诺如病毒P-partical-GnIH抗原制备及抗原性初步检测

doi:10.16431/j.cnki.1671-7236.2019.09.025

摘要/Abstract

摘要： 为探索调控家鹅繁殖性能的技术，本研究通过设计合成鹅促性腺激素抑制激素（gonadotropin-inhibitory hormone，GnIH）序列片段，酶切后连接到诺如病毒（Nov）P基因重组pEGX-4T-1载体，转化大肠杆菌DH5α感受态细胞，提取阳性菌重组质粒，并通过测序和酶切进行验证。将测序正确的质粒转化大肠杆菌BL21感受态细胞，利用IPTG诱导重组蛋白表达，SDS-PAGE及Western blotting检测重组蛋白表达。进一步在不同IPTG浓度及诱导时间条件下进行原核表达，确定最佳诱导条件，在最佳条件下的诱导菌液经过离心收集菌体，并超声波破碎离心后对重组蛋白可溶性进行分析。利用xTractor Buffer和Glutathione Superflow树脂对重组蛋白进行纯化，并使用自制的抗GnIH鹅血清及抗鹅二抗通过Western blotting检测重组蛋白。结果显示，重组质粒Nov P-partical-GnIH经双酶切鉴定获得大小约为96 bp的目的基因条带，进一步测序结果证明重组质粒构建成功。诱导表达的重组蛋白分子质量约为64 ku，与预期大小一致，且在37℃、1.0 mmol/L IPTG、诱导2 h条件下表达量最高；菌体超声破碎后经SDS-PAGE检测，重组蛋白主要表达在沉淀中。重组蛋白与GST单抗及抗GnIH阳性血清均能反应，表明此重组蛋白具有较好的反应原性和免疫原性。本研究通过对Nov P-partical-GnIH蛋白原核表达及纯化成功获得了大量纯度较高的重组蛋白，将有助于进一步建立家鹅的繁殖性能免疫调控方法，为提高繁殖性能及调控繁殖机制的研究奠定基础。

关键词: 诺如病毒P颗粒; 促性腺激素抑制激素(GnIH); 蛋白免疫原性; 疫苗

Abstract: In order to explore the technique of regulating the reproductive performance of domestic geese,this study designed a synthetic goose gonadotropin-inhibitory hormone (GnIH) sequence fragment,which was digested and ligated to Norovirus (Nov) P gene recombinant pEGX-4T-1 vector,transformed into E.coli DH5α competent cells,extracted positive bacterial recombinant plasmid,and verified by sequencing and enzyme digestion.The correctly sequenced plasmid was transformed into E.coli BL21 competent cells,and recombinant protein expression was induced by IPTG.The expression of recombinant protein was detected by SDS-PAGE and Western blotting.The prokaryotic expression was further carried out under different IPTG concentration and induction time conditions,and the optimal induction conditions were determined.Under the optimal conditions,the induced bacterial liquid was collected by centrifugation,and the protein was analyzed by ultrasonic disruption and centrifugation.The recombinant protein was purified using xTractor Buffer and Glutathione Superflow resin,and the recombinant protein was detected by Western blotting using self-made anti-GnIH goose serum and anti-goose secondary antibody.The results showed that the recombinant plasmid Nov P-partical-GnIH was identified by double restriction enzyme digestion to obtain the target gene band of about 96 bp.The sequencing results confirmed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant protein induced by expression was about 64 ku,which was consistent with the expected size,and the highest expression was obtained at 37℃,1.0 mmol/L IPTG and induction for 2 h.The recombinant protein was detected by SDS-PAGE after sonication.Mainly expressed in the precipitate.The recombinant protein reacted with both GST monoclonal antibody and anti-GnIH positive serum,indicating that the recombinant protein had good reactogenicity and immunogenicity.In this study,a large number of highly purified recombinant proteins were successfully obtained by prokaryotic expression and purification of Nov P-partical-GnIH protein,which would help to further establish the reproductive regulation of domestic geese and lay the foundation for studing on improving reproductive performance and regulation reproduction mechanism.

Key words: Nov P-partical; gonadotropin-inhibitory hormone(GnIH); protein immunogenicity; vaccine

中图分类号:

S853.74

黄雪冰, 杨培洁, 邓汝森, 杨博, 刘文俊, 黄运茂. 诺如病毒P-partical-GnIH抗原制备及抗原性初步检测[J]. 中国畜牧兽医, 2019, 46(9): 2699-2706.

