牦牛HMGN1多克隆抗体制备及应用OA北大核心CSTPCD
Preparation and Application of Polyclonal Antibody against Yak HMGN1 Protein
为了克隆牦牛高迁移率族核小体结合蛋白1(HMGN1)基因并制备牦牛HMGN1多克隆抗体,探究HMGN1在牦牛生殖生理研究过程中的作用,本研究通过基因克隆获得牦牛HMGN1基因序列并对其进行生物信息学分析,利用实时荧光定量PCR(qRT-PCR)检测HMGN1在牦牛子宫、卵巢、输卵管中的相对表达量.利用原核表达、亲和层析法纯化牦牛HMGN1重组蛋白,免疫10月龄新西兰大白兔,亲和层析法纯化所制备的牦牛HMGN1多克隆抗体;分别利用蛋白免疫印迹(WB)、免疫组织化学技术(IHC)检测HMGN1在牦牛生殖器官中的表达和定位;利用免疫荧光技术(IF)检测HMGN1蛋白在颗粒细胞中的表达.本试验成功克隆了牦牛HMGN1基因并制备了牦牛HMGN1多克隆抗体.WB结果显示,HMGN1蛋白在黄体期和卵泡期牦牛子宫的表达量高,妊娠期的表达量最低(P<0.05);卵巢组织中,妊娠期的表达量最高,卵泡期的表达量最低(P<0.05);输卵管中妊娠期的表达量最高,黄体期和卵泡期的表达量较低(P<0.05).IHC结果显示,HMGN1主要分布在子宫内膜和子宫腺、卵泡膜和卵泡颗粒层、输卵管黏膜上皮等部位.IF结果显示,HMGN1主要表达在卵巢颗粒细胞的细胞质中.综上,HMGN1在牦牛发情周期和妊娠过程中均参与调节.本研究结果为进一步研究HMGN1蛋白在调节牦牛生殖生理机制提供了基础资料.
The aim of this study was to clone the yak high mobility group nucleosome binding protein 1(HMGN1)gene and prepare a yak HMGN1 polyclonal antibody,so as to provide information for exploring the roles of HMGN1 in yak reproductive physiology.The HMGN1 gene sequence of yak was obtained by gene cloning and analyzed by bioinformatics.The relative expressions of HMGN1 in the yak uterus,ovaries and oviducts were detected by real-time fluorescence quantitative PCR(qRT-PCR).The recombinant protein of yak HMGN1 was purified by prokaryotic expression and affinity chromatography.10-month-old New Zealand white rabbits were immunized,and the polyclonal antibody against yak HMGN1 was purified by affinity chromatography.The expression and localization of HMGN1 in yak reproductive organs were detected by western blot(WB)and immunohistochemistry(IHC),respectively.The expression of the HMGN1 protein in granulosa cells was detected by immunofluorescence(IF).The HMGN1 gene of yaks was successfully cloned,and a polyclonal antibody against yak HMGN1 was prepared.The results show that the expressions of HMGN1 protein are higher in the uterus of the yak during the luteal phase and follicular phase than those in the gestation period(P<0.05).In ovarian tissues,the expressions of HMGN1 are the highest in the gestation period and lowest in the follicular phase(P<0.05).And in oviducts,the highest expression of HMGN1 was in the gestation period but the lowest in the luteal phase and follicular phase(P<0.05).The results of IHC show that HMGN1 is mainly located in the endometrium and uterine glands,theca and follicular granular layer,and mucosal epithelium of oviducts.And it has been found that HMGN1 is mainly expressed in the cytoplasm of granulosa cells by IF.It is stated that HMGN1 is involved in the regulation of the estrous cycle and pregnancy of the yak.The results of this study provide basic data for further study of the role of the HMGN1 protein in regulating the reproductive physiological mechanism of yaks.
李玉平;王立斌;潘阳阳;王萌;张晖;张瑞;贾利毅;余四九
甘肃农业大学动物医学院,甘肃 兰州 730070甘肃农业大学动物医学院,甘肃 兰州 730070||甘肃省牛羊胚胎工程技术研究中心,甘肃 兰州 730070
牦牛HMGN1原核表达多克隆抗体
yakHMGN1prokaryotic expressionpolyclonal antibody
《核农学报》 2024 (011)
2106-2115 / 10
国家自然科学基金(32273091),甘肃省自然科学基金(22JR5RA864)
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