CN109893659A - 一种复方硫洗剂助悬剂及其应用 - Google Patents
一种复方硫洗剂助悬剂及其应用 Download PDFInfo
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- CN109893659A CN109893659A CN201910052567.6A CN201910052567A CN109893659A CN 109893659 A CN109893659 A CN 109893659A CN 201910052567 A CN201910052567 A CN 201910052567A CN 109893659 A CN109893659 A CN 109893659A
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- mrpc
- albumen
- compound sulfur
- suspending
- lotion
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Abstract
本发明公开了一种复方硫洗剂助悬剂及其应用,该MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。本发明中的MrpC蛋白对复方硫洗剂的助悬能力远远大于甲基纤维素,可发挥优秀的助悬性能。
Description
技术领域
本发明属于助悬剂技术领域,具体涉及一种复方硫洗剂助悬剂及其应用。
背景技术
现有技术中的复方硫洗剂由升华硫、硫酸锌、樟脑醑、甘油为主原料制作,是一种治疗痤疮、疥疮、皮脂溢出及酒糟鼻的药物。该复方硫洗剂的传统处方为:硫酸锌30g升华硫30g樟脑醑250mL、甘油100mL、羧甲基纤维素钠5g和蒸馏水适量,共制1000mL。其中羧甲基纤维素钠的作用是助悬剂。但该羧甲基纤维素钠的助悬功能和效果仍然不尽理想。
发明内容
本发明的目的在于提供一种复方硫洗剂助悬剂。
本发明的另一目的在于提供MrpC蛋白在助悬复方硫洗剂中的应用。
本发明的再一目的在于提供一种复方硫洗剂组合物。
本发明的技术方案如下:
一种复方硫洗剂助悬剂,其有效成分包括MrpC蛋白,该MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。
在本发明的一个优选实施方案中,其有效成分为所述MrpC蛋白。
进一步优选的,所述MrpC蛋白的核苷酸序列如SEQ ID NO 02所示。
本发明的另一技术方案如下:
MrpC蛋白在助悬复方硫洗剂中的应用,该MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。
在本发明的一个优选实施方案中,所述MrpC蛋白的核苷酸序列如SEQ ID NO 02所示。
本发明的再一技术方案如下:
一种复方硫洗剂组合物,包括升华硫、硫酸锌、樟脑醑、甘油、MrpC蛋白和蒸馏水,其中MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。
在本发明的一个优选实施方案中,所述MrpC蛋白的核苷酸序列如SEQ ID NO 02所示。
本发明的有益效果是:本发明中的MrpC蛋白对复方硫洗剂的助悬能力远远大于甲基纤维素,可发挥优秀的助悬性能。
附图说明
图1为本发明实施例1中更换新鲜培养基前后藻细胞悬浮状态的照片,其中左侧为对照组,右侧为更换新鲜培养基后静置培养6天后的状况。
图2为本发明实施例1中MrpC蛋白分离纯化和MALDI-TOF-MS鉴定图。
图3为本发明实施例1中MrpC抑制细胞下沉的临界浓度检测图,其中,1.0μg/mL;2.5μg/mL;3.15μg/mL;4.30μg/mL;5.45μg/mL;6.60μg/mL;7.75μg/mL;8.90μg/mL;9.115μg/mL;10.155μg/mL;11.300μg/mL。
图4为本发明实施例1中不同生长时期培养基中的MrpC含量检测图。
图5为本发明实施例1中MrpC远紫外圆二色光谱图。
