CN105294849B - 小鼠精原干细胞特异性抗原4933427d06rik及其抗体和应用 - Google Patents
小鼠精原干细胞特异性抗原4933427d06rik及其抗体和应用 Download PDFInfo
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Abstract
本发明公开了一种小鼠精原干细胞特异性抗原4933427D06RIK及其抗体和应用。所述抗原4933427D06RIK由4933427d06rik基因(序列如SEQ ID NO.1所示)编码得到,其氨基酸序列如SEQ ID NO.2所示,并特异性表达于睾丸中精原干细胞表面。本发明所述4933427D06RIK蛋白特异性的表达于睾丸中精原干细胞表面,利用该蛋白制备出相应的抗体,可成功定位精原干细胞,进而可利用流式细胞术等方法高效分离出表达4933427D06RIK蛋白的SSCs细胞,从而实现SSCs细胞的识别、分离和纯化,为SSCs细胞的培养、诱导分化、移植等科研或临床应用提供支持。
Description
技术领域
本发明属于细胞分离纯化技术领域。更具体地,涉及小鼠精原干细胞特异性抗原4933427D06RIK及其抗体和应用。
背景技术
男性不育症的发生率为14%左右,是目前生殖医学面临的重要问题之一,精原干细胞(Spermatogonial stem cells,SSCs)是雄性动物体内一类具有自我更新和分化产生精子能力的细胞群;SSCs的研究发展为治疗男性不育提供了新的策略和方法。精原干细胞是维持精子发生的基础,也是成体哺乳动物体内唯一一种被证明能够将遗传信息传递给下一代的细胞。
成功识别、分离、纯化精原干细胞是进行细胞培养、诱导分化、移植等科研或临床应用的前提。利用抗体通过流式细胞术可以高效分离出表达某种蛋白的细胞,而该方法的关键与核心是蛋白的选择及其抗体的制备。
现有的研究显示,小鼠精原干细胞能够表达THY1、GFRa1、CD9等细胞表面蛋白,细胞表面分子是SSCs分选理想的标记物,但是,这些已知的分子并非在SSCs特异性表达;比如,THY1是熟知的淋巴细胞标记物,因此会严重影响SSCs的分离纯化效果。由于目前缺乏对精原干细胞的特异性分子标记物的认识,无法对SSCs进行准确鉴别、分离和纯化。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和不足,提供一种小鼠精原干细胞特异性抗原4933427D06RIK,该蛋白特异性的表达于睾丸中的精原干细胞表面,利用该蛋白制备出相应的抗体,可成功定位精原干细胞,进而可利用流式细胞术等方法高效分离表达4933427D06RIK蛋白的SSCs细胞,从而实现SSCs细胞的识别、分离和纯化,为SSCs细胞的培养、诱导分化、移植等科研或临床应用提供支持。
本发明的目的是提供一种小鼠精原干细胞特异性抗原4933427D06RIK。
本发明另一目的是提供利用所述抗原4933427D06RIK制备的精原干细胞特异性识别抗体。
本发明的再一目的是提供所述小鼠精原干细胞特异性抗原4933427D06RIK及其抗体在小鼠精原干细胞的特异性识别、分离、纯化中的应用。
本发明上述目的通过以下技术方案实现:
一种4933427d06rik基因,该基因特异性表达于小鼠睾丸中精原干细胞表面,该基因的序列如SEQ ID NO.1所示。
一种小鼠精原干细胞特异性抗原4933427D06RIK,由上述4933427d06rik基因编码得到,其氨基酸序列如SEQ ID NO.2所示,并特异性表达于睾丸中精原干细胞表面。
所述4933427D06RIK特异性的表达于睾丸中精原干细胞表面,因此可作为精原干细胞的标示物,应用于精原干细胞的识别、分离和/或纯化。
因此,所述抗原4933427D06RIK在作为精原干细胞的标示物中的应用,即所述抗原4933427D06RIK在特异性识别、分离和/或纯化精原干细胞方面的应用,均在本发明的保护范围之内。
另外,利用上述抗原4933427D06RIK制备得到的精原干细胞特异性识别抗体4933427D06RIK,也在本发明的保护范围之内。
具体优选地,所述精原干细胞特异性识别抗体4933427D06RIK为多克隆抗体。
作为一种可实施的优选方案,该抗体的制备方法包括如下步骤:
S1.原核表达和纯化4933427D06RIK蛋白;
S2.免疫家兔,获得含小鼠4933427D06RIK蛋白多克隆抗体的血清;
S3.纯化血清,获得纯化的小鼠4933427D06RIK蛋白多克隆抗体。
更具体地,制备方法如下;
S1.将4933427d06rik基因编码区克隆进pDEST17载体,转化BL21感受态细菌;待细菌生长至对数生长期前期,以0.5mM IPTG诱导表达6×His-4933427D06RIK融合蛋白,37℃诱导4小时后离心收集菌体;
S2.以PBS将菌体重悬,超声裂解BL21细菌,离心取裂解液上清。通过镍柱纯化6×His-4933427D06RIK融合蛋白,以包含咪唑溶液洗脱蛋白;以SDS-PAGE确定蛋白浓度与纯度;将纯度高于80%的蛋白用于下一步实验;
