China
Abstract：This study aimed to analyze the biological characteristics of glycogen phosphorylase (SmGP) from Sparganum mansoni by bioinformatics analysis, clone the gene and express the functional region of SmGP (fSmGP). Used bioinformatics software, the bioinformatics characteristics of SmGP were analyzed. fSmGP was amplified by PCR and cloned into the prokaryotic expression vector pET-28α. Expression of the functional region of SmGP was induced by isopropyl-β-D-thiolactoside, and the recombinant protein was analyzed by western blot. The GP was composed of 519 amino acids. The relative molecular weight and isoelectric point were 59.8 kDa and 6.95, respectively. The molecular components of SmGP comprised C, H, N, O and S atoms. Compared with GP from other species, the amino acid sequence similarity was greater than 70%. The amino acid sequence contained 32 phosphorylation sites, 10 potential B cell epitopes and 15 potential T cell epitopes. In the secondary structure, the α helix was the main structure. Three-dimensional structural analysis showed that the protein exists in dimeric form, and the functional sites are located in proximity in the molecule. A recombinant plasmid for fSmGP expression was successfully constructed by PCR, double enzyme digestion and sequencing analysis. Western blotting showed that fSmGP was expressed in E. coli. SmGP belongs to the glycosyltransferase-GTB-type supergene family and is a relatively conserved protein. The functional region and epitopes are found in the C-terminus of the molecule. The expression of this functional region should provide guidance for further immunological analysis and also lay a foundation for exploring glucose metabolism in Sparganum mansoni throughout its life cycle stages.
谭钦月, 王清吟, 符瑞佳, 周晓君, 刘亚妹, 王妹妹, 林于金, 吕刚, 梁培. 曼氏裂头蚴糖原磷酸化酶的生物学特性分析及功能域克隆、表达[J]. 中国人兽共患病学报, 2021, 37(4): 311-316.
TAN Qin-yue, WANG Qing-yin, FU Rui-jia, ZHOU Xiao-jun, LIU Ya-mei, WANG Mei-mei, LIN Yu-jin, LU Gang, LIANG Pei. Analysis of the biological characteristics, gene cloning, and protein expression of glycogen phosphorylase in functional domains from Sparganum mansoni. Chinese Journal of Zoonoses, 2021, 37(4): 311-316.
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2021, Vol. 37 
