CN116375844B - 一种抑制素抗原肽及其疫苗和制备方法 - Google Patents
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Abstract
本发明公开了一种抑制素抗原肽及其疫苗和制备方法。该疫苗包括偶联有载体蛋白的抑制素抗原以及佐剂；抑制素抗原的氨基酸序列如SEQ ID NO.1所示，其中增添第27位氨基酸作为偶联位点(如SEQ ID NO.2所示)与载体蛋白偶联。通过本发明制备得到的抑制素抗原/疫苗高效、效果稳定、成本低廉，能够进行大规模商业化生产。应用中，通过注射可显著提高母畜***率和产仔数。
Description
技术领域
本发明属于疫苗制备技术领域，具体涉及一种抑制素抗原肽及其疫苗和制备方法。
背景技术
低繁殖率一直以来都是制约和影响畜牧养殖业发展和经济效益的瓶颈性问题，特别对于羊、牛等单胎物种。卵巢卵泡发育和***数量的多少与母畜产仔数直接相关。垂体分泌的***(FSH)通过促进卵泡发育和抑制卵泡闭锁，在提高母畜***率和产仔数上发挥核心调控作用。垂体FSH的合成和分泌受到卵巢分泌的抑制素(inhibin,INH)的反馈抑制调控。大量研究表明，通过主动或被动免疫抑制素可显著促进母畜垂体FSH的合成和分泌，进而显著促进卵巢卵泡发育，从而大幅提高母畜***率和产仔数。抑制素主动免疫已成为一种提高母畜繁殖率的重要技术手段。
抑制素是一种由α和β两个亚基组成的异源二聚体糖蛋白。目前，学术圈在研制抑制素疫苗中主要采用两种思路制备抑制素抗原：其一，通过化学合成法合成抑制素α亚基N端1 32位氨基酸序列，例如牛(α1-25、α1-26、α1-29)、绵羊(α1-30)、猪(α1-26、α1-32)等多肽片段作为半抗原，再将半抗原与大分子载体蛋白偶联从而获得抑制素完全抗原；其二，利用基因工程技术表达重组抑制素α亚基作为抑制素抗原。通过以上述两种思路生产的抑制素抗原均存在明显的局限性。
其中，通过化学合成法合成抑制素α亚基N端氨基酸片段所制备的抑制素疫苗存在明显种属特异性，因而不具备广谱应用价值且效果不稳定、作用机制不明确；而通过基因工程技术生产的重组抑制素α亚基疫苗同样存在效果不理想、抗原分离纯化步骤繁琐及抗原不纯等问题。因此，亟需开发一种新的抑制素疫苗。
发明内容
针对现有技术中的上述不足，本发明提供一种抑制素抗原肽及其疫苗和制备方法，可显著促进卵泡发育，提高母畜***率和产仔数。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种抑制素抗原肽，该抗原肽的氨基酸序列如SEQ ID NO.1所示。
进一步地，抗原肽C端末尾通过偶联位点与载体蛋白偶联。
进一步地，在抗原肽C端末尾额外添加第27位氨基酸作为偶联位点。
进一步地，偶联位点为额外添加的半胱氨酸，其侧链-SH作为偶联位点与载体蛋白偶联(如SEQ ID NO.2所示)。
进一步地，载体蛋白为卵清蛋白、钥孔戚血蓝蛋白、破伤风类毒素、牛血清白蛋白或人血清白蛋白
一种抑制素疫苗，其包括上述抗原肽，以及佐剂。
进一步地，佐剂为商业化的兽用或人用佐剂。
进一步地，佐剂为Specol。
一种提高动物繁殖能力的试剂，包括上述抑制素疫苗，或由上述抑制素抗原肽、抑制素疫苗刺激产生的抗体。
一种制备上述抑制素抗原肽的方法，包括以下步骤：
