CN111235156B - 源于华西雨蛙的免疫调节肽及其基因在皮肤光损伤防护上的应用 - Google Patents
源于华西雨蛙的免疫调节肽及其基因在皮肤光损伤防护上的应用 Download PDFInfo
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Abstract
本发明属于生物化学领域中的多肽技术领域,尤其是涉及源于华西雨蛙的免疫调节肽及其基因,本发明还涉及该肽在制备抗炎药物等方面的应用。免疫调节肽的前体基因由609个核苷酸组成,其5’端至3’端的核苷酸序列为SED ID NO:1所示。两种免疫调节肽均含5个氨基酸残基。本发明中的华西雨蛙免疫调节肽FW1和FW2具有分子量小、人工合成简单、抗炎症作用高效、溶血活性低的优点,可以用于制备抗炎药物、保健品、化妆品,具有广泛的应用前景。
Description
技术领域
本发明属于生物化学领域中的多肽技术领域,尤其是涉及源于华西雨蛙的免疫调节肽及其基因,本发明还涉及该肽在制备抗炎药物等方面的应用。
背景技术
皮肤作为人体最大的器官,是机体防御体系的第一道屏障,直接暴露于多种外界环境因素之中,如机械损伤、病原微生物入侵以及紫外线辐射等。长时间大剂量的紫外辐射会对皮肤造成一系列损伤,包括引起炎症反应、促进表皮细胞凋亡、引起色素沉淀、光老化、破坏皮肤屏障以及诱发皮肤癌等等。此外,UVB辐射还会导致皮肤出现典型的急性炎症反应,包括表皮细胞内一系列促炎细胞因子如TNF-、IL-6以及IL-1的合成和分泌增加,这主要是由于辐射导致表皮角化细胞中活性氧自由基(ROS)的产生和DNA损伤的形成进而造成与炎症相关NF-B信号通路的激活。
为了抵抗活性氧自由基对机体的损害,在漫长的进化过程中生物体形成多种不同的自由基清除系统,包括非基因编码的小分子物质,如尿酸、维生素C、维生素E、胡萝卜烯类、硫辛酸和辅酶Q等;基因编码的大分子抗氧化酶类,如超氧化物歧化酶(SOD)、过氧化氢酶、过氧化物酶、硫氧还蛋白酶系统和谷胱甘肽酶系统;以及从高原两栖类蛙科动物皮肤中发现的各种基因编码的小分子抗氧化多肽。各种不同类型的自由基清除剂在医药和化妆品领域具有极大的应用潜力。
华西雨蛙为蛙科雨蛙属的两栖动物,是中国西南高原地区的特有物种,目前关于华西雨蛙来源的皮肤光损伤防护抗炎多肽的研究还没有报道。
发明内容
本发明的目的是提供两种源于华西雨蛙的抗炎免疫调节肽及其基因与应用。
本发明采取以下技术方案来实现这些内容:
一种源于华西雨蛙的免疫调节肽的前体基因,免疫调节肽的前体基因由609个核苷酸组成,其5’端至3’端的核苷酸序列为SED ID NO:1所示。
其5’端至3’端的核苷酸序列(SED ID NO:1):
atggcattgttgaagaaatcacttttccttgtactattccttggattagtctcactgtca 60
ttctgtgaagaagagaaaagagaggaagaagaagaagagaagaaaagagatggtagtgag 120
gaggaagaaaagagattttggccactgataggccgtcgtgatgaagaaaagcgattttgg 180
ccaatgataggtcgtcgtgatgaagaaaagcgattttggccactgataggccgtcgtgat 240
gaagaaaagagattttggccactgataggccgtcgtgatgaagagaagagattttggcca 300
atgataggccgtcgtgatgaagaaaagaaattttggccactgataggccgtcgtgatgaa 360
gaaaagagattttggccaatgataggccgtcgtgatgaagaaaagagattttggccaatg 420
ataggccgtcgtgatgaagaaaagagattttggccactgataggccgtcgtgatgaagaa 480