图6为本发明实施例1中MrpC冷冻电镜观察图。
图7为本发明实施例1中MrpC的TEM观察图。
图8为本发明实施例1中MrpC对不同细胞悬浮的影响,其中1∶8000g离心后静置;2:重悬于新BG-11;3:重悬于60μg/mL MrpC BG-11;A.Anabaena sp.PCC 7120;B.M.aeruginosa PPC 7820;C.Synechocystis sp.PCC 6803;D.Chlore sp.;E.Nostocflagelliforme FACHB 838;F.Gloeobacter violaceus PCC 7421;G.Synechococcussp.PCC 7942;H.E coli.BL21(DE3);I.巴斯德毕赤酵母GS 115.。
图9为本发明实施例1中MrpC对不同种类粉末的悬浮作用,其中,A.淀粉;B.琼脂糖;C.鱼粉;D.螺旋藻粉(Spirulina platensis)。
图10为本发明实施例1和实施例3中复方硫洗剂混悬剂沉降体积比图。
图11为本发明实施例2中提取的分泌蛋白的SDS-PAGE分析和anti-MrpC-IgG特异性检测图,其中,M:marker;1:提取的M.aeruginosa PCC7806分泌的蛋白组分;2:对去藻长时间培养物的western blotting检测。
图12为本发明实施例2中M.aeruginosa PCC7806分泌蛋白MALDI-TOF-MS肽指纹图谱。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1 MrpC蛋白的助悬功能的发现和鉴定
一、悬浮蛋白的发现(长时间的培养基和新鲜培养基的细胞培养状况对照如图1所示)
藻细胞悬浮的现象和其培养基中的分泌物有关,接下来鉴定出来悬浮因子来自于该藻分泌的一种蛋白质MrpC。
二、MrpC的分离鉴定
·藻细胞中分泌蛋白的分离和质谱鉴定,如图2所示
·MrpC抑制细胞下沉的临界浓度,结果如图3所示,显示MrpC蛋白浓度大于30ug/mL即可抑制铜绿微囊藻细胞下沉
·不同生长时期培养基中的MrpC含量检测,结果如图4所示,显示实验室培养60天左右MrpC便达到了对细胞沉降产生影响的浓度
结论:MrpC是藻细胞悬浮的决定因子
三、悬浮蛋白MrpC的理化特性
1.MrpC的基因和氨基酸序列
M.aeruginosa PCC 7806的基因组已于2007年由法国巴斯德研究所完成(Frangeul,Quillardet et al.2008)。通过genbank获取mrpC基因序列,基因全长465bp,起始密码子为ATG(下划线),终止子是TAG(下划线)。蛋白质的一级结构就是蛋白质多肽链中氨基酸残基的排列顺序,由基因上遗传密码的排列顺序所决定的。利用DNAman分析获得MrpC氨基酸序列全长154aa,与文献预测序列一致。MrpC为胞外分泌蛋白,N末端包含33aa信号肽(下划线),第34位aa谷氨酰胺被修饰为焦谷氨酸(Zilliges,Kehr et al.2008)。
mrpC基因序列(SEQ ID NO 02):
MrpC蛋白的氨基酸序列(SEQ ID NO 01):
2.MrpC蛋白理化性质
MrpC纯品呈白色,易溶于水和PBS,不溶于乙醇、甲醇、丙酮等有机溶剂。重金属盐:硝酸银,碘化镉,硝酸铅,硫酸锰,硝酸钴等都会使MrpC变性。
利用NDJ-1旋转粘度计,30r/min转速下室温测定1mg/mL MrpC的表观粘度为6mPa.S。粘度更小时,由于仪器的限制无法测定。当MrpC浓度达到30ug/mL时,就可保持多种藻细胞悬浮,推测此时的溶液的粘度几乎接近于水。
实验室提取的MrpC经SDS-PAGE测定分子量为14Kda。Zilliges Y等利用分子筛(体积排斥色谱)测得自然状态下MrpC分子量为120kDa-170kDa。多数纤维蛋白质由于其分子形状的高度不对称性,无法使用超速离心。对MrpC进行超速离心,当浓度为1.2mg/mL时发现MrpC无法被离心下来,将其稀释至60ug/mL时测得沉降系数为168S,而对1.2mg/mLMrpC超声(f=20kHz,I=10±5%W)60s时测得沉降系数为68S,初步推测自然状态下MrpC为高聚物。