S3.纯化后的蛋白用于免疫家兔,多次免疫一个半月后,获得含小鼠4933427D06RIK蛋白多克隆抗体的血清;
S4.将6×His-4933427D06RIK融合蛋白共价偶联于AminoLink Plus CouplingResin;以等体积TBS稀释血清与抗原柱孵育纯化血清,最后以pH2.50.2M甘氨酸洗脱得到抗体。
得到的抗体立刻以1M Tris-HCl pH9.0中和至pH8.0;浓缩并添加等体积甘油于-20℃保存。
另外,取上述获得的纯化后的6×His-4933427D06RIK融合蛋白进行SDS-PAGE,转印至NC膜;用S4中得到的纯化抗体进行孵育,以IRDye 680驴抗兔IgG(H+L)抗体作为二抗,用Odyssey双色红外激发光成像系统检测并记录。
本发明利用该抗体,通过流式细胞术等方法,可分离出表达4933427D06RIK蛋白的精原干细胞(该阳性细胞类群高表达精原干细胞自我更新的标志基因,而低丰度表达精原干细胞分化的标志基因)。
因此,所述精原干细胞特异性识别抗体4933427D06RIK在特异性的识别、分离和/或纯化精原干细胞方面的应用,也都在本发明的保护范围之内。
本发明具有以下有益效果:
本发明公开了一种小鼠精原干细胞特异性抗原4933427D06RIK及其抗体和应用。所述抗原4933427D06RIK由4933427d06rik基因(序列如SEQ ID NO.1所示)编码得到,其氨基酸序列如SEQ ID NO.2所示,并特异性表达于睾丸中精原干细胞表面。
本发明提供的4933427D06RIK蛋白特异性的表达于睾丸中精原干细胞表面,利用该蛋白制备出相应的抗体,可成功定位精原干细胞,进而可利用流式细胞术等方法,可高效分离表达4933427D06RIK蛋白的SSCs细胞,分离得到的SSCs细胞类群可以用于培养、扩增等,从而实现SSCs细胞的识别、分离和纯化,为SSCs细胞的培养、诱导分化、移植等科研或临床应用提供支持,进而SSCs的研究发展为治疗男性不育提供新的策略和方法。
附图说明
图1为4933427d06rik基因(A)及其蛋白的表达情况;图中,A为4933427d06rik基因在各个组织的表达情况,B为蛋白在各个组织的表达情况。
图2为4933427d06rik基因和plzf基因的表达随小鼠日龄增长的变化趋势;图中。
图3为4933427D06RIK蛋白、PLZF蛋白、GFRa1蛋白、GATA4蛋白阳性细胞在曲细精管上的定位;图中,A为4933427D06RIK以及PLZF蛋白、B为GFRa1蛋白、C为GATA4蛋白,D为其细胞重叠性统计。
图4为利用4933427D06RIK抗体进行流式细胞分选4933427D06RIK阳性细胞的结果。
图5为4933427D06RIK阳性细胞类群的plzf,bcl6b,etv5等精原干细胞自我更新相关基因表达情况。
图6为4933427D06RIK阳性细胞类群的c-kit,sohlh1,sohlh2等精原细胞分化基因表达情况。
图7为4933427D06RIK阳性细胞类群在体外培养体系中的生长情况。
图8为抗体用Odyssey双色红外激发光成像系统检测的记录图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1 4933427d06rik基因及其蛋白的表达情况
1、实验方法
取小鼠睾丸组织于Trizol中,苯-氯仿法提取RNA,反转录为cDNA。设计上下游引物进行PCR,产物进行凝胶电泳分析。
取小鼠睾丸组织于蛋白裂解中裂解,离心后取上清,加入上样缓冲液,进行SDS-PAGE电泳。随后将蛋白质样品转印至NC膜上,孵育对应的一抗、二抗,用Odyssey双色红外激发光成像系统检测并记录。
2、结果如图1和2所示,4933427d06rik基因及其蛋白在睾丸中特异性表达,4933427d06rik基因和plzf基因的表达随小鼠日龄增长的变化趋势近似。
实施例2免疫荧光实验
1、利用免疫荧光实验检测4933427D06RIK阳性细胞在曲细精管上的定位,以及该细胞类群与其他标志蛋白的关系。
免疫荧光实验的方法如下:
将小鼠睾丸组织——曲细精管片段置于4%多聚甲醛中固定15min,再用PBS洗两次,置于水平摇床5min,加入10%FBS封闭1h;孵一抗4℃过夜,实验第二天用封闭液清洗三次,每次10min,再在室温下孵育二抗1-2h;PBST洗一次,置于水平摇床5min,Hoechst33343染色5min,PBS洗一次,置于水平摇床5min;加封片液,涂指甲油封片,荧光显微镜检测。
2、结果如附图3所示,所述的4933427D06RIK蛋白,其标记的细胞类群,与小鼠精原干细胞标记蛋白PLZF,在不同发育时期均高度重叠。
4933427D06RIK蛋白标记的细胞类群,与As、Apr精原细胞标记蛋白GFRa1,随小鼠周龄增大而重叠比例下降
4933427D06RIK蛋白标记的细胞类群,与睾丸体细胞标记蛋白GATA4,几乎没有重叠。
实施例3制备4933427D06RIK多克隆抗体