aagagaaggcctggccgtcgttaataaaatgtcatgtttcatctgtaaggagcacaatta 540
ctattagttgttccagatctataataaagcatattaaactgaaaaaaaaaaaaaaaaaaa 600
aaaaaaaaa 609
一种1所述前体所得源于华西雨蛙的免疫调节肽,源于华西雨蛙的免疫调节肽分别为华西雨蛙免疫调节肽FW-1和华西雨蛙免疫调节肽FW-2;其,华西雨蛙免疫调节肽FW-1的编码基因的多拷贝点分别位于SED ID NO:1的第136-150,214-228,253-267,331-345,448-462位核苷酸;华西雨蛙免疫调节肽FW-2的编码基因的多拷贝点分别位于SED ID NO:1的第176-189,292-306,370-384,409-423位核苷酸。
所述华西雨蛙免疫调节肽FW-1分子量为673.8Da,等电点5.52,由5个氨基酸残基组成,全序列的一级结构为:Phe1Trp2Pro3Leu4Ile5,其中C末端酰胺化修饰;
所述华西雨蛙免疫调节肽FW-2分子量为691.9Da,等电点5.52,由5个氨基酸残基组成,全序列的一级结构为:Phe1Trp2Pro3Met4Ile5,其中C末端酰胺化修饰。
一种所述源于华西雨蛙的免疫调节肽的应用,所述华西雨蛙免疫调节肽FW-1和/或华西雨蛙免疫调节肽FW-2在抗紫外辐射引起的皮肤炎症反应中的应用。
所述调节肽在制备抗紫外辐射引起的皮肤炎症中免疫调节功能的药物、保健品或化妆品中的应用。
一种重组蛋白,含至少一种所述华西雨蛙免疫调节肽。
一种重组表达载体,含所述的重组表达载体。
一种重组细胞,含所述的重组表达载体。
一种组合药物,为含至少一种所述华西雨蛙免疫调节肽、重组蛋白、重组表达载体、重组细胞中一种或几种。
本发明通过UVB照射小鼠皮肤引起急性炎症反应的动物模型实验检测抗炎免疫调节肽FW-1,FW-2抗炎活性,并通过免疫调节肽FW-1,FW-2分别与动物红细胞悬液混合,检测溶血活性。
本发明的优点是:
本发明中的华西雨蛙免疫调节肽FW-1及FW-2具有分子量小、人工合成简单的特点,其所述华西雨蛙免疫调节肽具有抗紫外辐射引起的皮肤炎症反应的免疫调节功能。
蛙类皮肤提取的小分子抗炎多肽具有极高的抗炎活性和极低的溶血性,且有热稳定性强、水溶性好、无毒副作用,在人体内可被降解消化的优势,在制备抗炎药物、保健品、化妆品具有很好的应用前景。
附图说明
图1是华西雨蛙免疫调节肽FW-1及FW-2抗紫外引起的表皮增厚的活性图;
图2是华西雨蛙免疫调节肽FW-1及FW-2抗紫外引起的炎性细胞浸润活性图。
具体实施方式
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合附图详细说明如后。
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
本发明首先通过基因克隆得到免疫调节肽FW-1与FW-2的编码基因,再通过化学合成的方法得到两种免疫调节肽的一级结构序列,两种免疫调节肽均含5个氨基酸残基。本发明中的华西雨蛙免疫调节肽FW1和FW2具有分子量小、人工合成简单、抗炎症作用高效、溶血活性低的优点,可以用于制备抗炎药物、保健品、化妆品,具有广泛的应用前景。
实施例1华西雨蛙免疫调节肽(FW-1/2)前体基因克隆:
1)华西雨蛙皮肤总RNA提取:
①取200mg华西雨蛙背部皮肤组织,放入研钵中加入液氮研磨成粉末,转移到EP管中,加入1ml总RNA提取缓冲液(Trizol,美国Life公司产品),充分混匀,而后于4℃,12000rpm离心10min。
②离心取上清,加入0.2ml氯仿溶液,剧烈混匀,室温放置10分钟,而后以4℃,12000rpm离心10分钟,弃除沉淀。
③上清加入等体积的异丙醇,室温放置10分钟,以4℃,12000rpm离心10分钟,收集沉淀用75%(V/V)乙醇洗一次,晾干,管底沉淀物即为华西雨蛙皮肤总RNA。
