CN116948028A - 一种抗抑制素抗体及其应用 - Google Patents
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Abstract
本发明提供了一种抗抑制素抗体及其应用，其是以重组人抑制素α链为免疫原制备得到抗抑制素抗体，该抗体可以有效促进哺乳动物排卵，提高雌性哺乳动物的受孕率和产仔量。可用于开发促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖、诱导母畜发情的药物。同时，本发明的抗抑制素抗体对抑制素具有较高的亲和力，可应用于抑制素的检测分析，具有较高的灵敏性和检测下线。
Description
技术领域
本发明涉及抗抑制素抗体技术领域，更具体地，涉及一种抗抑制素抗体及其应用。
背景技术
抑制素是糖蛋白激素，由α与β亚单位经过两个硫键相连组成。于1932年，McCullagh在睾丸提取物中发现。抑制素源于睾丸支柱细胞的一种大分子多肽，具有强烈的抑制FSH分泌的作用，但对LH的分泌仅具轻微的抑制作用。抑制素由卵巢颗粒细胞分泌，相对分子质量为32000。抑制素可以反馈抑制垂体前叶促卵泡激素的释放，调节卵泡的生成。
目前，大量研究发现通过主动或被动免疫中和抑制素，促使内源FSH的产生和分泌，均能刺激雌性动物卵泡发育以及增加排卵数，表明这种两种方法都具有调控家畜繁殖性能的潜力，对畜牧生产和经济动物繁殖具有重要价值。
提高家畜产仔数的方法通常是对家畜注射FSH或FSH类似的药物，拮抗抑制素发挥作用，但是该方法却存在胚胎利用率低的问题。因此，如何有效提高家畜的繁殖能力对于当下家畜市场而言至关重要。
发明内容
针对现有技术所存在的技术问题，本发明提供了一种抗抑制素抗体及其应用。本研究以重组人抑制素α链为免疫原，并制备得到抗抑制素抗体，并发现该抗体可以有效促进哺乳动物排卵，提高雌性哺乳动物的受孕率和产仔量。
具体而言，本发明首先是提供了一种抗抑制素抗体，其特征在于，所述抗抑制素抗体重链可变区序列如SEQ ID NO.1所示，轻链可变区序列如SEQ ID NO.2所示。
进一步地，所述抗抑制素抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示，所述抗抑制素抗体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ ID NO.6-8；
进一步地，本发明还提供了一种荧光标记的抗抑制素抗体，进一步地，本发明提供的所述荧光标记为FITC、FAM、Rhodamine B、TAMRA、Cy3、Cy5等荧光基团标记。
进一步地，本发明提供的所述荧光标记为FITC。
进一步地，本发明还提供了一种非诊断目的抑制素直接免疫荧光检测方法，该方法包括采用所述荧光标记的抗抑制素抗体接触待检样本，孵育结束后，在荧光显微镜下观察结果或酶标仪读取荧光值。
进一步地，本发明还提供了上述荧光标记的单克隆抗体在制备检测抑制素检测试剂盒中的应用。
进一步地，本发明还提供了抗抑制素抗体在制备用于刺激雌性哺乳动物卵泡发育、增加排卵数、增加受孕率或提高产仔量药物中的用途。
进一步地，所述雌性哺乳动物是选择鼠、猪、牛、羊等。
进一步地，本发明还提供了一种刺激雌性哺乳动物卵泡发育、增加排卵数、增加受孕率或提高产仔量的药物，其特征在于，所述药物包括所述的抗抑制素抗体和药学上可接受的辅料。
本发明的优点如下：本发明提供了以重组人抑制素α链为免疫原制备得到抗抑制素抗体，并发现该抗体可以有效促进哺乳动物排卵，提高雌性哺乳动物的受孕率和产仔量。可用于开发促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖、诱导母畜发情的药物。同时，本发明的抗抑制素抗体对抑制素具有较高的亲和力，可应用于抑制素的检测分析，具有较高的灵敏性和检测下线。
附图说明
图1为抗抑制素抗体对抑制素检测灵敏度分析。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明，以使本领域的技术人员更加清楚地理解本发明。