制备方法
S1.将4933427d06rik基因编码区克隆进pDEST17载体,转化BL21感受态细菌。待细菌生长至对数生长期前期,以0.5mM IPTG诱导表达6×His-4933427D06RIK融合蛋白,37℃诱导4小时后离心收集菌体。
S2.以PBS将菌体重悬,超声裂解BL21细菌,离心取裂解液上清。通过镍柱纯化6×His-4933427D06RIK融合蛋白,以包含咪唑溶液洗脱蛋白。以SDS-PAGE确定蛋白浓度与纯度。将纯度高于80%的蛋白用于下一步实验。
S3.纯化后的蛋白用于免疫家兔,多次免疫一个半月后,获得含小鼠4933427D06RIK蛋白多克隆抗体的血清。
S4.将6×His-4933427D06RIK融合蛋白共价偶联于AminoLink Plus CouplingResin。以等体积TBS稀释血清与抗原柱孵育纯化血清,最后以pH2.50.2M甘氨酸洗脱得到抗体。得到的抗体立刻以1M Tris-HCl pH9.0中和至pH8.0。浓缩并添加等体积甘油于-20℃保存。
S5.取纯化后的6×His-4933427D06RIK融合蛋白进行SDS-PAGE,转印至NC膜;用S4中得到的纯化抗体进行孵育,以IRDye 680驴抗兔IgG(H+L)抗体作为二抗,用Odyssey双色红外激发光成像系统检测并记录。得到的图如附图8所示。
实施例4流式细胞术分离4933427D06RIK阳性细胞
1.利用流式细胞术,分离4933427D06RIK阳性细胞,表达各种精原干细胞标志基因,而不表达分化精原细胞的标志基因。
流式细胞分选实验方法如下:
(1)用0.25%胰酶和DNaseI将小鼠睾丸组织消化成单细胞悬液,10%FBS终止消化;
(2)离心去除上清,在冰上孵育一抗30min;
(3)10%FBS洗两次,冰上孵育二抗30min;
(4)10%FBS洗两次;
(5)流式检测前5min加入Hoechst33343,进行流式检测;
(6)流式分选时,以SSC/FSC设门,去除细胞碎片和部分死细胞;以Hoechst33343通道设门,去除死细胞;以PE-A通道设门,分离4933427D06RIK阳性或阴性细胞类群。
2、结果如附图4所示,利用4933427D06RIK抗体进行流式细胞分选,分离出4933427D06RIK阳性细胞。
3、对分离出的4933427D06RIK阳性细胞类群的相关基因表达情况进行检测。结果如附图5和6所示,分离得到的4933427D06RIK阳性细胞类群表达plzf,bcl6b,etv5等精原干细胞自我更新相关基因(图5),而低表达c-kit,sohlh1,sohlh2等精原细胞分化基因(图6)。
实施例5 4933427D06RIK阳性细胞类群的体外培养
1、检测4933427D06RIK阳性细胞类群在体外培养体系中的生长情况。具体方法如下:
(1)按照下表配制SSCs无血清培养基:
(2)选择对数生长期的MEF,在培养基中加入丝裂霉素C处理2~3h。处理结束后,用PBS清洗细胞两次后,加入4933427D06RIK阳性细胞群,以SSCs无血清培养基培养。
2、结果如附图7所示,该细胞类群可以在体外培养体系中形成克隆。
综上所述研究表明,4933427D06RIK蛋白特异性的表达于睾丸中的精原干细胞表面,利用该蛋白制备出相应的抗体,可成功定位精原干细胞,进而可利用流式细胞术等方法,可高效分离表达4933427D06RIK蛋白的SSCs细胞,从而实现SSCs细胞的识别、分离和纯化,为SSCs细胞的培养、诱导分化、移植等科研或临床应用提供支持。
SEQUENCE LISTING
<110> 中山大学
<120> 小鼠精原干细胞特异性抗原4933427D06RIK及其抗体和应用
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 2621
<212> DNA
<213> 4933427d06rik基因序列
<400> 1
gaatcccgct ctccttgcac ggaggacttc cctttcgcta gggcagactc accggaagct 60
cactcaggca caagttctct ctagctgagc tgccgctgct cctcctcctc ctcttcccag 120
gtgattgatt ccagcttgtg tcaagttgac agacagacaa acaaacaaaa acaggagaga 180
agaatctgac ggagacctca tggcagaagg cccaagtcca agtcagcttc cctctgagat 240
gcccgatcaa gactcagatg agcgacctgg agagcaaggt gatccctgtg tggaacaaca 300
agacccagtg gaagtagacg agcctaaaga gtcaggccag cctgccagac ccattgctta 360
tgtacgttcg cagaggtctg agcccacaga gcccacagag ccagccggag ctgcaggacg 420
