牦牛Fas相关因子1多克隆抗体制备及初步应用

Preparation and Preliminary Application of Yak(Bos Grunniens)Fas-associated Factor 1 Polyclonal Antibody

Fas相关因子1(Fas-associated factor-1,FAF1)为Fas家族成员,含有数个多功能结构域,可参与细胞凋亡、炎症、坏死、细胞增殖和蛋白质稳态等生物学过程。本研究旨在制备牦牛FAF1多克隆抗体,探索FAF1在牦牛不同年龄睾丸组织及不同繁殖周期卵巢的生物学作用。试验扩增出牦牛FAF 1基因全序列,构建牦牛pET28a-FAF1蛋白原核表达载体,优化诱导条件,通过Ni-NTA柱纯化后,免疫试验动物,获得兔源FAF1多克隆抗体。选取幼年(1、2周岁)、成年(3、4、6、7周岁)、老年(11周岁)三个年龄段雄性牦牛睾丸,选取不同繁殖周期(卵泡期、黄体期和妊娠期)雌性牦牛卵巢,采用实时荧光定量、Western blot、免疫组织化学(IHC)方法分别检测不同年龄雄性牦牛睾丸及不同繁殖周期雌性牦牛卵巢中FAF1 mRNA、蛋白相对表达量及蛋白的表达定位。结果表明,成功构建原核表达载体pET28a-FAF1,25℃、IPTG浓度为0.5 mmol·L^(-1)诱导5 h为最优诱导条件,该重组蛋白主要存在于包涵体中,免疫试验动物后获得兔源FAF1多克隆抗体,抗体效价为1∶1024000,特异性良好。睾丸组织实时荧光定量结果显示,FAF1 mRNA在3、4、7周岁雄性牦牛睾丸组织中相对表达量显著高于其他年龄段;睾丸组织Western blot结果显示,FAF1蛋白在11周岁时相对蛋白表达量显著高于其他年龄段,6、7周岁次之,3、4周岁FAF1相对蛋白表达量最低;睾丸组织免疫组织化学结果显示,FAF1主要表达于精子、精子细胞、精原细胞、初级精母细胞、支持细胞、睾丸间质细胞和管周肌样细胞中。卵巢组织实时荧光定量结果显示,雌性牦牛黄体期卵巢FAF1 mRNA相对表达量显著高于卵泡期和妊娠期,妊娠期卵巢次之,卵泡期卵巢最低;卵巢Western blot结果显示,卵泡期和妊娠期卵巢FAF1蛋白相对表达量显著高于黄体期,卵泡期卵巢表达量最高,妊娠期卵巢次之,黄体期卵巢中表达量最低;卵巢IHC结果显示FAF1主要表达于卵巢生殖上皮细胞、颗粒细胞、卵丘细胞、卵泡膜细胞和黄体细胞。FAF1 mRNA和蛋白在不同年龄睾丸及不同繁殖周期卵巢组织存在显著差异化表达,综上推测,FAF1可能与雄性牦牛的睾丸发育、精子发生及睾酮分泌密切相关,与雌性牦牛的卵泡闭锁、发育、成熟和黄体溶解生理过程相关,本研究为更进一步探索FAF1在牦牛生殖生理功能方面提供依据。

Fas-associated factor-1(FAF1)is a member of the Fas family and contains several multifunctional domains.It can participate in biological processes such as apoptosis,inflammation,necrosis,cell proliferation and protein homeostasis.The purpose of this study was to prepare the polyclonal antibody of yak FAF1,and to explore the biological role of FAF1 in testicular tissues of different ages,and ovaries of different reproductive cycles.The whole sequence of yak FAF1 gene was amplified,and the prokaryotic expression vector pET28a-FAF1 was constructed.The induction conditions were optimized.After purification by Ni-NTA column,the experimental animals were immunized to obtain rabbit FAF1 polyclonal antibody.The testes of male yaks in three age groups of juvenile(1-year-old,2-year-old),adult(3-year-old,4-year-old,6-year-old,7-year-old)and elderly(11-year-old)were selected,and the ovaries of female yaks in different breeding cycles(follicular phase,luteal phase and pregnancy)were selected.Real-time fluorescence quantitative,Western blot and immunohistochemical(IHC)methods were used to detect the relative expression of FAF1 mRNA,protein and protein expression localization in testes of different ages and ovaries of different breeding cycles.The results showed that the prokaryotic expression vector pET28a-FAF1 was successfully constructed,and the optimal induction conditions were 25℃,IPTG concentration of 0.5mmol·L^(-1)and induction for 5 hours.The recombinant protein was mainly present in the inclusion body.After immunizing experimental animals,rabbit-derived FAF1 polyclonal antibody was obtained,with a antibody titer of 1∶1024000 and good specificity.Real-time fluorescence quantitative results of testicular tissue showed that the relative expression of FAF1 mRNA in testicular tissue of 3,4 and 7 years old was significantly higher than that of other ages.The Western blot results of testicular tissue showed that the relative protein expression of FAF1 protein at 11 years old was significantly higher than that at other ages,followed by 6 and 7 years old,and the relative protein expression of FAF1 at 3 and 4 years old was the lowest.Immunohistochemistry showed that FAF1 was mainly expressed in sperm,sperm cells,spermatogonia,primary spermatocytes,Sertoli cells,Leydig cells,peritubular myoid cells and seminiferous tubules.The real-time fluorescence quantitative results of ovarian tissue showed that the relative expression of FAF1 mRNA in the luteal phase was significantly higher than that in the follicular phase and pregnancy,followed by the pregnancy ovary,and the follicular phase ovary was the lowest.The results of ovarian Western blot showed that the relative expression of FAF1 protein in ovarian follicular phase and pregnancy were significantly higher than that in luteal phase,followed by pregnancy,and the lowest expression in luteal phase.Ovarian IHC results showed that FAF1 was mainly expressed in ovarian reproductive epithelial cells,granulosa cells,cumulus cells and luteal cells.The expression of FAF1 mRNA and protein in testis of different ages and ovarian tissues of different reproductive cycles is significantly different.It is speculated that FAF1 may be closely related to the development of testis,spermatogenesis and testosterone secretion of male yaks,as well as the physiological processes of follicular atresia,development,maturation and luteolysis of female yaks.The current study provides the basis for further exploring the reproductive physiological function of FAF1 in yaks.