实施例1抗抑制素抗体的制备
1.1杂交瘤细胞系制备
以150μg/mL重组人抑制素α链（购自齐一生物科技（上海）有限公司）为免疫原对6只6-8周龄雌性BALB/c小鼠进行免疫注射，每次皮下注射150μL，间隔时间1周。经连续三次间隔免疫后，对小鼠断尾采血，用间接 ELISA 方法监测血清抗体效价，并选择效价最高的免疫小鼠，用纯的抗原液尾静脉加强免疫，3天后无菌采取小鼠脾细胞与 SP2/0 细胞融合，两种细胞之比为 1:10，将培养板放置 37℃ 5%的 CO2培养箱中培养。经6天的连续培养后，取上清进行抗体检测。
1.2杂交瘤细胞系筛选
用间接ELISA方法检测杂交瘤细胞分泌抗体的情况，以筛选出阳性细胞株。具体方法为取杂交瘤细胞培养上清150μL加入前日包被好的酶标板孔中，同时设立阳性、阴性对照和空白对照。判定标准为OD490值阳性孔与对照孔比值≥2.0判定为阳性。阴性孔3d后再检测一次，如仍为阴性则弃之。
1.3单克隆抗体免疫球蛋白重链和轻链类型的鉴定
取杂交瘤细胞株培养上清，采用IgG抗体检测试剂盒(上海抚生实业有限公司)测定抗体重链、轻链类型。
经鉴定，本发明所述抗抑制素抗体的重链可变区序列如SEQ ID NO.1所示，轻链可变区序列如SEQ ID NO.2所示。所述抗抑制素抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示，所述抗抑制素抗体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ ID NO.6-8。
1.4单克隆抗体的特性鉴定
单抗效价的测定
用间接 ELISA 方法测定单克隆抗体的效价。以重组人抑制素α链为包被抗原，将抗抑制素抗体以 1:10 开始倍比稀释，同时以同等稀释度的注射 SP2/0 细胞的小鼠腹水作为阴性对照。以平行稀释的单抗 OD490值/对照OD490≥2.1 的最高稀释倍数为单抗的效价。结果显示：三免后小鼠的血清效价均在107以上，达到融合要求。
实施例2抗抑制素抗体对小鼠排卵影响
选取6-8周龄雌性小鼠，随机分成5组，每组5只小鼠，其中处理组1是腹腔注射1μg/kg抗抑制素抗体，处理组2是腹腔注射5μg/kg抗抑制素抗体，处理组3是腹腔注射10μg/kg抗抑制素抗体，阴性对照组是腹腔注射同等体积的生理盐水，阳性对照组是腹腔注射10IUPMSG。3天后进行二次腹腔注射，经二次注射后的第4天对参与实验小鼠进行解剖取卵计数。结果如表1所示。
表1. 抗抑制素抗体对小鼠排卵影响
| 阴性对照组 | 阳性对照组 | 处理组1 | 处理组2 | 处理组3 | |
| 排卵个数 | 0 | 17.6±1.23 | 21.8±1.62 | 26.2±2.51 | 32.7±2.85 |
从结果可以看出，腹腔注射本发明所述抗抑制素抗体后可以明显提高小鼠的排卵数量，且随着抗抑制素抗体的注射剂量的加大，小鼠排卵数量也呈现上升现象。
实施例3抗抑制素抗体对小鼠受胎率、产仔数的影响
选取6-8周龄雌性小鼠，随机分成5组，每组10只小鼠，其中处理组1是腹腔注射1μg/kg抗抑制素抗体，处理组2是腹腔注射5μg/kg抗抑制素抗体，处理组3是腹腔注射10μg/kg抗抑制素抗体，阴性对照组是腹腔注射同等体积的生理盐水，阳性对照组是腹腔注射10IUPMSG，腹腔注射后24h对每只小鼠注射10IU HCG并配种。25天后统计受胎率，产仔后统计产仔数。
结果如表2所示：在1μg/kg的抗抑制素抗体用量情况下，小鼠的受胎率(90％)明显高于阳性对照(60％)，其均产仔数平均可达到13.5只，且发现伴随着抗体剂量的增加，均产仔数也呈现上升趋势。同时可以看出，抗抑制素抗体在受胎率和均产仔数上均表现出色。
表2抗抑制素抗体对小鼠受胎率、产仔数的影响
| 组别 | 小鼠数量（只） | 受胎小鼠（只） | 受胎率（%） | 产仔数（只） | 均产仔数（只） |
| 阴性对照 | 10 | 3 | 30 | 22 | 7.3±0.89 |
