鹅VDR基因克隆及其多克隆抗体制备与鉴定

doi:10.16431/j.cnki.1671-7236.2024.03.031

摘要/Abstract

摘要： 【目的】维生素D₃(vitamin D₃,VD₃)是一类重要的类固醇激素,参与调控动物体内多种生理活动,具有生物活性的VD₃代谢物1,25(OH)₂D₃通过与维生素D受体(vitamin D receptor,VDR)结合发挥生物学功能。试验旨在克隆鹅VDR基因CDS序列并制备鹅VDR多克隆抗体,为后续开展VDR介导VD₃调控鹅体内多种生理活动的分子机制研究提供基础支撑。【方法】通过基因克隆方法获得鹅VDR基因CDS序列,并采用生物信息学方法对其编码蛋白进行结构和功能预测分析。通过基因合成和亚克隆方法构建鹅VDR基因重组表达质粒,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达和镍柱纯化后获得鹅VDR重组蛋白。以纯化的鹅VDR重组蛋白作为免疫原,制备兔抗鹅VDR多克隆抗体,采用间接ELISA法检测抗体效价,采用Western blotting检测抗体特异性。【结果】试验成功克隆了鹅VDR基因编码区序列,长度为1 356 bp,编码451个氨基酸残基。该编码蛋白与鸡VDR蛋白的相似性高达91.4%,无信号肽和跨膜结构域,N-端含有核定位序列,且含有DNA结合域和配体结合域2个保守结构域,属于类固醇/甲状腺激素受体超家族成员。试验成功构建了pET-30a(+)-VDR重组质粒,并诱导表达鹅VDR重组蛋白。纯化后的重组蛋白纯度高达90%以上,且大小符合预期(52 ku)。试验制备了兔抗鹅VDR多克隆抗体,其抗体效价＞1∶1 093 500;除肝脏组织外,该多克隆抗体在产蛋鹅肾脏、十二指肠、空肠和回肠组织中均能够识别天然VDR蛋白的2种亚型。【结论】试验成功克隆了鹅VDR基因CDS序列,并制备了兔抗鹅VDR多克隆抗体,该多克隆抗体特异性高,可用于后续鹅体内VDR介导的生物学功能研究。

关键词: 鹅; VDR基因; 克隆; 多克隆抗体

Abstract: 【Objective】 Vitamin D₃ (VD₃) is an important steroid hormone involved in the regulation of various physiological activities in animals.Its bioactive metabolite 1,25 (OH)₂D₃ performs the biological function by binding to vitamin D receptor (VDR).Cloning of goose VDR gene CDS and preparation of its polyclonal antibody would provide the basic support for further research on the molecular mechanism of VDR-mediated VD₃ regulation of various physiological activities in geese.【Method】 The CDS sequence of VDR gene in geese was obtained by the gene cloning method,and the structure and function of the encoded protein were predicted by bioinformatic methods.The recombinant plasmid of VDR gene in geese was constructed by gene synthesis and sub-cloning methods,and then was transformed into Escherichia coli BL21(DE3) competent cells.The recombinant VDR protein in geese was obtained by induction with IPTG and purification with a nickel column.The rabbit anti-goose VDR polyclonal antibody was prepared with the purified recombinant VDR protein as an immunogen.The antibody titer was detected by indirect ELISA,and the antibody specificity was detected by Western blotting.【Result】 The CDS sequence of VDR gene in geese was successfully cloned with a length of 1 356 bp and was predicted to encode 451 amino acid residues.The coding protein shared a high identity with chicken VDR protein (91.4%),and no signal peptide and transmembrane domain were observed.The coding protein contained an N-terminal nuclear localization sequence and two conserved domains of DNA binding domain and ligand binding domain,suggesting that it belonged to the steroid/thyroid hormone receptor superfamily.The recombinant plasmid pET-30a(+)-VDR was successfully constructed and the recombinant VDR protein in geese was expressed.The purity of the purified recombinant protein was more than 90% and its size was 52 ku as expected.The rabbit anti-goose VDR polyclonal antibody was successfully prepared,and the titer was greater than 1∶1 093 500.Except for the liver tissue,the polyclonal antibody was able to recognize two isoforms of native VDR protein in kidney,duodenum,jejunum and ileum in laying geese.【Conclusion】 The CDS region of VDR gene in geese was successfully cloned,and the rabbit anti-goose VDR polyclonal antibody was prepared.The polyclonal antibody had a high specificity and could be used for further study of VDR-mediated biological function in geese.