3.圆二色光谱(CD)测定MrpC二级结构
蛋白质中氨基酸的碳原子是不对称碳原子,具有光学活性,当平面圆偏振光通过这些光活性的生色基团时,光活性中心对平面圆偏振光中的左、右圆偏振光的吸收不相同,产生吸收差值,由于这种吸收差的存在,造成偏振光矢量的振幅差,圆偏振光变成了椭圆偏振光,这就是蛋白质的圆二色性。圆二色光谱是研究稀溶液中蛋白质构象的一种快速、简单、较准确的方法。
图5所示为MrpC溶液的圆二色光谱,MrpC浓度为0.1mg/mL,波长范围190-260nm。从图5中可以看出,MrpC在193nm左右有一个正峰,222nm左右处存在弱峰,说明MrpC以α-螺旋为主。通过曲线拟合软件CD Pro计算得到经拟合计算得到MrpC溶液二级结构为:α-螺旋结构占87.7%,β-转角结构占12.3%。
4.冷冻电镜观察MrpC
冷冻电镜技术是对含水生物大分子进行快速冷冻到液氮或液氦温度(冷冻速率>104℃/s),使样品处于或接近于其生理活性状态,并在低温条件下采用低电子剂量成像,从而可在高分辨水平上研究生物大分子的三维结构。将MrpC蛋白稀释浓度至25-50μg/mL,快速冷冻后于电镜观察蛋白形态。电镜结果如图6显示MrpC为直径约8.5nm的纳米级长线状物,即蛋白纳米纤维。纳米纤维的排列未发现规律,有的多束纳米纤维并排形成簇,不同空间排列的纤维丝或平行或纵横交错,从而形成网络结构。
直接将MrpC(100ug/mL)于铜网自然干燥后,透射电镜下观察。图7显示出直径为纳米级而长度可达好几微米的长线状,这些长线状物纵横交错形成网状结构,网孔的大小与线状物的密集程度有关。观察到的网状结构与冷冻电镜结果推测相符合,但普通电镜的纤维丝直径则大于冷冻电镜,可能是多束纤维丝组成了簇。这些结果证实了溶液中MrpC聚集成纳米级长纤维状,并随着MrpC浓度增加形成网状结构。
四、MrpC蛋白作为新型悬浮分散剂的应用潜力
正是由于上面发现的纳米网状结构,可以推断MrpC蛋白的悬浮作用是非特异性的。
为验证MrpC对不同细胞的悬浮是否都有作用,结果发现以60μg/mL的浓度即可阻止Anabaena sp.PCC 7120、M.aeruginosa PPC 7820、Synechocystis sp.PCC 6803、Chloresp.、Nostoc flagelliforme FACHB 838、Gloeobacter violaceus PCC 7421和Synechococcus sp.PCC 7942等蓝藻细胞的下沉,而MrpC对E coli.DH5α和毕赤酵母GS 115要产生同样的效果则需要大于100μg/mL的浓度(图8)。虽然MrpC具有抑制不同细胞的作用,但是对蓝藻细胞的产生的作用要更明显。
另外,MrpC在500μg/mL下也能抑制鱼粉、螺旋藻粉(Spirulina platensis)和淀粉等的下沉;尽管MrpC对琼脂糖的悬浮影响较小,但是琼脂糖在水中会吸胀比重增大,可能需要MrpC浓度远大于500μg/mL的才能抑制其的下沉(图9)。因此,MrpC不仅可以抑制不同细胞下沉,而且也可以抑制某些粉末下沉,说明MrpC抑制细胞下沉的作用是非特异性的。
以上结果表明MrpC蛋白有非特异性的悬浮功能,可替代传统的化学分散剂和稳定剂。
五、MrpC蛋白对复方硫洗剂助悬效果的考察
复方硫磺洗剂由升华硫、硫酸锌、樟脑醑、甘油为主原料制作,是一种治疗痤疮、疥疮、皮脂溢出及酒糟鼻的药物。
传统处方:硫酸锌30g升华硫30g樟脑醑250mL甘油100mL羧甲基纤维素钠5g蒸馏水适量共制1000mL。其中羧甲基纤维素钠的作用是助悬剂。
本发明利用MrpC蛋白替代甲基纤维素的助悬剂,实验证明可以替代,并且悬浮效果更好。通过测定由MrpC助悬的混悬剂与原处方混悬剂的沉降体积比、重新分散等性质,评价MrpC为助悬剂时,制剂处方的质量稳定性(并且以已有的助悬剂为对照),实验数据证明MrpC为助悬剂优于现有的助悬剂。
不同时间测得的高度及沉降体积比见下表。
硫磺洗剂沉降体积比