HUANG Xuebing, YANG Peijie, DENG Rusen, YANG Bo, LIU Wenjun, HUANG Yunmao. Preparation of P-partical-GnIH Antigen of Norovirus and Preliminary Detection of Its Antigenicity[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2699-2706.

参考文献

[1] UBUKA T,SON Y L,TOBARI Y,et al.Central and direct regulation of testicular activity by gonadotropin-inhibitory hormone and its receptor[J].Frontiers in Endocrinolog,2014,5:8.
[2] 张蕊,常玲玲,穆春宇,等.GnIH/GnRH对鸟类排卵的调控作用[J].中国家禽,2018,40(1):42-45. ZHANG R,CHANG L L,MU C Y,et al.Regulation of GnIH/GnRH in avian ovulation[J].China Poultry,2018,40(1):42-45.(in Chinese)
[3] TSUTSUI K,UBUKA T,BENTLEY G E,et al.Review:Regulatory mechanisms of gonadotropin-inhibitory hormone (GnIH) synthesis and release in photoperiodic animals[J]. Frontiers in Neuroscience,2013,7:60.
[4] 张蕊,常玲玲,穆春宇,等.鸽GnIH和GnRH基因克隆及其在不同繁殖阶段下丘脑中的表达[J].中国畜牧兽医,2018,45(9):2478-2485. ZHANG R,CHANG L L,MU C Y,et al.Cloning and expression of GnIH and GnRH genes in hypothalamus of pigeons at different breeding stages[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2478-2485.(in Chinese)
[5] DAN X,LIU X,HAN Y,et al.Effect of the novel DNA vaccine fusing inhibin alpha (1-32) and the RF-amide related peptide-3 genes on immune response,hormone levels and fertility in Tan sheep[J]. Animal Reproduction Science,2016,164:105-110.
[6] 何宗亮.鸡GnRH、GnIH和VIP基因表达与繁殖性能间关系的研究[D].南京:南京农业大学,2009. HE Z L.Study on the relationship between GnRH, GnIH and VIP gene expression and reproductive performance in chicken[D].Nanjing:Nanjing Agricultural University,2009.(in Chinese)
[7] 程军军.GnIH基因疫苗制备及其免疫效果研究[D].武汉:华中农业大学,2017. CHEN J J.Construction of GnIH gene vaccine and its immunization effect[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[8] 张玲玲,杨博辉,岳耀敬,等.GnIH与INH表位多肽疫苗主动免疫对甘肃高山细毛羊生殖激素的影响[J].中国畜牧兽医,2016,43(4):1039-1044. ZHANG L L,YANG B H,YUE Y J,et al.Effect of active immunization with GnIH and INH epitope peptide vaccine on reproductive hormones of Gansu Alpine Fine-wool sheep[J].China Animal Husbandry & Veterinary Medicine,2016,43(4):1039-1044.(in Chinese)
[9] 张玲玲.促性腺激素抑制激素-C3d DNA疫苗的构建及免疫作用[D].北京:中国农业科学院,2016. ZHANG L L.Construction and immunization of gonadotropin-inhibiting hormone-C3d DNA vaccine[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[10] DAN X,HAN L,RIAZ H,et al.Construction and evaluation of the novel DNA vaccine harboring the inhibin alpha (1-32) and the RF-amide related peptide-3 genes for improving fertility in mice[J].Experimental Animals,2016,65(1):17-25.
[11] 淡新刚.GnIH对小鼠繁殖的调控及其分子机制和基因免疫技术研究[D].武汉:华中农业大学,2015. DAN X G.Regulation and molecular mechanism of GnIH on murine repeoduction and study of genetic immunization and study of genetic[D].Wuhan:Huazhong Agricultural University,2015.(in Chinese)
[12] 龙丹丹,嵇辛勤,段志强,等.犬细小病毒VP2蛋白的原核表达与纯化[J].中国畜牧兽医,2018,45(3):643-649. LONG D D,JI X Q,DUAN Z Q,et al.Prokaryotic expression and purification of VP2 protein of canine parvovirus[J]. China Animal Husbandry & Veterinary Medicine.2018,45(3):643-649.(in Chinese)
[13] 戴迎春.基于P颗粒的诺如病毒GⅡ.4型流行株进化机制及免疫保护研究[D].广州:南方医科大学,2013. DAI Y C.Evolution mechanism of major epidemic norovirus GⅡ.4 strain and immunization based on P particle[D].Guangzhou:Southern Medical University,2013.(in Chinese)
