CN116239668A - 一种hCGβ亚基突变体及其在PDRN提取中的应用 - Google Patents
一种hCGβ亚基突变体及其在PDRN提取中的应用 Download PDFInfo
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Abstract
本发明提供一种hCGβ亚基突变体及其在PDRN提取中的应用，属于生物技术领域。采用本发明所述的hCGβ亚基突变体处理鱼类精巢，可获得纯度、产量更高的多聚脱氧核糖核苷酸（PDRN），为PDRN的大规模制备或工业化生产提供了一种新的思路或方法，具有广泛的应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种人绒毛膜促性腺激素（hCG）β亚基突变体及其在高纯度多聚脱氧核糖核苷酸（PDRN）提取中的应用。
背景技术
多聚脱氧核糖核苷酸（PDRN）是一种来自于人胎盘，长度介于50到2000bp的线性脱氧核苷酸多聚物。它可以高效地激活细胞腺苷A2A受体，而且由于PDRN对5'-核苷酸外切酶具有一定抗性，所以体内稳定性好，不易降解为小分子片段，从而避免了对其他腺苷和嘌呤受体的激活作用。PDRN通过激活A2A受体，对缺血缺氧性疾病具有一定治疗作用，如心肌梗塞、脑梗塞、周围动脉闭塞性疾病等。研究发现PDRN可以显著增加创面血管和肉芽形成，提高创面愈合速度，改善慢性软组织溃疡症状，上述作用可以被A2A受体的特异性拮抗剂所阻断。另外来源于三文鱼的PDRN为小分子线性脱氧核苷酸片段，其组成与人体DNA的碱基组成相似，在人体内不产生免疫原性，不引起过敏反应。大量急性和慢性毒性研究结果显示，全身和腹腔给药后，在生理剂量范围内不会造成小鼠的死亡或病理反应，且肝脏、脑、肺脏、骨骼肌和心脏等组织未发现病理学变化。
为了获得大量的PDRN，从鱼类的精液或精巢中提取PDRN逐渐成为热点。例如，发明专利申请CN201610497384.1公开了一种从鱼类的精液分离PDRN的方法，包括：（1）将鱼类的精液进行第1次离心分离，获取包含精液细胞的沉淀物；（2）将所述沉淀物添加于细胞溶解缓冲液，制造细胞溶解物后，使所述细胞溶解物消化；（3）将步骤（2）的细胞溶解物进行第2次离心分离，获取上清液；（4）在步骤（3）获取的上清液中投入饱和氯化钠水溶液并混合后，进行第3次离心分离，获取包含DNA的上清液；（5）在步骤（4）获取的上清液中投入乙醇并使DNA沉淀后，收集所述沉淀的DNA并进行干燥；（6）用物理方法破碎步骤（5）获得的DNA，获取多脱氧核糖核苷酸。
人绒毛膜促性腺激素（hCG），是一种糖蛋白激素，由胎盘合体滋养层细胞所分泌，由α和β两个亚基经由非共价二聚组成，α亚基与β亚基的分子量分别为10.2kD和15.5kD，其中，α-链结构与LH（黄体生成素）相似，其他结构有整分子hCG、高糖基化hCG、游离β-hCG等。hCG的主要功能是促进黄体细胞产生孕酮，同时，hCG的检查对早期妊娠诊断有重要意义，对与妊娠相关疾病、滋养细胞肿瘤等疾病的诊断、鉴别和病程观察等有一定价值。
如发明专利申请CN202110961715.3公开了一种从鲑鱼精巢中提取高纯度聚脱氧核糖核苷酸的方法，所述的方法包括以下步骤：1）从鲑鱼的精巢中分离出精液和未成熟精巢区，2）温和地研磨所述未成熟精巢区，然后用人工精浆稀释，3）用预定浓度的人绒毛膜促性腺激素（hCG）处理稀释液，以诱导精巢细胞人工性成熟为精子，4）在hCG处理预定时间后，离心精巢稀释液并收集潜在的活动精子，5）从收集的精子中提取PDRN。
目前，亟需提供一种从鱼类精巢中提取PDRN的产量更高的方法。
发明内容
为了解决上述问题，本发明提供了一种人绒毛膜促性腺激素（hCG）β亚基突变体，采用本发明所述的hCGβ亚基突变体处理鱼类精巢，可获得纯度、产量更高的多聚脱氧核糖核苷酸（PDRN）。
为了实现上述发明目的，本发明的技术方案如下：
一方面，本发明提供了一种人绒毛膜促性腺激素（hCG）β亚基突变体，所述的人绒毛膜促性腺激素（hCG）β亚基突变体为人绒毛膜促性腺激素（hCG）β亚基野生型氨基酸发生K20D、V56T和/或K104D突变。
具体地，所述的人绒毛膜促性腺激素（hCG）β亚基野生型氨基酸序列在GeneBank中的编号为AAI06724.1，氨基酸序列如SEQ ID NO.2所示。
所述的人绒毛膜促性腺激素（hCG）β亚基突变体的氨基酸序列如SEQ ID NO.1所示。
另一方面，本发明提供了上述人绒毛膜促性腺激素（hCG）β亚基突变体在制备多聚脱氧核糖核苷酸（PDRN）中的应用。
又一方面，本发明提供了一种多聚脱氧核糖核苷酸（PDRN）的制备方法，所述的方法包括以下步骤：采用上述人绒毛膜促性腺激素（hCG）β亚基突变体处理鱼类精巢，诱导精巢细胞成熟后提取多聚脱氧核糖核苷酸（PDRN）。
具体地，所述的人绒毛膜促性腺激素（hCG）β亚基突变体的浓度为0.5-5mg/mL。
进一步具体地，所述的人绒毛膜促性腺激素（hCG）β亚基突变体的浓度为1-3mg/mL，优选为2mg/mL。