(1)以CTC Resin树脂为原料，采用基于Fmoc固相多肽合成法合成氨基酸序列如SEQ ID NO.2所示的INHα13AA-T-C寡肽；寡肽合成后采用反相高效液相进行纯化。由此，制备得到的INHα13AA-T-C寡肽，其具体序列如下：
NH2-VRTTSDGGYSFKY-VRTTSDGGYSFKY-C-COOH；
(2)采用MBS(3-马来酰亚胺基苯甲酸琥珀酰亚胺酯)将合成肽与载体蛋白(OVA)进行偶联，其中合成肽第27位半光氨酸的-SH为合成肽与载体蛋白偶联的位点。
本发明机理如下：
本发明以β聚糖与抑制素A的结合位点VRTTSDGGYSFKY(位于抑制素α亚基上第108-120位氨基酸位点，其在哺乳动物中高度保守，见图1中虚线框内)为起始片段，最终获得了如SEQ ID NO.2所示的抗原肽序列。将该INH抗原制备成抑制素疫苗主动免疫母畜，刺激其免疫***产生抗INH抗体，该抗体特异性与动物体内内源性抑制素A结合，阻断内源性抑制素A与β聚糖(betaglycan)辅助受体结合，使抑制素A失去拮抗激活素(Activin)与激活素II型受体(ACTR II)结合的能力，从而促进垂体FSH合成和分泌，达到促进母畜卵泡发育和增加其***率和产仔数的目的。
本发明的有益效果：
本发明中合成的INHα13AA-T寡肽成本低，效果稳定；同时合成INHα13AA串联体，并进一步与大分子载体蛋白(OVA)偶联，使其获得抗原免疫原性，从而真正成为INH抗原。该技术方案使INH抗原及疫苗制备切实可行；且INH抗原/疫苗生产质量高度可控、效果稳定，成本低廉，极具商业化应用价值。在应用中，注射该疫苗可促进卵泡发育，从而显著增加母畜***率和产仔数。
附图说明
图1为β聚糖与抑制素A的结合位点在哺乳动物中高度保守；
图2为SD雌鼠***率和产仔数变化量检测图，注射抑制素疫苗显著增强SD雌鼠繁殖力；
图3为抑制素疫苗通过促进垂体FSH合成增强卵泡发育。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1INH抗原及疫苗的制备
1、制备INH抗原
(1)以CTC Resin树脂为原料，采用基于Fmoc固相多肽合成法合成氨基酸序列如SEQ ID NO.2所示的INHα13AA-T-C寡肽；寡肽合成后采用反相高效液相进行纯化。由此，制备得到的INHα13AA-T-C寡肽，其具体序列如下：
NH2-VRTTSDGGYSFKY-VRTTSDGGYSFKY-C-COOH；
(2)采用MBS(3-马来酰亚胺基苯甲酸琥珀酰亚胺酯)将合成肽与载体蛋白(OVA)进行偶联，其中合成肽第27位半光氨酸的-SH为合成肽与载体蛋白偶联的位点。
2、制备INH疫苗
采用Specol佐剂乳化制备得到的INH抗原，制得INH疫苗。
实施例2提高SD大鼠繁殖力效果评价(模型动物实验)
1、设置A、B两组SD雌鼠(rat)为模型，每组均包括45只雌性SD大鼠。
2、在8周龄时，B组(INHα-immunized)SD雌鼠主动免疫INH疫苗(腿部肌肉注射1mL疫苗，含300μg INH抗原)，A组(Placebo-immunized)注射不含INH抗原的乳化剂，以作为对照。
B组雌鼠分别在首次注射4周和8周后进行2次加强免疫，免疫方法和注射剂量与首次等同；A组雌鼠在首次注射4周和12周后分别注射不含INH抗原的乳化剂。第二次加强免疫后4周，所有试验组和对照组雌鼠均随机分为三个亚组，每个亚组15只，其中第一亚组鼠，与雄鼠成功交配后处死检测雌鼠自然***率；第二亚组鼠与雄鼠成功交配后，饲喂至产仔并统计产仔数；第三亚组雌鼠，进行麻醉处死采样，检测疫苗免疫对雌鼠生殖激素分泌、卵泡发育及卵泡发育相关基因表达的影响，以解析疫苗免疫提高雌性动物繁殖力的分子机制。其检测结果见图2、图3和表1。
