CN107188954B - 一种与生殖支原体MgPa特异结合的受体蛋白及其分离方法和用途 - Google Patents
一种与生殖支原体MgPa特异结合的受体蛋白及其分离方法和用途 Download PDFInfo
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Abstract
本发明提供了一种与生殖支原体MgPa特异结合的受体蛋白及其分离方法和用途,本发明首先筛选得到了位于人尿道上皮细胞膜表面的受体蛋白——亲环蛋白。所述蛋白分离自人尿道上皮细胞的细胞膜蛋白,其核苷酸序列如SEQ ID NO.1所示,由165个氨基酸组成。本发明利用改进的VOPBA方法筛选rMgPa在人尿道上皮细胞膜上的受体,为进一步阐明Mg可能的黏附和致病机制,为进一步设计受体模拟分子和拮抗分子阻挡Mg黏附入侵宿主细胞,为Mg感染防治提供前期实验基础。因此所述受体蛋白CypA能够用于制备治疗或预防生殖支原体感染性疾病的靶向药物中。
Description
技术领域
本发明涉及一种与生殖支原体MgPa特异结合的受体蛋白——亲环蛋白(Cyclophilin A,CypA),还提供了该蛋白的分离方法和用途,属于生物工程和疾病诊断与防治技术领域。
背景技术
生殖支原体(Mycoplasma genitalium,Mg)是首次从一名非淋菌性尿道炎患者尿道分泌物中分离出的一种泌尿生殖道感染支原体。临床研究表明,女性感染Mg可引起宫颈炎、盆腔炎和不孕等;而男性感染则可引起急慢性非淋菌性尿道炎和不育;另外,Mg感染还与人类免疫缺陷病毒(HIV)的机会感染密切相关,是HIV的协同因子,故称之为艾滋病相关支原体。
病原体和宿主细胞膜上相应的受体蛋白的结合是病原体感染宿主的第一步,对其能否感染宿主细胞至关重要。研究表明,Mg必须先黏附于泌尿生殖道或呼吸道黏膜上皮细胞才能感染宿主并定植于感染部位或侵入细胞内。
支原体没有细胞壁,但在Mg的膜上存在较多的膜蛋白。Mg通过细胞膜上的膜蛋白介导其定植到宿主细胞表面而感染致病。生殖支原体黏附蛋白(Mycoplasma genitaliumprotein of adhesion,MgPa)是Mg含量最多的一种膜蛋白,且是一种最主要的黏附蛋白,在介导Mg黏附甚至侵入宿主上皮细胞的过程中发挥着至关重要的作用。自发性的无细胞黏附活性的Mg突变株则缺乏MgPa。Iverson-Cabral SL等的研究也表明在Mg的持续性感染过程中,机体产生的抗体主要针对于MgPa保守的C端可变区,说明MgPa的C端与其免疫原性密切相关。Dehon PM等研究表明针对MgPa C端的抗体能部分抑制Mg对Hep-2细胞的黏附,说明MgPa是一种重要的黏附蛋白,且主要通过其C端黏附于宿主上皮细胞。既然MgPa与Mg的粘附、感染致病有关,因此在宿主细胞上可能存在与MgPa相互作用的受体蛋白。然而,迄今为止,关于Mg通过MgPa与宿主细胞膜上什么受体分子结合,从而导致其黏附或入侵到宿主上皮细胞,尚未见相关报道。
宿主细胞表面的受体是决定生殖支原体对宿主细胞是否易感的关键因素,且是决定生殖支原体的入侵途径、传播方式、和宿主的病理特征的主要因素之一。因此,对生殖支原体受体的研究有助于从分子水平上阐明生殖支原体的致病机理及其与宿主细胞的相互作用的关系,利用人工表达的受体分子或其模拟分子与拮抗分子阻挡生殖支原体与其受体的结合,是防治生殖支原体感染的重要途径。
发明内容
本发明利用超声破碎法提取人尿道上皮细胞(SV-HUC-1cell)的膜蛋白,应用改进的病毒铺覆蛋白印迹技术(VOPBA),即利用原核表达并经纯化的MgPa直接与转印至PVDF膜上的细胞膜蛋白组分进行孵育,筛选得到了位于人尿道上皮细胞膜表面的受体蛋白——亲环蛋白。所述蛋白分离自人尿道上皮细胞的细胞膜蛋白,其核苷酸序列如SEQ ID NO.1所示,由165个氨基酸组成。
本发明还提供了上述受体蛋白CypA的分离制备方法,包括以下步骤:
(1)对生殖支原体的膜蛋白MgPa进行表达、纯化,制得重组蛋白rMgPa;
(2)采用超声破碎法提取人尿道上皮细胞的膜蛋白;