| 阳性对照 | 10 | 6 | 60 | 59 | 9.8±0.96 |
| 处理组1 | 10 | 9 | 90 | 130 | 13.5±1.25 |
| 处理组2 | 10 | 10 | 100 | 145 | 14.5±1.05 |
| 处理组3 | 10 | 10 | 100 | 148 | 14.8±1.15 |
实施例4抗抑制素抗体对抑制素的检测分析
4.1荧光抗体的制备
取一定量的纯单抗溶液，在1.5 mol/L pH9.0碳酸盐缓冲液透析过夜，按荧光素与抗抑制素抗体质量比1:100称取适量FITC，加入二甲亚砜（DMSO），使终浓度为10mg FITC/1mL DMSO；将单抗溶液置于电磁搅拌器上，轻轻搅拌，以不起沫为准。按上述比例将FITC-DMSO溶液逐滴加入单抗溶液中，磁力搅拌器4℃避光搅拌24h；加完后，随时用试纸测定搅拌液的pH值，若低于9.0，则应以碳酸钠溶液调整。置4℃搅拌24h即可。用Sephadex G25柱过滤标记的荧光抗体，以去除游离荧光素。
4.2荧光抗体的最低检测线
在96孔微量培养板中加入依次加入浓度为40μg/mL、20μg/mL、10μg/mL、1.0μg/mL、0.10μg/mL、0.01μg/mL的重组人抑制素α链，每个浓度分别做8个孔，加入100μg/mL荧光抗体，100μL/孔，37℃孵育30min，PBS洗涤3次。最后用三蒸水洗2min，于酶标仪下记录，激发波长460nm，发射波长520nm。
结果显示：经荧光检测分析可知，如图1所示，本发明提供荧光标记的抗抑制素抗体能很好地实现对重组人抑制素α链的检测，且其在0.01-40μg/mL之间存在线性相关，可实现高灵敏地检测抑制素，能满足高通量，快速检测的要求，具有潜在推广应用价值。
在此有必要指出的是，以上实施例仅限于对本发明的技术方案做进一步的阐述和说明，并不是对本发明的技术方案的进一步的限制，本发明的方法仅为较佳的实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1. 一种抗抑制素抗体，其特征在于，所述抗抑制素抗体重链可变区序列如SEQ IDNO.1所示，轻链可变区序列如SEQ ID NO.2所示。
2. 如权利要求1所述的抗抑制素抗体，其特征在于，所述抗抑制素抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示，所述抗抑制素抗体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ ID NO.6-8。
3.一种荧光标记的抗抑制素抗体，其特征在于，采用荧光标记基团对权利要求1或2任一项所述的抗抑制素抗体进行标记。
4. 如权利要求3所述的荧光标记的抗抑制素抗体，其特征在于，所述荧光标记选自FITC、FAM、Rhodamine B、TAMRA、Cy3或Cy5。
5.如权利要求4所述的荧光标记的抗抑制素抗体，其特征在于，所述荧光标记为FITC。
6.一种非诊断目的抑制素直接免疫荧光检测方法，其特征在于，该方法包括采用权利要求3-5中任一项所述的荧光标记的抗抑制素抗体进行接触待检样本，在孵育结束后，在荧光显微镜下观察结果或酶标仪读取荧光值。
7.权利要求3-5中任一项所述的荧光标记的抗抑制素抗体在制备检测抑制素检测试剂盒中的应用。
8.权利要求1或2所述的抗抑制素抗体或权利要求3-5中任一项所述的荧光标记的抗抑制素抗体在制备用于刺激雌性哺乳动物卵泡发育、增加排卵数、增加受孕率或提高产仔量的药物中的应用。
9.如权利要求8所述的应用，其特征在于，所述雌性哺乳动物选择鼠、猪、牛、羊。
10.一种刺激雌性哺乳动物卵泡发育、增加排卵数、增加受孕率或提高产仔量的药物，其特征在于，所述药物包括权利要求1或2所述的抗抑制素抗体或权利要求3-5中任一项所述的荧光标记的抗抑制素抗体、和药学上可接受的辅料。
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