注:Ho为混悬液的高度,Hu为沉降物的高度,下同
根据上表数据,以Hu/Ho(沉降体积比)为纵坐标,时间为横坐标,绘制各处方的沉降曲线,见图10,比较两种助悬剂的助悬能力。
通过图10可以明显的看出,MrpC对升华硫的助悬能力远远大于甲基纤维素,可发挥优秀的助悬性能。MrpC在复方硫磺洗剂中的助悬效果明显优于甲基纤维素的助悬效果。
实施例2 MrpC蛋白的分离提纯
1.藻细胞的培养
将铜绿微囊藻Microcystis aeruginosa PCC7806按1∶50接种于新鲜配制BG-11培养基,28℃持续光照摇床培养。
2.硫酸铵盐析法沉淀蛋白
(1)取培养时间大于两个月藻液高速离心去藻;
(2)得到的去藻培养物再过滤一次;
(3)滤液中定量缓慢加入研磨成细粉状并干燥无水的硫酸铵,并不停搅拌,可用磁
力搅拌,直到有蛋白沉淀析出;
(4)12000g离心蛋白沉淀,上清继续加入硫酸铵沉淀蛋白,分批获得蛋白,方法同上,直至溶液中硫酸铵的溶解度为100%。
(5)蛋白沉淀用一定体积的PBS溶解,再高速离心一次,弃去沉淀杂质,上清即为蛋白溶液。
硫酸铵盐析法沉淀去藻培养基中的蛋白,在加入硫酸铵的量约为其溶解度的5%时得到的蛋白沉淀对抑制藻沉底效果显著,该蛋白沉淀用PBS溶解,-20℃保存。如图11所示,通过SDS-PAGE检测只有一条带,说明得到蛋白质较纯;如图12所示,经MALDI-TOF-MS鉴定只有1077.461m/z和949.434m/z两个峰,在matrix数据库中搜索的结果得分较低,但与已经发现的M.aeruginosa PCC 7806的一个分泌蛋白质MrpC的MALDI-TOF-MS峰谱一致,证明该提取蛋白为Mrp C。
实施例3 MrpC应用于复方硫洗剂
现有的复方硫洗剂的处方组成:升华硫0.12g,硫酸锌0.12g,樟脑醑1mL,甘油0.4mL,甲基纤维素0.02g,蒸馏水加至4mL。
操作:
(1)将硫酸锌溶于0.8mL水中;将升华硫过6号筛,加甘油研磨成细糊状;将甲基纤维素用0.8mL水制成胶浆并在搅拌下缓缓加入,随后在搅拌下加入硫酸锌溶液、樟脑醑(0.1g樟脑溶于1mL无水乙醇)最后加蒸馏水至全量,搅匀,即得第一复方硫洗剂。
(2)用MrpC蛋白溶液代替上述第一复方硫洗剂中的助悬剂(甲基纤维素),制成第二复方硫洗剂,MrpC蛋白在该第二复方硫洗剂中的浓度为0.5mg/mL。
(3)测第一复方硫洗剂和第二复方硫洗剂的沉降体积比,对比MrpC蛋白与传统助悬剂的助悬效果差别。通过图10可以明显的看出,MrpC对升华硫的助悬能力远远大于甲基纤维素,可发挥优秀的助悬性能。MrpC在复方硫磺洗剂中的助悬效果明显优于甲基纤维素的助悬效果。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
序列表
<110> 厦门大学
厦门科厦技术有限公司
<120> 一种复方硫洗剂助悬剂及其应用
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 154
<212> PRT
<213> Microcystis aeruginosa
<400> 1
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Gly Gly Leu Ala Ala Ala Gly Val Leu Ser Val Gly Glu Gly Ala Gln
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Ala Gln Phe Ile Gly Thr Ala Ser Gln Ser Arg Val Ser Ala Ala Thr
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Thr Ser Ile Leu Thr Asp Gly Asn Thr Ala Ala Thr Asn Ser Phe Ala
50 55 60
Val Glu Gln Val Leu Pro Ala Ala Asp Tyr Val Phe Ser Ser Pro Ile