(3)采用改进的病毒铺覆蛋白印迹技术筛选步骤(2)中制得的膜蛋白中与重组蛋白rMgPa特异结合的膜蛋白,筛选得到一条约17KDa的蛋白;
(4)将步骤(3)中得到的约17KDa蛋白进行质谱分析鉴定,即得上述的受体蛋白CypA。
步骤(2)中,取培养形态良好的人尿道上皮细胞,胰蛋白酶消化5min,加入培养基终止消化,离心6min后收集细胞沉淀;然后加入PBS轻轻吹打细胞沉淀,洗三次;再加入细胞裂解液冰浴20min,加入蛋白酶抑制剂PMSF,超声破碎,10W,5s/次,间歇15s,共计30次,然后2500rpm/min离心10min,取上清离心30min,沉淀用PBS重悬,-80℃保存,即获得人尿道上皮细胞的膜蛋白提取物。
步骤(3)的具体步骤为:
a、SDS-PAGE
将步骤(2)中提取的人尿道上皮细胞膜蛋白,测定浓度后加入上样缓冲液(5×),混匀后煮沸5min,1000g离心1min,取10μL上样进行SDS-PAGE分离,分离胶为12%,浓缩胶为5%,电泳条件为:浓缩胶80V,分离胶120V;同时设置对照组;
b、转膜与封闭
(i)PVDF膜的处理及凝胶的平衡:裁剪一张比凝胶片段略大的PVDF膜,将其用甲醇浸泡10-15s后再用去离子水漂洗10s,经转膜缓冲液平衡10min,电泳结束后,将目的片段放置于转膜缓冲液中平衡10min;
(ii)在半干转膜仪中,将已经平衡好的滤纸、PVDF膜、凝胶、滤纸从下到上依次叠加,每层均用玻璃棒将气泡排除,在恒压条件进行转膜:条件为15V,30min;
(iii)PVDF膜浸入封闭液中,4℃过夜,或者室温摇床2h,TBST漂洗5次,每次5min;
c、免疫印迹
(i)rMgPa蛋白孵育:加入rMgPa蛋白,在4℃孵育6h或过夜,TBST漂洗5次,每次5min;
(ii)孵育一抗:加入稀释1:1000的兔抗rMgPa的多克隆抗体,37℃孵育2h,TBST漂洗5次,每次5min;
(iii)孵育二抗:加入稀释1:5000的HRP标记的羊抗兔IgG,37℃孵育1h,TBST漂洗5次,每次5min;
(iv)显影。
受体是生殖支原体感染宿主细胞时遇到的第一个分子,揭示受体的种类和结构,有助于进一步阐明生殖支原体的致病机制。本发明利用改进的VOPBA方法筛选rMgPa在人尿道上皮细胞膜上的受体,为进一步阐明Mg可能的黏附和致病机制,为进一步设计受体模拟分子和拮抗分子阻挡Mg黏附入侵宿主细胞,为Mg感染防治提供前期实验基础。因此所述受体蛋白CypA能够用于制备治疗或预防生殖支原体感染性疾病的靶向药物中。
附图说明
图1为SDS-PAGE对人尿道上皮细胞膜蛋白分析图;
图2为改进的VOPBA技术筛选到的rMgPa结合蛋白图;
图3为间接ELISA鉴定rMgPa与CypA的特异性结合图;
图4为Western blotting鉴定rMgPa与细胞膜蛋白中CypA的结合图;
图5为CyPA在人尿道上皮细胞上的定位图;
图6为rMgPa被CypA处理后的间接免疫荧光黏附抑制实验图;
图7为Mg被CypA处理后的间接免疫荧光黏附抑制实验图。
具体实施方式
目前,质谱分析技术已在蛋白质与蛋白质组学研究中发挥重要作用。它可以利用蛋白质分子荷质比(M/Z)的不同而将不同的蛋白质分离开来,从而分析鉴定未知蛋白质。本发明专利中对约17kDa蛋白进行质谱分析,结果表明17kDa受体蛋白与CypA蛋白有较强的相关性。
获得受体蛋白和受体蛋白抗体有助于研究受体的特性和功能,本发明中进一步利用CypA蛋白及CypA抗体,经ELISA、Far-western blotting方法研究rMgPa和受体蛋白结合的特异性。结果表明rMgPa能与CypA特异结合;约17kDa的目标条带里的特定蛋白也能与rMgPa结合,说明约17KDa蛋白中含有CypA蛋白。本发明利用间接免疫荧光试验检测CypA在人尿道上皮细胞细胞的定位。结果显示,一部分荧光信号在膜上,一部分在细胞质内,说明CypA在人尿道上皮细胞膜上有分布,由于病原体的受体主要集中在宿主细胞膜表面,因此免疫荧光试验的结果从侧面反映了CypA蛋白可能是rMgPa的受体。
