CN114934089B - 一种用于瘦肉猪育种的生物制剂 - Google Patents
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Abstract
本发明提供了一种用于瘦肉猪育种的生物制剂,属于猪育种技术领域。本发明从小青叶蝉中提取出一种小青叶蝉寡肽,本发明所制备的小青叶蝉寡肽能够显著的抑制猪前体脂肪细胞的脂滴形成和成脂分化相关蛋白PPARγ和C/EBPα的表达,因此可以将其用于瘦肉猪育种来筛选脂肪沉积少的瘦肉猪。
Description
本案为分案申请,原申请的发明名称为:一种抑制猪前体脂肪细胞分化的提取物,原申请的申请日为:2020-10-15,原申请的申请号为:CN 202011100432.1。
技术领域
本发明属于猪育种技术领域,尤其涉及一种用于瘦肉猪育种的生物制剂。
背景技术
随着人民生活水平和消费水平的提高,对于肉类品质的要求也越来越高,作为世界上最大的肉类生产和消费的国家,猪肉消费占我国肉类消费中的第一位。肉质是猪肉生产中极其重要的经济指标,脂肪细胞分化和脂质沉积对猪肉的肉质具有重要的调控作用,抑制脂前体脂肪细胞的分化来减少皮下脂肪的生成对于提高猪肉的瘦肉率具有重要的价值。其次,皮下脂肪沉积也是影响猪生长和肉质的重要因素,在瘦肉猪育种方面也备受关注。因此,寻找能够有效抑制猪前体脂肪细胞分化的物质对于提高猪肉瘦肉率和猪育种具有重要的研究价值。
昆虫是蕴含着大量高质量蛋白的生物资源,昆虫的虫体中富含多种氨基酸,肽和蛋白质,且现有研究已证明昆虫肽具有多种功能,因此,从昆虫中寻找可用于猪前体脂肪细胞的寡肽或多肽是切实可行的。同时,同蝉亚目中的金蝉的蝉蜕及其多肽已被用于疾病的治疗,而同样含有丰富蛋白质的小青叶蝉则很少有研究涉及,因此本发明选取小青叶蝉对其进行研究。
发明内容
本发明的目的在于提供一种能够抑制猪前体脂肪细胞成脂分化的提取物
为实现上述目的,本发明提供了如下技术方案:
本发明提供了一种小青叶蝉提取物在抑制猪前体脂肪细胞成脂分化中的应用,所述小青叶蝉提取物为小青叶蝉寡肽。
优选地,所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,PH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
此外,本发明提供了小青叶蝉提取物在制备抑制猪前体脂肪细胞成脂分化的抑制剂中的应用,所述小青叶蝉提取物为小青叶蝉寡肽。
优选地,所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,PH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
优选地,所述应用包括小青叶蝉在制备抑制脂肪转化相关基因PPARγ和C/EBPα中的应用。
此外,本发明提供了一种抑制猪前体脂肪细胞成脂分化的抑制剂,所述抑制剂为小青叶蝉寡肽。
优选地,所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,PH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
除此之外,本发明提供了小青叶蝉寡肽在瘦肉猪育种中的应用,所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,PH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
本发明的有益效果在于
本发明从小青叶蝉中制备出小青叶蝉寡肽实现了害虫小青叶蝉的再利用。
其次,本发明所制备的小青叶蝉寡肽可以显著的抑制猪前体脂肪细胞的脂滴形成并且能够显著的抑制成脂分化相关蛋白PPARγ和CEBPα蛋白表达量,因此可将其用于制备抑制猪前体脂肪细胞成脂分化的抑制剂或药物。同时,可将其用于瘦肉猪育种来筛选脂肪沉积少的瘦肉猪。
附图说明
图1 对照组,0ug/ml小青叶蝉寡肽组,150ug/ml小青叶蝉寡肽组油红O染色结果。
图2 对照组,0ug/ml小青叶蝉寡肽组,150ug/ml小青叶蝉寡肽组脂肪转化相关蛋白PPARγ和CEBPα蛋白表达结果。
具体实施方式
为了能够清楚的说明本方案的技术特点,下面通过具体的实施方式,对本方案进行阐述。
实施例1
(1)将小青叶蝉去翅洗净后,置于烘干机中烘干,使用破碎机破碎为粉末;
(2)加入5倍量的水,充分搅拌均匀后得到小青叶蝉匀浆液;
(3)在匀浆液中接种5% wt的枯草芽孢杆菌,PH7, 30℃,发酵48h;
(4)121℃,103.4kPa高温高压灭菌30min,离心取发酵上清,
(5)加入0.02倍量的木瓜蛋白酶,PH8.5,55℃,发酵48h;
(6)95℃灭酶10min,离心,得到小青叶蝉肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽液;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
实施例2
制备猪前体脂肪细胞
(1)取7日龄的仔猪背部皮下脂肪组织,使用含有双抗的PBS洗涤3次,使用剪刀将脂肪组织剪成大约1mm3的组织块;