Key words: goose; VDR gene; cloning; polyclonal antibody

中图分类号:

S835

秦逸飞, 陈蓉, 施振旦. 鹅VDR基因克隆及其多克隆抗体制备与鉴定[J]. 中国畜牧兽医, 2024, 51(3): 1194-1202.

QIN Yifei, CHEN Rong, SHI Zhendan. Cloning of VDR Gene and Preparation and Identification of Its Polyclonal Antibody in Geese[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 1194-1202.

参考文献

[1] MORA J R, IWATA M, VON ANDRIAN U H.Vitamin effects on the immune system:Vitamins A and D take centre stage[J].Nature Reviews Immunology, 2008, 8(9):685-698.
[2] FLEET J C.Vitamin D-mediated regulation of intestinal calcium absorption[J].Nutrients, 2022, 14(16):3351.
[3] GRZESIAK M, TCHURZYK M, SOCHA M, et al.An overview of the current known and unknown roles of vitamin D₃ in the female reproductive system:Lessons from farm animals, birds, and fish[J].International Journal of Molecular Sciences, 2022, 23(22):14137.
[4] HAUSSLER M R, WHITFIELD G K, KANEKO I, et al.Molecular mechanisms of vitamin D action[J].Calcified Tissue International, 2013, 92(2):77-98.
[5] TAKEYAMA K, KATO S.The vitamin D₃ 1alpha-hydroxylase gene and its regulation by active vitamin D₃[J].Bioscience Biotechnology and Biochemistry, 2011, 75(2):208-213.
[6] HAUSSLER M R, HAUSSLER C A, JURUTKA P W.Genomically anchored vitamin D receptor mediates an abundance of bioprotective actions elicited by its 1, 25-dihydroxy vitamin D hormonal ligand[J].Vitamins and Hormones, 2023, 123:313-383.
[7] LU Z, HANSON K, DELUCA H F.Cloning and origin of the two forms of chicken vitamin D receptor[J].Archives of Biochemistry and Biophysics, 1997, 339(1):99-106.
[8] YOSHIMURA Y, OHIRA H, TAMURA T.Immunocytochemical localization of vitamin D receptors in the shell gland of immature, laying, and molting hens[J].General and Comparative Endocrinology, 1997, 108(2):282-289.
[9] HUI Q, ZHAO X, LU P, et al.Molecular distribution and localization of extracellular calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) at three different laying stages in laying hens (Gallus gallus domesticus)[J].Poultry Science, 2021, 100(5):101060.
[10] 田晓晓,王志跃,杨海明,等.不同水平维生素D₃对扬州鹅仔鹅钙磷代谢和免疫机能的影响[J].中国农业科学, 2013, 46(15):3206-3211. TIAN X X, WANG Z Y, YANG H M, et al.Effect of different levels of vitamin D₃ on metabolism of calcium and phosphorus and immune function of Yangzhou goslings[J].Scientia Agricultura Sinica, 2013, 46(15):3206-3211.(in Chinese)
[11] 田晓晓,王志跃,杨海明,等.饲粮维生素D₃添加量对扬州鹅生长性能、屠宰性能和血清生化指标的影响[J].动物营养学报, 2013, 25(5):978-984. TIAN X X, WANG Z Y, YANG H M, et al.Effects of dietary vitamin D₃ supplemental level on growth performance, slaughter performance and serum biochemical indexes of Yangzhou geese[J].Chinese Journal of Animal Nutrition, 2013, 25(5):978-984.(in Chinese)
[12] 王迪,王宝维,葛文华,等.维生素D₃对鹅生长性能、屠宰性能、胴体品质和胫骨发育的影响[J].中国农业科学, 2013, 46(7):1470-1480. WANG D, WANG B W, GE W H, et al.Effects of vitamin D₃ on growth performance, slaughter performance, carcass quality and tibia quality of geese[J].Scientia Agricultura Sinica, 2013, 46(7):1470-1480.(in Chinese)