65 70 75 80
Ser Val Gln Ile Ser Tyr Asp Thr Leu Thr Leu Ala Lys Asp Lys Asn
85 90 95
Phe Val Ala Ile Thr Gly Ala Val Leu Thr Ala Thr Val Glu Val Asn
100 105 110
Ser Gly Thr Gln Leu Thr Val Glu Arg Ala Thr Ala Asp Ala Ile Ser
115 120 125
Gln Ala Ala Ala Ala Ser Gln Phe Gly Asp Val Ser Gly Ile Val Arg
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Ala Trp Thr Ala Gly Thr Ser Val Leu Asp
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<210> 2
<211> 465
<212> DNA
<213> Microcystis aeruginosa
<400> 2
atgaaggcct ctaattacca agaaatcgcc agaggagcta ttctagccgg tggtctcgcc 60
gctgcgggtg tattatccgt tggcgaaggg gcccaagccc aatttatcgg tacagcatcc 120
caatcccgag tgtccgctgc caccactagc attctcaccg acggcaacac tgcagccacc 180
aatagctttg ccgttgagca ggttttgcct gctgctgact atgttttcag ttctcctatt 240
tcagtacaaa taagttacga tacccttacc ctagctaagg acaaaaactt cgttgctatt 300
acgggcgcag tattgaccgc tacggtggag gtaaactcag gtacgcaact aacagttgag 360
cgtgccactg ctgatgctat ttcgcaggct gctgctgcca gtcagtttgg ggatgtgtct 420
ggtatcgtcc gggcttggac tgctggtact tccgtcttag attag 465
Claims (7)
1.一种复方硫洗剂助悬剂,其特征在于:其有效成分包括MrpC蛋白,该MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。
2.如权利要求1所述的一种复方硫洗剂助悬剂,其特征在于:其有效成分为所述MrpC蛋白。
3.如权利要求1或2所述的一种复方硫洗剂助悬剂,其特征在于:所述MrpC蛋白的核苷酸序列如SEQ ID NO 02所示。
4.MrpC蛋白在助悬复方硫洗剂中的应用,该MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。
5.如权利要求4所述的应用,其特征在于:所述MrpC蛋白的核苷酸序列如SEQ ID NO 02所示。
6.一种复方硫洗剂组合物,其特征在于:包括升华硫、硫酸锌、樟脑醑、甘油、MrpC蛋白和蒸馏水,其中MrpC蛋白的氨基酸序列如SEQ ID NO 01所示。
7.如权利要求6所述的复方硫洗剂组合物,其特征在于:所述MrpC蛋白的核苷酸序列如SEQ ID NO 02所示。
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尤玲 等: "复方硫洗剂的制备", 《中国医院药学杂志》, vol. 17, no. 10, pages 477 * |
陈新梅,郭喜红,杨轲: "几种助悬剂对复方硫洗剂助悬性能的考察", 中国医院药学杂志, no. 12 * |
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