如图2所示，注射本INH疫苗后，能刺激SD雌鼠机体产生显著的抗体反应(图2A)；与对照雌鼠(Placebo-immunized)相比，INH疫苗免疫显著促进雌鼠卵泡发育(图2B-C)，显著增加雌鼠***率(图2D)和产仔数(图2E-F)。进一步发现，INH疫苗注射显通过著促进雌鼠垂体Fshb基因表达(图3A)，提高血清FSH浓度(图3B)，从而促进卵巢卵泡发育相关基因表达(图3C)，以达到增加雌鼠***率和产仔数。该结果证实：该抑制素疫苗免疫能显著提高雌性动物繁殖力。图2A箭头表示疫苗注射时间点；图2A、C、D、F和图3A-C中*:P<0.05，**:P<0.01；图2B：箭头表示窦状卵泡，CL表示黄体；图2D：COCS表示卵丘-***复合体(代表***数)。
实施例3提高绵羊繁殖力的效果评价
选取1～1.5岁的28只凉山半细毛羊，分为A、B两组实验。B组绵羊(n＝16)主动免疫抑制素疫苗(颈部肌肉注射2mL抑制素疫苗，含300μg抑制素抗原)，在首次注射4周及8周后分别再次注射抑制素疫苗，免疫方法和注射剂量与首次相同。A组绵羊(n＝12)注射不含抑制素抗原的乳化剂，以作为对照。第二次加免后4周(11月中旬)，所有绵羊与雄性绵羊交配，然后检测绵羊产糕数，其检测结果见表1。
如表1所示，与对照组(Control)绵羊平均产糕数为1.08±0.08只，抑制素疫苗后雌性绵羊平均产糕数为增加1.63±0.15只，平均每只雌性绵羊产糕数显著增加0.55只(P<0.05)。该结果证实：本抑制素疫苗免疫能显著提高雌性绵羊繁殖力。
表1抑制主动免疫对雌性绵羊繁殖力的影响
| 组别 | 动物数量 | 平均产糕数 |
| 对照组 | 12 | 1.08±0.08b |
| 免疫组 | 16 | 1.63±0.15a |
注：a,b不同字母表示差异显著(P<0.05)。
最后应说明的是，以上具体实施方式仅用以说明本发明的技术方案而非限制，尽管参照实例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (6)
1.一种抑制素抗原肽，其特征在于，所述抗原肽的氨基酸序列如SEQ ID NO.1所示。
2.一种抑制素抗原肽，其特征在于，所述抗原肽的氨基酸序列如SEQ ID NO.2所示，并以该抗原肽C端末尾的半胱氨酸作为偶联位点与载体蛋白偶联。
3.根据权利要求2所述的抑制素抗原肽，其特征在于，所述载体蛋白为卵清蛋白、钥孔戚血蓝蛋白、破伤风类毒素、牛血清白蛋白或人血清白蛋白。
4.一种抑制素疫苗，其特征在于，所述抑制素疫苗包括权利要求1~3中任一项所述的抗原肽。
5.一种提高动物繁殖能力的试剂，其特征在于，包括权利要求4所述的抑制素疫苗。
6.一种制备权利要求2或3所述的抑制素抗原肽的方法，其特征在于，包括以下步骤：
（1）以CTC树脂为原料，采用Fmoc固相多肽合成法合成氨基酸序列如SEQ ID NO.2所示的INHα13AA-T-C寡肽序列；
（2）采用MBS将合成肽与载体蛋白偶联，并以抗原肽中C端末尾的氨基酸作为偶联位点。
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