Abstract：Transferrin receptor (TfR) is a type Ⅱ transmembrane dimer glycoprotein located on the cell membrane, which is the receptor for the invasion of dog (Canis lupus familiaris), cat (Felinae) and other Carnivorous parvovirus into host cells. This study aimed to establish a TaqMan real-time fluorescent quantitative PCR assay for detecting the expression of TfR in carnivore, and to detect and analyze the expression of TfR gene in different tissues of carnivores and passage cells. A pair of primers and a specific TaqMan probe were designed and synthesized by referencing to the gene sequence of the TfR extracellular region protein published in GenBank. The reaction system and conditions of qPCR were optimized, a positive plasmid was constructed, a standard curve was established, and the sensitivity and repeatability test were carried out. The expression of TfR gene in different tissues of carnivores and passage cells were detected and analyzed by the established method. The results showed that the assay had a good linear relationship, sensitivity and stability with a correlation coefficient of 0.998 7, the detection limit was 67 copies/μL, the coefficient of variation of intra-assay and inter-assay evaluation test was less than 2.0%. This study provides a qPCR method for detecting the expression of TfR gene in carnivore with good sensitivity and stability, and it can be used for detecting the expression of TfR gene in different tissues and passage cells of carnivore.
[1] 董巍, 王强, 张剑波. 2003. 免疫细胞化学法检测大鼠骨髓细胞转铁蛋白受体[J]. 临床检验杂志, (S1): 80-81. (Dong W, Wang Q, Zhang J B. 2003, Detecting transferrin receptors in rat bone marrow cells by immunocytochemistry[J]. Chinese Journal of Clinical Laboratory Science, (S1): 80-81.) [2] 贺英, 曹旺斌, 史秋梅, 等. 2011. 基于转铁蛋白受体对犬细小病毒原代细胞组织嗜性的研究[C]. Proceedings of 2011 International Conference on Biomedicine and Engineering (ISBE 2011 V3), 315-318. (He Y, Cao W B, Shi Q M, et al. 2011. Study on the tissue tropism of Canine parvovirus based on the distribution of the transferrin receptors[C]. Proceedings of 2011 International Conference on Biomedicine and Engineering (ISBE 2011 V3), 315-318.) [3] 刘波, 方国安, 金秀国, 等. 2003. 胃癌细胞转铁蛋白受体的表达与化疗药物敏感性的关系[J] .浙江实用医学, (06): 6-7. (Liu B, Fang G A, Jin X G, et al. 2003, Relationship between the expression of transferrin receptor and chemotherapeutic drug sensitivity in gastric cancer cells[J]. Zhejiang Practical Medicine, (06): 6-7.) [4] 秦源, 郭永红, 贾战生, 等. 2017. 转铁蛋白受体(TfR)在丙型肝炎病毒感染中的作用机制研究进展[J]. 细胞与分子免疫学杂志, 33(02): 274-277. (Qin Y, Guo Y H, Jia Z S, et al.2017. Research progress on the mechanism of transferrin receptor in Hepatitis C virus infection[J]. Chinese Journal of Cellular and Molecular Immunology, 33(02): 274-277.) [5] 文雪. 2016. 转铁蛋白受体的结构及功能[J]. 科技视界, (15): 98. (Wen X. 2016. Structure and function of transferrin receptors[J]. Science & Technology Vision, (15): 98.) [6] 吴冬燕, 颜慕霞, 钟玉葵, 等. 2018. 血清可溶性转铁蛋白受体测定在地中海贫血复合铁缺乏诊断与治疗中的意义[J]. 中国优生与遗传杂志, 26(09): 13-15. (Wu D Y, Yan M X, Zhong Y K, et al.2018. Significance of serum soluble transferrin receptor in diagnosis and treatment of thalassemia combined with iron deficiency[J]. Chinese Journal of Birth Health & Heredity, 26(09): 13-15.) [7] 杨颖, 郜振彦, 颜崇淮. 2018. 血清可溶性转铁蛋白受体诊断儿童铁缺乏的研究进展[J]. 中国儿童保健杂志, 26(09): 969-972. (Yang Y, Gao Z Y, Yan C H.2018. Research progress of serum soluble transferrin receptor in diagnosis of iron deficiency in children[J]. Chinese Journal of Child Health Care, 26(09): 969-972.) [8] 张薇, 闫琰, 白鲁根, 等. 2017. 稳定表达犬转铁蛋白受体CHO细胞系的建立和鉴定[J]. 四川大学学报(医学版), 48(01): 57-60, 80. (Zhang W, Yan Y, Bai L G, et al.2017. The establishment and identification of stable CHO cell line with canine transferrin receptor expression[J]. Journal of Sichuan University (Medical Sciences), 48(01): 57-60, 80.) [9] 邹翔宇, 胡楠, 鲁承. 2016. 肉食兽细小病毒研究进展[J]. 吉林畜牧兽医, 37(06): 16-18, 20. (Zou X Y, Hu N, Lu C, et al.2016. Advances in research on Carnivorous parvovirus[J]. Jilin Animal Husbandry and Veterinary Medicine, 37(06): 16-18, 20.) [10] Chan L N, Gerhardt E M.1992. Transferrin receptor gene is hyperexpressed and transcriptionally regulated in differentiating erythroid cells[J]. Biological Chemistry, 267(12): 8254-8259. [11] Kerstin W, Bernd H, Donata K, et al.2011. Development and validation of a triplex realtime PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of bovine herpesvirus type 1[J]. Journal Virology Methods, 174: 77-84. [12] Hueffer K, Palermo L M, Parrish C R.2004. Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization,and capsid-binding domains[J]. Journal of Virology, 78(11): 5601-5611. [13] Park G S, Best S M, Bloom M E.2005. Two mink parvoviruses use different cellular receptors for entry into CRFK cells[J]. Virology, 340(1): 1-9. [14] Parker J S L, Murphy W J, Wang D, et al.2001. Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells[J]. Journal of Virology, 75(8): 3896. [15] Schneider C, Owen M J, Banville D, et al.1984. Primary structure of human transferrin receptor deduced from the mRNA sequence[J]. Nature, 311(5987): 675-678. [16] Trowbridge I S, Omary M B.1981. Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin[J]. Proceedings of the National Academy of Sciences of the USA, 78(5): 3039-3043. [17] Webb T L, Burnett R C, Avery A C, et al.2010. Cloning and tissue expression of the equine transferrin receptor[J]. Veterinary Clinical Pathology, 39(4): 424-432.