[13] 王宝维,王迪,葛文华,等.维生素D₃对14周龄青农灰鹅血清生化指标、免疫性能及维生素D受体mRNA表达量的影响[J].动物营养学报, 2014, 26(7):1760-1768. WANG B W, WANG D, GE W H, et al.Effects of vitamin D₃ on serum biochemical parameters, immune function and vitamin D receptor mRNA expression level of Qingnonghui geese aged from 1 to 4 weeks[J].Chinese Journal of Animal Nutrition, 2014, 26(7):1760-1768.(in Chinese)
[14] WANG H, WANG X Y, ZHAN Y C, et al.Dietary vitamin D₃ requirement of magang goslings from 1-21 days of age[J].Animal Feed Science and Technology, 2023, 304:115757.
[15] 邢月,王宝维,葛文华,等.不同水平维生素D₃对产蛋期种鹅繁殖性能、血清生殖激素及抗氧化指标的影响[J].中国畜牧杂志, 2021, 57(5):183-187. XING Y, WANG B W, GE W H, et al.Effects of different levels of vitamin D₃ on reproductive performance, serum reproductive hormones and antioxidant indexes of breeding goose during laying period[J].Chinese Journal of Animal Science, 2021, 57(5):183-187.(in Chinese)
[16] 邢月,王宝维,葛文华,等.维生素D₃对产蛋期种鹅蛋品质、血液生化指标及蛋壳超微结构的影响[J].中国畜牧杂志, 2022, 58(1):173-178. XING Y, WANG B W, GE W H, et al.Effect of vitamin D₃ on egg quality, blood biochemical indexes and eggshell ultrastructure of breeding goose during laying period[J].Chinese Journal of Animal Science, 2022, 58(1):173-178.(in Chinese)
[17] ZHANG B, ZHANG M, XING Y, et al.Dietary cholecalciferol and 25-hydroxycholecalciferol supplementation interact to modulate reproductive performance, egg quality, serum antioxidant capacity, intestinal morphology and tibia quality of breeder geese[J].British Poultry Science, 2023, 64(5):625-633.
[18] GARCIA L A, KING K K, FERRINI M G, et al.1, 25(OH)₂ vitamin D₃ stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells[J].Endocrinology, 2011, 152(8):2976-2986.
[19] CHENG M, SONG Z, GUO Y, et al.1alpha, 25-dihydroxy vitamin D₃ improves follicular development and steroid hormone biosynthesis by regulating vitamin D receptor in the layers model[J].Current Issues in Molecular Biology, 2023, 45(5):4017-4034.
[20] YAO X, ZHANG G, GUO Y, et al.Vitamin D receptor expression and potential role of vitamin D on cell proliferation and steroidogenesis in goat ovarian granulosa cells[J].Theriogenology, 2017, 102:162-173.
[21] CARLBERG C, MUNOZ A.An update on vitamin D signaling and cancer[J].Seminars in Cancer Biology, 2022, 79:217-230.
[22] JIA B, JEON C O.High-throughput recombinant protein expression in Escherichia coli:Current status and future perspectives[J].Open Biology, 2016, 6(8):160196.
[23] SINGHVI P, PANDA A K.Solubilization and refolding of inclusion body proteins[J].Methods in Molecular Biology, 2022, 2406:371-387.
[24] 何赛,周欢庆,陈昱希,等.鸡生长激素的原核表达、纯化及活性研究[J].中国家禽, 2016, 38(5):5-9. HE S, ZHOU H Q, CHEN Y X, et al.Prokaryotic expression, purification and activity study of chicken growth hormone[J].China Poultry, 2016, 38(5):5-9.(in Chinese)