CN111733148A - 一种重组spam1蛋白及其应用 - Google Patents

一种重组spam1蛋白及其应用 Download PDF

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CN111733148A
CN111733148A CN202010541299.7A CN202010541299A CN111733148A CN 111733148 A CN111733148 A CN 111733148A CN 202010541299 A CN202010541299 A CN 202010541299A CN 111733148 A CN111733148 A CN 111733148A
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顾亚云
曾旭辉
陈晨
彭利忠
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Abstract

本发明公开了一种重组SPAM1蛋白及其应用，属于医学技术领域。所述重组SPAM1蛋白的氨基酸序列如SEQ ID NO:1所示。本发明的重组SPAM1蛋白具有较高的降解透明质酸的酶活力。与不含有GPI修饰结构域SPAM1‑W相比，加入含有GPI锚修饰结构域的SPAM1重组蛋白后，精子胞内钙信号明显提高且其顶体反应发生率显著增加。完整SPAM1蛋白的成功制备将更有效的提高体外受精作用，提高其临床应用度，为男性不育的治疗提供有效的手段。

Description

一种重组SPAM1蛋白及其应用
技术领域
本发明属于医学技术领域，具体涉及一种重组SPAM1蛋白及其应用。
背景技术
特异性人精子黏附分子（SPAM1）是通过羧基末端的糖基磷脂酰肌醇（GPI）锚定至精子头部浆膜和顶体内膜，但不跨越膜磷脂双层的单链膜蛋白，即人透明质酸酶PH-20。SPAM1的509个氨基酸序列主要由三部分组成：信号肽（1-35氨基酸）、SPAM1结构区（36-483氨基酸）及GPI锚修饰结构域（484-509氨基酸）。GPI修饰锚是真核生物翻译后修饰所产生，主要由磷酸氨基乙醇联结子、核心聚糖和糖脂链组成，其中在聚糖核心的磷酸肌醇、葡萄糖胺甘露醇残基可以被磷酸氨基乙醇组和其他糖类不同程度的修饰，这种复杂的结构具有编码细胞膜表面上不同功能的能力。GPI锚定蛋白SPAM1的生理功能具有多样性，如：发挥位于头部浆膜透明质酸酶中性活性和顶体内膜酶酸性、中性活性，有效降解卵细胞周质；识别并结合透明带，促进精卵结合；作为透明质酸受体，调节Ca2+信号而介导精子顶体胞吐作用。
近年来，针对SPAM1的特异酶活性，科研人员将检测SPAM1的透明质酸酶活性的高低作为预测人类精子受精潜能的重要指标。虽然研究表明无GPI锚修饰结构区的重组SPAM1已被成功体外表达并应用于体外受精（IVF）和胞浆内单精子注射（ICSI）技术，也可用于解决大分子药物难以渗透肿瘤细胞外基质的问题中，但该重组蛋白缺乏因 GPI 结合肽段（484-509 氨基酸区域），限制了 SPAM1 进行更好的临床应用。
现有研究表明不育男性精子SPAM1活性与精子密度、活力、形态及精卵结合等因素密切相关，精子顶体内透明质酸含量及活性的降低是导致男性不育的重要原因之一，检测透明质酸活性可作为临床评价精子功能的有效指标。因此，完整的SPAM1蛋白的成功制备将有助于深入理解受精过程，并为男性不育的诊断与治疗提供新策略。
发明内容
本发明的目的是提供一种重组SPAM1蛋白，该重组SPAM1蛋白含有GPI修饰结构域，具有较高的降解透明质酸的酶活力，可用于治疗弱精症。
为了实现上述发明目的，本发明采用以下技术方案：
一种重组SPAM1蛋白，其氨基酸序列如SEQ ID NO:1所示；编码所述重组SPAM1蛋白的核苷酸序列如SEQ ID NO:2所示。
上述重组SPAM1蛋白的制备方法，包括以下步骤：
步骤1，利用Hind III/Xho I双酶切位点将SEQ ID NO:2所示序列插入至表达载体pcDNA3.1(+)中，获得C端含有His标签的重组质粒；
步骤2，将重组质粒转染至293T细胞进行表达，并对获得的蛋白进行纯化，得到重组SPAM1蛋白。
上述重组SPAM1蛋白在制备男性不育治疗药物中的应用。
上述重组SPAM1蛋白在制备男性不育诊断药物/试剂中的应用。
本发明通过体外重组并优化纯化条件获得含有GPI修饰结构域的SPAM1蛋白，该重组蛋白具有较高的降解透明质酸的酶活力。与不含有GPI修饰结构域SPAM1-W相比，加入含有GPI锚修饰结构域的SPAM1重组蛋白后，精子胞内钙信号明显提高且其顶体反应发生率显著增加。完整SPAM1蛋白的成功制备将更有效的提高体外受精作用，提高其临床应用度，为男性不育的治疗提供有效的手段。
附图说明
图1为实施例1中SPAM1重组蛋白的纯化结果，（a）为AKTA纯化系统分析阴离子交换层析曲线，(b) 为SDS-PAGE 检测结果。
图2为实施例1中SPAM1重组蛋白降解透明质酸活力的标准曲线。
图3为实施例2中重组蛋白SPAM1对人精子细胞内 Ca2+浓度（[Ca2+]i）的影响。
图4为实施例3中重组蛋白SPAM1 对人精子顶体反应的影响结果。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
GPI锚定SPAM1蛋白体外重组及纯化
利用Hind III/Xho I双酶切位点将全基因合成的SPAM1/ SPAM1-W的目的基因插入至表达载体pcDNA3.1(+)中，获得C端含有His标签的重组质粒Spam1-pcDNA3.1(+)和Spam1-W-pcDNA3.1(+)，转染至细胞密度高达80%-90%的293T细胞中，37℃、5% CO2培养箱中培养24 -48 h，收集细胞。利用试剂盒分离细胞膜蛋白与浆蛋白，16000 rpm离心30 min，抗SPAM1抗体进行western blot检测。
上述重组SPAM1蛋白，其氨基酸序列如SEQ ID NO.1所示，编码所述重组SPAM1蛋白的核苷酸序列如SEQ ID NO.2所示。