tcatgctggt agaggaaacg atgaagaaca aagagcttac cctggaaggc agagacggag 480
gtggaggcag aggcagaggc agaggcagtg ggcagagaca ggcagccact cacaggcagc 540
cttccaggag tggcaggaac cctctgagga actggttcta cagacatact tcagccacag 600
tgaagaaaca ggggctgcca tgaacctgta tcccaccttg actccagtag tccctgtgcc 660
agcagggatg gcccagtggc cacctatagt ttatgggtac tgggctgcca tggctaccta 720
cccctatgtt accttcatcc ctgtggccaa catagtcccg tgcttcatgt cattcagggc 780
ccccatgttc ttagcctaca tcccttggtg actggtatcc cttggtgctg gccctccctg 840
gctatgtttg tttcccctga ggaggaggaa agcttacagt tgaaaaggtt tattctttct 900
catagtttag gttccagttc atcccagcca ggaaaccagg tacccagcac ttacagctgg 960
tcttgacaca ccccaaatca gaagcacgaa tgcatcctag gtccccagat ggctttagat 1020
ttggtgacat tgacagttaa cactaaccat tagactattt gtccctcctg ggtttattat 1080
ttgaaaaaaa atatttttat ggtccagtgg ccccttaaca cttccccttc ccacttcctc 1140
tctctattct atttttttaa aaatctgtca tacagcctca aatgcatgac tctgggatca 1200
tccactagag tatggataac taatactgca gaaaaatgac tctattctat ttttttaaaa 1260
atctgtcata cagcctcaaa tgcatgactc tgggatcatc cactagagta tggataacta 1320
atactgcaga aaaatgactc ttcctcccta gcaaccatca ttcaccacca gcaacttagc 1380
tgggggtgga gcctcatgag ccccctcccc atccatgctg caatggtgac tggcttgaac 1440
ttgcagcata aggctttccc aggaatctta taccctcagg gaaatggagt gatgctgtac 1500
ccctgaaatc gtgtatttgt gtatagaaag agcaagttac agagctcagg tggaattggg 1560
atgcaagatt acagatttga accaacaacg gatgggactg aacatctgat agccccgact 1620
atcacagagg ggagccagga aggaagcaaa cggccccaga agccgaccca gccgtgccta 1680
gagctgggga ggatgagtgt atgtatgtat gaagttctct gaagcaggat atcctgatgt 1740
ggacccttat agtttggagg cagcagtagc tgttgctgaa tagtagtagt agtagtagta 1800
gtagtagtag tagtagtagt agtactagtt gttgttgttg tttttactat gatatcttat 1860
tgtgtgtatc tgtgtgggca tgtatgtggc acagtctgtc tatggaggtc agaggacaac 1920
ttgcagagac agctcttaat ttttaccctg tgcagacccg agccttgaac ccaggcctgt 1980
aggcttggca gaacgttttt accggaagca ctgtcccacc agcccaggat ccccaagtga 2040
gttgaaaggc actagaagtt cagactagat tgaaaatgag taagtcaaag aaaattggac 2100
aaattgaaca aaaggcacgg gggtttatga aaggaaacaa agagagtgag ccacatttaa 2160
aacaccgagt aagaggctgg gcgatagtgg cgcacacctt taatcccagc acttgtgagg 2220