具体地，所述的人绒毛膜促性腺激素（hCG）β亚基突变体的处理时间为10-60min。
进一步具体地，所述的人绒毛膜促性腺激素（hCG）β亚基突变体的处理时间为25-45min，优选为30min。
具体地，所述的鱼类为鲑科，所述的鱼类包括虹鳟、鲑鱼或大马哈鱼，优选为大马哈鱼。
又一方面，本发明提供了一种采用上述方法制备得到的多聚脱氧核糖核苷酸（PDRN）。
又一方面，本发明提供了上述多聚脱氧核糖核苷酸（PDRN）在制备药物和/或化妆品中的应用。
与现有技术相比，本发明的积极和有益效果在于：
本发明提供了一种人绒毛膜促性腺激素（hCG）β亚基突变体，采用本发明所述的hCG β亚基突变体处理鱼类精巢，可获得纯度、产量更高的多聚脱氧核糖核苷酸（PDRN），为PDRN的大规模制备或工业化生产提供了一种新的思路或方法，具有广泛的应用前景。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1.一种人绒毛膜促性腺激素（hCG）β亚基突变体
本发明所述的hCGβ亚基突变体为hCGβ亚基野生型氨基酸发生K20D、V56T和K104D突变，所述的hCGβ亚基野生型氨基酸序列在GeneBank中的编号为AAI06724.1，野生型氨基酸序列为SEQ ID NO.2，突变体序列为SEQ ID NO.1。
实施例2.一种多聚脱氧核糖核苷酸（PDRN）的制备方法
取大马哈鱼未成熟的精巢，将其研磨并采用人工精浆（取NaCl 7.60g、KCl 2.98g、CaCl2·2H2O 0.37g、MgCl2·6H2O 0.31g、NaHCO30.21g用蒸馏水溶解，定容至1L，pH8.4）进行稀释。分别在上述稀释液中加入浓度为2mg/mL的hCGβ亚基突变体或野生型，缓慢搅拌均匀后，静置30min。静置完成后取样本检测精子活力、提取PDRN。
（1）精子活力检测结果如下表1所示。
表1
（2）PDRN提取
PDRN提取方法如下：
1）取上述hCGβ亚基处理后的样本，离心取沉淀100g，加入600mL裂解液（0.1mol/LEDTA，2mol/L NaOH，0.5wt% SDS），颠倒混匀，95℃恒温20min。
2）降温至40℃，加入2mol/L的Tris-HCl（pH8.0），加入体积量为步骤1）反应体系体积的1/2，颠倒混匀。
3）加入1mol/L的HCl，加入体积量占步骤2）反应体系体积的1/5，颠倒混匀。
4）0-4℃条件下，6000rpm离心15min，收集上清。
5）用DNA打断仪6000焦耳/s破碎30s。
6）加入10mol/L NH4AC溶液，其体积占步骤5）反应体系体积的1/10，再加入-20℃预冷的无水乙醇，颠倒混匀，-20℃放置2h。
7）0-4℃条件下，10000rpm离心15min，倾去上清，加入70%的乙醇，轻轻吹打，使沉淀泛起，轻缓颠倒数次，然后10000rpm离心15min，倾去上清，残留乙醇用枪头吸除。
8）用生理盐水悬浮沉淀步骤7）获得的沉淀，置于真空冷冻干燥仪中干燥，获得PDRN。
9）纯度和产率计算：
产率=产物重量/精巢重量；
产物溶于生理盐水后使用分光光度计分别检测260nm和280nm下的吸光度，根据以下公式计算产物中多聚脱氧核糖核苷酸的纯度：纯度=OD260/OD280。
PDRN提取产率及纯度检测结果如下表2所示。
表2
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围，本发明的保护范围以所附权利要求为准。
Claims (11)
1.一种人绒毛膜促性腺激素β亚基突变体，其特征在于：氨基酸序列为SEQ ID NO.1。
2.权利要求1所述的人绒毛膜促性腺激素β亚基突变体在制备多聚脱氧核糖核苷酸中的应用。
3.一种多聚脱氧核糖核苷酸的制备方法，其特征在于：所述的制备方法包括以下步骤：采用权利要求1所述的人绒毛膜促性腺激素β亚基突变体处理鱼类精巢，诱导精巢细胞成熟后提取多聚脱氧核糖核苷酸。
4.根据权利要求3所述的制备方法，其特征在于：所述的人绒毛膜促性腺激素β亚基突变体的浓度为0.5-5mg/mL。
5.根据权利要求4所述的制备方法，其特征在于：所述的人绒毛膜促性腺激素β亚基突变体的浓度为1-3mg/mL。
6.根据权利要求3所述的制备方法，其特征在于：所述的人绒毛膜促性腺激素β亚基突变体的处理时间为10-60min。
7.根据权利要求6所述的制备方法，其特征在于：所述的人绒毛膜促性腺激素β亚基突变体的处理时间为25-45min。
8.根据权利要求3所述的制备方法，其特征在于：所述的鱼类为鲑科。
9.根据权利要求8所述的制备方法，其特征在于：所述的鱼类包括虹鳟、鲑鱼或大马哈鱼。
10.一种权利要求3-9任一项所述的制备方法制备得到的多聚脱氧核糖核苷酸。
11.权利要求10所述的多聚脱氧核糖核苷酸在制备药物和/或化妆品中的应用。
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