2)华西雨蛙皮肤cDNA二链合成:采用CLONTECH公司In-Fusion SMARTerTMDirectional cDNA Library Construction Kit合成。
(1)cDNA第一链合成(mRNA反转录):
①RNase-free的PCR管中加入1μl华西雨蛙皮肤总RNA、1μl 3’端一链合成引物(3’In-Fusion SMARTer CDS Primer)(5’-CGGGGTACGATGAGACACCATTTTTTTTTTTTTTTTTTTTVN-3’)和2.5μl RNase-free水使总体积达到4.5μl,混匀后短暂离心(2000rpm,30s),离心后于72℃保温3分钟;保温后再将离心管在42℃孵育2分钟。
②在上述离心管中加入以下试剂(均为CLONTECH公司In-Fusion SMARTerTMDirectional cDNA Library Construction Kit建库试剂盒中配备),2.0μl 5×第一链缓冲液、0.25μl 100mM DTT、1.0μl 10mM dNTP Mix、1.0μl SMARTer V Oligonucleotide、0.25μl RNase Inhibitor和1.0μl SMARTScribe Reverse Transcriptase反转录酶,混合离心管中试剂并短暂离心(2000rpm,30s),在42℃保温90min,然后68℃保温10min。保温处理后将离心管置于冰上中止第一链的合成。从离心管取2μl所合成的cDNA第一链备用。
(2)采用长末端聚合酶链式反应(LD-PCR)方法扩增第二链(所用试剂均为CLONTECH公司In-Fusion SMARTerTM Directional cDNA Library Construction Kit建库试剂盒中配备)
①将2μl cDNA第一链(mRNA反转录)、80μl去离子水、10μl 10×Advantage 2PCR缓冲液、2μl50×dNTP混合物、2μl 5’PCR引物、2μl3’In-Fusion SMARTer PCR引物(5’-CGGGGTACGATGAGACACCA-3’)以及2μl50×Advantage 2Polymerase Mix在95℃预热的PCR管中进行混合。
②在PCR仪中按以下程序扩增:
95℃,1min;18个循环:95℃,15sec,65℃,30sec,68℃,6min。循环结束后,将离心管中合成的cDNA双链-80℃保存。
(3)华西雨蛙免疫调节肽FW-1/FW-2编码基因克隆:
根据雨蛙科已鉴定的活性多肽cDNA序列信号肽前约50个核苷酸序列的保守区域,设计上游特异性正向引物,其序列为5’-GCACTCACTCACCCAAACAT-3’,反向引物为CLONTECH公司In-Fusion SMARTer TM Directional cDNA Library Construction Kit中的3’-PCR引物,其序列为5’-CGGGGTACGATGAGACACCAT-3’。PCR反应在如下条件下进行:95℃4min,95℃30sec,57℃30sec和72℃1min,30个循环。扩增完成后用胶回收试剂盒(天根生物)进行目的片段回收。将回收的目的片段连接到pMD19-T载体(Takara,大连),转化DH5α感受态细胞。涂板并进行氨苄青霉素筛选,挑取单菌落用M13引物PCR检测插入片段大小。挑取阳性菌落,摇菌提取质粒,使用Applied Biosystems DNA sequencer,model ABI PRISM 377进行核苷酸测序。
测定结果:
编码华西雨蛙免疫调节肽(FW-1/2)前体的基因自5’端至3’端序列为:
atggcattgttgaagaaatcacttttccttgtactattccttggattagtctcactgtca 60
ttctgtgaagaagagaaaagagaggaagaagaagaagagaagaaaagagatggtagtgag 120