cagaggcagg tggatctttg ttaattctat gccaacctgg tctgcagagt tacatgatga 2280
gacctgtctc aacagaaggc taagtgtgga atagtttggt aataatagcc ttttggcctc 2340
agtgagaata ttttaaatgt cttcttcttc tttttttttt tttaattatc attaatggga 2400
atttcctgtc agttttctca tggatgtctc tgatcaagtt aaaatagttc ctgtcttcag 2460
tactcctaaa ggtgaattat aaattggatt ttgtatttat gaagtttgta tttgtttcta 2520
agaaaaatgt ccaagaagag tttttatcat gaatctttgt atgtgtgtgt gtgtgtgtag 2580
ttaactatcc ttttaaataa atcctcctct tgggctttgg t 2621
<210> 2
<211> 203
<212> PRT
<213> 4933427D06RIK蛋白氨基酸序列
<400> 2
Met Ala Glu Gly Pro Ser Pro Ser Gln Leu Pro Ser Glu Met Pro Asp
1 5 10 15
Gln Asp Ser Asp Glu Arg Pro Gly Glu Gln Gly Asp Pro Cys Val Glu
20 25 30
Gln Gln Asp Pro Val Glu Val Asp Glu Pro Lys Glu Ser Gly Gln Pro
35 40 45
Ala Arg Pro Ile Ala Tyr Val Arg Ser Gln Arg Ser Glu Pro Thr Glu
50 55 60
Pro Thr Glu Pro Ala Gly Ala Ala Gly Arg His Ala Gly Arg Gly Asn
65 70 75 80
Asp Glu Glu Gln Arg Ala Tyr Pro Gly Arg Gln Arg Arg Arg Trp Arg
85 90 95
Gln Arg Gln Arg Gln Arg Gln Trp Ala Glu Thr Gly Ser His Ser Gln
100 105 110
Ala Ala Phe Gln Glu Trp Gln Glu Pro Ser Glu Glu Leu Val Leu Gln
115 120 125
Thr Tyr Phe Ser His Ser Glu Glu Thr Gly Ala Ala Met Asn Leu Tyr
130 135 140
Pro Thr Leu Thr Pro Val Val Pro Val Pro Ala Gly Met Ala Gln Trp
145 150 155 160
Pro Pro Ile Val Tyr Gly Tyr Trp Ala Ala Met Ala Thr Tyr Pro Tyr
165 170 175
Val Thr Phe Ile Pro Val Ala Asn Ile Val Pro Cys Phe Met Ser Phe
180 185 190
Arg Ala Pro Met Phe Leu Ala Tyr Ile Pro Trp
195 200
Claims (6)
1.4933427D06RIK蛋白在作为小鼠精原干细胞特异性抗原方面的应用,其特征在于,所述4933427D06RIK蛋白的氨基酸序列如SEQ ID NO.2所示,所述应用是指用于制备精原干细胞特异性识别抗4933427D06RIK蛋白抗体。
2.权利要求1中所述4933427D06RIK蛋白在制备能够特异性识别、分离和/或纯化精原干细胞的制剂中的应用。
3.一种精原干细胞特异性识别抗小鼠4933427D06RIK蛋白的抗体,其特征在于,是利用权利要求1中所述4933427D06RIK为抗原制备得到。
4.根据权利要求3所述抗体,其特征在于,所述抗体为多克隆抗体。
5.根据权利要求4所述抗体,其特征在于,其制备方法包括如下步骤:
S1.原核表达和纯化4933427D06RIK蛋白;
S2.免疫家兔,获得含抗小鼠4933427D06RIK蛋白的多克隆抗体的血清;
S3.纯化血清,获得纯化的抗小鼠4933427D06RIK蛋白的多克隆抗体。
6.权利要求3~5任一所述抗体在制备能够特异性的识别、分离和/或纯化精原干细胞的制剂方面的应用。
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unnamed protein product [Mus musculus];Carninci,P. et al.;《GenBank: BAC26859.1》;20101006;全文 * |
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