[14] TAN M,JIANG X.The P domain of norovirus capsid protein forms a subviral particle that binds to histo-blood group antigen receptors[J].Journal of Virology,2005,79(22):14017-14030.
[15] NASSAL M,SKAMEL C,KRATZ P A,et al.A fusion product of the complete Borrelia burgdorferi outer surface protein A (OspA) and the hepatitis B virus capsid protein is highly immunogenic and induces protective immunity similar to that seen with an effective lipidated OspA vaccine formula[J].European Journal of Immunology,2005,35(2):655-665.
[16] 钟罗华,邓舜洲,胡杨,等.表达猪圆环病毒2型衣壳蛋白的重组猪痘病毒的构建及鉴定[J].中国畜牧兽医,2018,45(2):279-290. ZHONG L H,DENG S Z,HU Y,et al.Construction and identification of recombinant swinepox virus expressing porcine circovirus type 2 capsid protein[J]. China Animal Husbandry & Veterinary Medicine,2018,45(2):279-290.(in Chinese)
[17] 魏德强.以植物病毒为载体建立新型的载体——半抗原免疫体系[D].哈尔滨:东北林业大学,2014. WEI D Q.Establishment of a novel carrier——Hapten immune system using plant virus as a vector[D].Harbin:Northeast Forestry University,2014.(in Chinese)
[18] 金浩,付璐,史晶莹,等.重组诺如病毒P颗粒的表达纯化及免疫原性[J].吉林大学学报(理学版),2017,55(4):1025-1030. JIN H,FU L,SHI J Y,et al.Expression,purification and immunogenicity of recombinant norovirus P particles[J].Journal of Jilin University (Science Edition),2017,55(4):1025-1030.(in Chinese)
[19] 张婷,龙艳,张绪富,等.我国GⅡ.4型诺如病毒Sydney株P颗粒的表达及其组织血型抗原结合特征[J].中国人兽共患病学报,2016,32(4):344-348. ZHANG T,LONG Y,ZHANG X F,et al.Expression of Nov GⅡ.4 Sydeny strain P particle and analysis of its binding activity with HBGAs receptor in China[J].Chinese Journal of Zoonoses,2016,32(4):344-348.(in Chinese)
[20] XIA M,TAN M,WEI C,et al.A candidate dual vaccine against influenza and noroviruses[J].Vaccine,2011,29(44):7670-7677.
[21] 宋维,李善爽,杨桂华,等.用大肠杆菌(Escherichia coli)表达诺如病毒P粒子[J].上海交通大学学报(农业科学版),2011,29(2):37-42. SONG W,LI S S,YANG G H,et al.Express recombinant norovirus P particles in Escherichia coli[J].Journal of Shanghai Jiaotong University (Agricultural Science),2011,29(2):37-42.(in Chinese)
[22] ZHU H,SHAO X,CHEN Z,et al.Induction of out-of-season egg laying by artificial photoperiod in Yangzhou geese and the associated endocrine and molecular regulation mechanisms[J].Animal Reproduction Science,2017,180:127-136.
[23] 张克山,胡彦竞科,韩笑哲,等.鹅不同繁殖时期GnRH和GnIH基因表达和激素浓度变化分析[J].畜牧兽医学报,2016,47(8):1720-1726. ZHANG K S,HU Y J K,HAN X Z,et al.Analysis of the serum concentrations and mRNA expression levels of GnRH and GnIH in geese during different reproductive periods[J].Acta Veterinaria et Zootechnica Sinica,2016,47(8):1720-1726.(in Chinese)
[24] ZHU H X,LIU X Q,HU M D,et al.Endocrine and molecular regulation mechanisms of the reproductive system of Hungarian White geese investigated under two artificial photoperiodic programs[J].Theriogenology,2019,123:167-176.
[25] 王增,马会勤,张文,等.包涵体蛋白的分离和色谱法体外复性纯化研究进展[J].中国生物工程杂志,2009,29(7):102-107. WANG Z,MA H Q,ZHANG W,et al.The progress of inclusion body isolation and chromatograpgic refolding purification methods[J].Chinese Journal of Bioengineering,2009,29(7):102-107.(in Chinese)