gaggaagaaaagagattttggccactgataggccgtcgtgatgaagaaaagcgattttgg 180
ccaatgataggtcgtcgtgatgaagaaaagcgattttggccactgataggccgtcgtgat 240
gaagaaaagagattttggccactgataggccgtcgtgatgaagagaagagattttggcca 300
atgataggccgtcgtgatgaagaaaagaaattttggccactgataggccgtcgtgatgaa 360
gaaaagagattttggccaatgataggccgtcgtgatgaagaaaagagattttggccaatg 420
ataggccgtcgtgatgaagaaaagagattttggccactgataggccgtcgtgatgaagaa 480
aagagaaggcctggccgtcgttaataaaatgtcatgtttcatctgtaaggagcacaatta 540
ctattagttgttccagatctataataaagcatattaaactgaaaaaaaaaaaaaaaaaaa 600
aaaaaaaaa 609
华西雨蛙免疫调节肽(FW-1,FW-2)前体的编码基因核苷酸序列表为:序列长度为609个碱基,序列类型:核酸,链数:单链,拓扑学:直链状,序列种类:cDNA,来源:华西雨蛙皮肤。
实施例2华西雨蛙免疫调节肽FW-1/2的制备:
(1)华西雨蛙免疫调节肽FW-1/2的化学合成方法:根据上述实施例1获得前体编码基因,推导的氨基酸序列,用自动多肽合成仪(433A,Appl ied Biosystems)合成其全序列,通过HPLC反相柱层析脱盐纯化。
(2)分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)。(3)纯化的免疫调节肽FW-1/2用高效液相色谱HPLC方法鉴定其纯度,分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF),等电聚焦电泳测定等电点,用自动氨基酸测序仪测定氨基酸序列结构。
华西雨蛙免疫调节肽FW-1/2是基因编码的两种小分子多肽,均分别由5个氨基酸残基组成,分子量依次分别是673.8Da和691.9Da,等电点均为5.52。华西雨蛙免疫调节肽FW-1和FW-2全序列分别依次为:
Phe1Trp2Pro3Leu4Ile5和Phe1Trp2Pro3Met4Ile5,其中C末端酰胺化修饰。
实施例3华西雨蛙免疫调节肽FW-1/2的抗紫外引起的皮肤损伤活性检测:
C57BL/6小鼠则于UVB辐照前进行脱毛处理暴露出皮肤,裸露皮肤处记号笔画一1cm直径圆圈,在此圆圈内给药。之后分为4组:正常组(不做UVB辐照及任何处理)、生理盐水组(UVB辐照前,皮内注射100ul生理盐水)、UVB辐照及样品处理组(于UVB辐照前,皮内注射5mg/kg剂量的多肽样品,每天一针,样品分别为调节肽FW-1或FW-2)以及仅样品处理组(不做UVB辐照仅以5mg/kg剂量皮内注射相应剂量多肽样品)。
样品注射后立即进行相应剂量UVB(500mJ/cm2)辐照,之后48hr于记号笔画圈区域皮肤打下1-2块6mm孔径皮肤组织,置于PBS中漂洗干净血渍后置于10%福尔马林(4%甲醛)中固定后进行石蜡包埋、切片及HE染色后观察并定量皮肤表皮厚度以及炎症细胞浸润情况(参见图1和图2)。
由图1可见正常的小鼠皮肤在经过一定剂量UVB照射后会出现明显的表皮增厚的现象,而调节肽FW-1和FW-2处理能明显抑制紫外照射引起的表皮增厚;
由图2可见正常的小鼠皮肤在经过一定剂量UVB照射后会出现明显的炎性细胞浸润增强的现象,而调节肽FW-1和FW-2处理能明显抑制紫外照射引起的炎症反应。综合图1及图2,说明华西雨蛙免疫调节肽FW-1/2具有一定的抗紫外辐射引起的皮肤光损伤和炎症反应的功能。
实施例4华西雨蛙免疫调节肽FW-1/2的溶血活性测定:
将采集的新鲜C57BL/6小鼠血与阿氏液混合抗凝,生理盐水洗涤2次,并经生理盐水重悬成107-108cell/ml的悬浮液。上述稀释好的红细胞悬液与溶解于生理盐水的华西雨蛙免疫调节肽FW-1或FW-2样品终浓度均为200μg/ml,而后混合,37℃保温30min,再于1000rpm离心5min,上清液于540nm测吸收值。阴性对照使用生理盐水,阳性对照使用TritonX-100,溶血百分比按以下公式计算:溶血百分比(H%)=A样品-A阴性对照/A阳性对照×100%。
结果表明FW-1和FW-2浓度为200μg/ml时,溶血百分比分别为1.97%和1.82%。说明免疫调节肽FW-1/2对哺乳动物红细胞具有极低的溶血活性。
序列表
<110> 温州医科大学
中国科学院昆明动物研究所
<120> 源于华西雨蛙的免疫调节肽及其基因在皮肤光损伤防护上的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 609
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcattgt tgaagaaatc acttttcctt gtactattcc ttggattagt ctcactgtca 60
ttctgtgaag aagagaaaag agaggaagaa gaagaagaga agaaaagaga tggtagtgag 120
gaggaagaaa agagattttg gccactgata ggccgtcgtg atgaagaaaa gcgattttgg 180
ccaatgatag gtcgtcgtga tgaagaaaag cgattttggc cactgatagg ccgtcgtgat 240
gaagaaaaga gattttggcc actgataggc cgtcgtgatg aagagaagag attttggcca 300
atgataggcc gtcgtgatga agaaaagaaa ttttggccac tgataggccg tcgtgatgaa 360
gaaaagagat tttggccaat gataggccgt cgtgatgaag aaaagagatt ttggccaatg 420
ataggccgtc gtgatgaaga aaagagattt tggccactga taggccgtcg tgatgaagaa 480
aagagaaggc ctggccgtcg ttaataaaat gtcatgtttc atctgtaagg agcacaatta 540
ctattagttg ttccagatct ataataaagc atattaaact gaaaaaaaaa aaaaaaaaaa 600
aaaaaaaaa 609
Claims (6)
1.一种源于华西雨蛙的免疫调节肽的前体基因,其特征在于:免疫调节肽的前体基因由609个核苷酸组成,其5’端至3’端的核苷酸序列为SED ID NO:1所示。
2.一种权利要求1所述前体基因所得源于华西雨蛙的免疫调节肽,其特征在于:源于华西雨蛙的免疫调节肽为华西雨蛙免疫调节肽FW-2,华西雨蛙免疫调节肽FW-2的编码基因的多拷贝点分别位于SED ID NO:1的第175-189, 292-306,370-384,409-423位核苷酸;
所述华西雨蛙免疫调节肽FW-2分子量为691.9 Da,等电点5.52,由5个氨基酸残基组成,全序列的一级结构为:Phe1Trp2Pro3Met4Ile5,其中C末端酰胺化修饰。
3.按权利要求2所述的源于华西雨蛙的免疫调节肽在制备抗紫外辐射引起的皮肤炎症的免疫调节功能药物中的应用。
4.一种重组蛋白,其特征在于,含权利要求2所述的源于华西雨蛙的免疫调节肽。
5.一种重组表达载体,其特征在于:含编码权利要求4所述的重组蛋白的基因。
6.一种重组细胞,其特征在于:含权利要求5所述的重组表达载体。
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