SPAM1-W蛋白为缺乏GPI结合结构域的SPAM1蛋白，其氨基酸序列如SEQ ID NO.3所示，编码所述重组SPAM1-W蛋白的核苷酸序列如SEQ ID NO.4所示。
采用 Ni Sepharose Excel柱进行亲和层析，并SDS-PAGE检测纯度，根据纯度和杂蛋白情况再借助离子交换柱进行进一步优化纯化制备条件，检测蛋白纯度。
如图1所示，所制得的SPAM1重组蛋白电泳条带清晰，纯度高。
利用将SPAM1重组蛋白按一定比例进行稀释置于96孔板中，加入适量温热的透明质酸底物，震荡后37℃，反应10 min，血清工作液加入上述反应液中，震荡混匀15 min后测量样品在640 nm处的吸收度并计算重组蛋白活性。
如图2所示，利用浊度法测定纯化的重组蛋白SPAM1及 SPAM1-W体外降解透明质酸底物的比活力分别为420.21±1.19 U/mg 和340.43±0.25 U/mg。
实施例2
高通量测定精子胞内钙信号
5 μM荧光探针Fluo-4 AM和0.05% 表面活性剂Pluronic F-127加入percoll纯化的精子中，37℃、5% CO2培养箱中避光染色30 min。2000 rpm离心5 min，弃上清，HS缓冲液重悬，重复两次，即获得荧光染色的精子溶液。人精子Ca2+浓度的变化通过FlexStation3全自动钙流检测工作站进行检测：以HS缓冲液为阴性对照、孕酮P4为阳性对照，20 μL不同浓度的SPAM1或SPAM1-W蛋白分别自动加入至80 μL荧光染色后的精子中，490 nm激发波长/525 nm发射波长检测精子胞内Ca2+离子变化情况。
如图3所示，含有GPI锚定重组蛋白SPAM1对人精子[Ca2+]i 产生高达 80% 的增强效应，而缺乏GPI结合结构域的SPAM1-W则对[Ca2+]i 无影响，进一步说明了GPI锚修饰结构域对人精子胞内钙信号转导起关键调节作用。
实施例3
金霉素染色法（CTC）考察SPAM1对人精子顶体反应的作用
取1 mL液化好的精液进行percoll梯度纯化，适量HTF重悬沉淀。取50 μL重悬精子混悬液，加入阳性对照孕酮P4、阴性对照HTF缓冲液及重组蛋白SPAM1/SPAM1-W，37℃、5% CO2孵育4 h。按等比例加入40 μL CTC染色液，染色30 s，再加入3.5 μL 10% 福尔马林固定液封片，室温过夜。正置荧光显微镜下对精子数目（每个实验组不少于200个）进行统计，判断精子发生获能及发生顶体反应情况。荧光显微镜下观察精子可将其分为未获能型（F型）、获能但未发生顶体反应型（B型）及发生顶体反应（AR型），其中“B+AR”型精子所占比例为精子获能百分率，“AR”型精子所占比例为精子发生顶体反应率。
如图4所示，与SPAM1-W相比，加入含有GPI锚修饰结构域的SPAM1重组蛋白后，顶体反应发生率显著增加。
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Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro
20 25 30
Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala
35 40 45
Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr
50 55 60
Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro
65 70 75 80
Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile
85 90 95
Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp
100 105 110
Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val
115 120 125
Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu
130 135 140
Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala
145 150 155 160
Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg
165 170 175
Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His
180 185 190
His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile
195 200 205
Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu
210 215 220
Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr
225 230 235 240
Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile
245 250 255
Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val
260 265 270
Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr
275 280 285
Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp
290 295 300
Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp
305 310 315 320
Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu
325 330 335
Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys
340 345 350
Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp
355 360 365
Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly
370 375 380
Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys
385 390 395 400
Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp
405 410 415
Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala
420 425 430
Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn
435 440 445
<210> 4
<211> 1344
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctgaatttca gagcacctcc tgttattcca aatgtgcctt tcctctgggc ctggaatgcc 60
ccaagtgaat tttgtcttgg aaaatttgat gagccactag atatgagcct cttctctttc 120
ataggaagcc cccgaataaa cgccaccggg caaggtgtta caatatttta tgttgataga 180
cttggctact atccttacat agattcaatc acaggagtaa ctgtgaatgg aggaatcccc 240
cagaagattt ccttacaaga ccatctggac aaagctaaga aagacattac attttatatg 300
ccagtagaca atttgggaat ggctgttatt gactgggaag aatggagacc cacttgggca 360
agaaactgga aacctaaaga tgtttacaag aataggtcta ttgaattggt tcagcaacaa 420
aatgtacaac ttagtctcac agaggccact gagaaagcaa aacaagaatt tgaaaaggca 480
gggaaggatt tcctggtaga gactataaaa ttgggaaaat tacttcggcc aaatcacttg 540
tggggttatt atctttttcc ggattgttac aaccatcact ataagaaacc cggttacaat 600
ggaagttgct tcaatgtaga aataaaaaga aatgatgatc tcagctggtt gtggaatgaa 660
agcactgctc tttacccatc catttatttg aacactcagc agtctcctgt agctgctaca 720
ctctatgtgc gcaatcgagt tcgggaagcc atcagagttt ccaaaatacc tgatgcaaaa 780
agtccacttc cggtttttgc atatacccgc atagttttta ctgatcaagt tttgaaattc 840
ctttctcaag atgaacttgt gtatacattt ggcgaaactg ttgctctggg tgcttctgga 900
attgtaatat ggggaaccct cagtataatg cgaagtatga aatcttgctt gctcctagac 960
aattacatgg agactatact gaatccttac ataatcaacg tcacactagc agccaaaatg 1020
tgtagccaag tgctttgcca ggagcaagga gtgtgtataa ggaaaaactg gaattcaagt 1080
gactatcttc acctcaaccc agataatttt gctattcaac ttgagaaagg tggaaagttc 1140
acagtacgtg gaaaaccgac acttgaagac ctggagcaat tttctgaaaa attttattgc 1200
agctgttata gcaccttgag ttgtaaggag aaagctgatg taaaagacac tgatgctgtt 1260
gatgtgtgta ttgctgatgg tgtctgtata gatgcttttc taaaacctcc catggagaca 1320
gaagaacctc aaattttcta caat 1344

Claims (5)

1.一种重组SPAM1蛋白，其氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的重组SPAM1蛋白，其特征在于：编码所述重组SPAM1蛋白的核苷酸序列如SEQ ID NO:2所示。
3.权利要求1所述重组SPAM1蛋白的制备方法，其特征在于：包括以下步骤：
步骤1，利用Hind III/Xho I双酶切位点将SEQ ID NO:2所示序列插入至表达载体pcDNA3.1(+)中，获得C端含有His标签的重组质粒；
步骤2，将重组质粒转染至293T细胞进行表达，并对获得的蛋白进行纯化，得到重组SPAM1蛋白。
4.权利要求1所述重组SPAM1蛋白在制备男性不育治疗药物中的应用。
5.权利要求1所述重组SPAM1蛋白在制备男性不育诊断药物/试剂中的应用。
CN202010541299.7A 2020-06-15 2020-06-15 一种重组spam1蛋白及其应用 Pending CN111733148A (zh)

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