(2)将剪碎后的脂肪组织转移至离心管中,加入I型胶原蛋白酶,37℃恒温水浴锅中消化,消化90min后加入完全培养基终止消化;
(3)将消化液分别使用80目和200目细胞筛过滤,收集滤液至新的无菌离心管中;
(4)将离心管置于离心机中,1500r/min离心10min,弃去上清,在沉淀中加入红细胞裂解液悬浮细胞沉淀,然后1500r/min离心5min;
(5)弃去上清,加入PBS洗涤细胞,1500r/min离心5min;
(6)弃去上清,加入DMEM完全培养基重悬细胞,接种于细胞培养皿中,37℃,5% CO2细胞培养箱中培养,即可得到猪前体脂肪细胞。
实施例3
(1)使用DMEM溶解实施例1所制备的小青叶蝉寡肽,制成150ug/ml的小青叶蝉寡肽液,使用0.22um过滤膜进行除菌过滤。
(2)将猪前体脂肪细胞接种于96孔板中,对照组不添加小青叶蝉寡肽,实验组加入150ug/ml小青叶蝉寡肽液;
(3)培养48h后,参照CCK-8说明书测定两组细胞的OD值。
实验结果如下,对照组OD值为0.841±0.029,150小青叶蝉寡肽组的OD值为0.875±0.025,P值为0.426,二者差异无统计学意义,因此,小青叶蝉寡肽对于猪前体脂肪细胞的增殖没有显著的影响。
实施例4
油红O染色实验
(1)实验分组:对照组不进行处理;0ug/ml小青叶蝉寡肽组加入成脂诱导培养基Ⅰ和成脂诱导培养基Ⅱ进行成脂诱导,但不加入小青叶蝉寡肽;150ug/ml小青叶蝉寡肽组使用溶解有150ug/ml的小青叶蝉寡肽的成脂诱导培养基Ⅰ和成脂诱导培养基Ⅱ进行成脂诱导;
(2)将猪前体脂肪细胞接种于细胞培养板中,按照实验分组进行成脂诱导培养,首先使用成脂诱导培养基I诱导3天,之后使用成脂诱导培养基Ⅱ诱导2天,之后更换为完全培养基;
(3)成脂诱导14天后,弃去培养基,加入PBS清洗细胞3次,吸干多余的PBS后加入4%的多聚甲醛室温进行细胞固定;
(4)固定20min后,加入PBS清洗细胞3次,加入油红O染色液,室温孵育10min;
(5)使用PBS漂洗细胞,置于显微镜下观察并拍照。
实验结果如图1所示,从图中可以看出,加入小青叶蝉寡肽后,相较于0ug/ml小青叶蝉寡肽组脂滴的数量明显减少,说明小青叶蝉寡肽能够抑制猪前体脂肪细胞的脂滴形成,即能够抑制猪前体脂肪细胞转化为脂肪细胞。
实施例5
(1)细胞分组和成脂转化同实施例4;
(2)培养结束后,去除培养基,加入1mlPBS清洗剩余的培养基,弃去PBS加入100ul细胞裂解液并使用细胞刮刀将细胞刮下,收集裂解混合液到EP管中;
(3)超声破碎细胞,置于离心机中,4℃,12000rpm/min,离心15min;
(4)离心结束后,转移上清至新的离心管中,使用BCA法测量蛋白浓度,加入5×SDS上样缓冲液调整蛋白样品浓度为2ug/ml;
(5)配置12%分离胶,5%浓缩胶,加入10ul蛋白样品和蛋白marker,恒压90V,到分离胶后改为恒压120v,当溴酚蓝跑出分离胶时停止电泳;
(6)取出凝胶按照‘三明治’模型按照转膜夹,将转膜夹置于电转槽中,室温恒流250mA电转1.5h;
(7)电转完成后,将PVDF膜取出,放入5%的脱脂奶粉中,室温封闭1h;
(8)按照蛋白大小进行裁膜,孵育PPARγ,CEBPα和β-actin,4℃孵育过夜;
(9)吸取一抗,使用TBST洗膜3次,每次10min,进行二抗孵育,室温孵育1h;
(10)弃去二抗,使用TBST洗膜3次,进行显影。
实验结果如图2所示,从图中可以看出,小青叶蝉寡肽能够显著的逆转成脂转化所导致的PPARγ和CEBPα的蛋白表达量升高。因此,小青叶蝉寡肽能够有效的抑制前体脂肪细胞向脂肪细胞转化。
Claims (3)
1.小青叶蝉寡肽在瘦肉猪育种中的应用,其特征在于,所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,pH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽;
所述小青叶蝉寡肽可用于筛选脂肪沉积少的瘦肉猪。
2.一种抑制猪前体脂肪细胞成脂分化的抑制剂,其特征在于,所述抑制剂为小青叶蝉寡肽;
所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,pH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
3.小青叶蝉寡肽在制备抑制猪前体脂肪细胞中成脂分化相关蛋白表达的抑制剂中的应用,其特征在于,所述成脂分化相关蛋白为PPARγ和C/EBPα;
所述小青叶蝉寡肽的制备方法如下:
(1)去除小青叶蝉翅膀,干燥后将其粉碎为粉末;
(2)加入5倍量的水,得到小青叶蝉匀浆液;
(3)接种5% wt的枯草芽孢杆菌发酵48h;
(4)高温高压灭菌,离心取发酵上清;
(5)加入0.02倍量的木瓜蛋白酶,pH8.5,55℃,发酵48h;
(6)95℃ 灭酶10min,离心,得到小青叶蝉寡肽液;
(7)使用截留分子量1KDa的超滤膜进行过滤,得到小青叶蝉寡肽;
(8)冷冻干燥,得到成品小青叶蝉寡肽。
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