本发明中还为了进一步验证CypA是rMgPa的受体,即CypA能介导rMgPa和Mg与人尿道上皮细胞黏附,并用黏附抑制试验证明商业途径购买的CypA能抑制rMgPa和Mg对人尿道上皮细胞的黏附,说明CypA能用于治疗Mg引起的感染性疾病。
以上内容所述的具体实施例如下:
1、制备重组蛋白rMgPa
根据发明人前期摸索的条件,对重组蛋白rMgPa进行表达、纯化,超滤浓缩、鉴定及去内毒素处理。经BCA试剂盒测定蛋白浓度约为2000μg/mL。
2、超声破碎法提取人尿道上皮细胞膜蛋白
(1)取培养形态良好的人尿道上皮细胞约107/mL,胰蛋白酶消化5min,加入培养基终止消化,1000g,离心6min后收集细胞沉淀;
(2)加入PBS轻轻吹打细胞沉淀,洗三次;
(3)加入细胞裂解液冰浴20min,加入蛋白酶抑制剂PMSF,超声破碎(10W,5s/次,间歇15s,共计30次)然后2500rpm/min离心10min,取上清16 000g离心30min,沉淀用300μL的PBS重悬,-80℃保存;
使用超声破碎法提取人尿道上皮细胞膜蛋白后进行SDS-PAGE分析,结果如图1所示,在12~55KDa范围内,皆有明显的条带出现,超速离心后重悬的膜蛋白,经BCA法测定其浓度为600μg/mL,说明成功提取到人尿道上皮细胞的膜蛋白。
3、改进的VOPBA法筛选与rMgPa特异结合的细胞膜蛋白
3.1 SDS-PAGE
将提取的人尿道上皮细胞膜蛋白,测定浓度后取20μL加入5μL蛋白电泳上样缓冲液(5×),混匀后煮沸5min,1000g离心1min,取10μL上样进行SDS-PAGE分离。分离胶为12%,浓缩胶浓度为5%,电泳条件为:浓缩胶80V,分离胶120V;同时设置对照组。
3.2转膜与封闭
(1)PVDF膜的处理及凝胶的平衡:裁剪一张比凝胶片段略大的PVDF膜,将其用甲醇浸泡10-15s后再用去离子水漂洗10s,随后将PVDF膜用转膜缓冲液平衡10min。电泳结束后,将分离胶中的目的片段放置于转膜缓冲液中平衡10min。
(2)在半干转膜仪中,将已经平衡好的滤纸、PVDF膜、凝胶、滤纸从下到上依次叠加,用玻璃棒将气泡排除。在恒压条件进行转膜:条件为15V,30min。
(3)PVDF膜浸入封闭液中,4℃过夜,或者室温摇床2h,TBST漂洗5次,每次5min。
3.3免疫印迹
(1)rMgPa蛋白孵育:加入rMgPa蛋白,在4℃孵育6h或过夜,TBST漂洗5次,每次5min;
(2)孵育一抗:加入稀释1:1000的兔抗rMgPa的多克隆抗体,37℃孵育2h,TBST漂洗5次,每次5min;
(3)孵育二抗:加入稀释1:5000的HRP标记的羊抗兔IgG,37℃孵育1h,TBST漂洗5次,每次5min;
(4)显影。
结果如下:
提取的膜蛋白经SDS-PAGE分析并转膜后,将其与rMgPa孵育,rMgPa抗体为一抗,HRP标记的羊抗兔IgG为二抗。结果表明,经与rMgPa孵育的泳道在分子量约为17KDa处有一明显的条带出现,而未与rMgPa孵育的泳道没有明显条带出现,如图2所示,说明17KDa蛋白可能为与rMgPa特异结合的靶蛋白。
4、质谱分析鉴定目标条带
(1)取测定浓度后的人尿道上皮细胞膜蛋白10μL加入2.5μL(5×蛋白电泳上样缓冲液),混匀后煮沸5min,1000g离心1min,取10μL上样进行SDS-PAGE分离。分离胶为12%,浓缩胶浓度为5%,电泳条件为:浓缩胶80V,分离胶120V。
(2)带上口罩和薄膜手套进行拆胶;
(3)考马斯亮蓝染色液染色3h,脱色液脱色2h,至目标条带清晰可见;
(4)将切胶用的刀片洗干净,切下来的条带用去离子水洗刷好;
(5)将目标条带放入进口EP管内,用封口膜将EP管封好,常温运输;
(6)由辉骏生物科技进行LC-MS鉴定。
质谱鉴定结果
通过LC-MS的鉴定,检索NCBI数据库,同时把所测得的蛋白做二级质谱分析,经过蛋白质比对匹配分析,CypA蛋白得分很高,可能是能与rMgPa特异结合的蛋白。5、间接ELISA鉴定rMgPa与CypA的结合情况
(1)TBS稀释rMgPa至100μg/mL,取150μL包被ELISA板,4℃湿盒过夜;同时设置对照组;
(2)甩掉包被液,用TBST清洗3次,加满封阻液,4℃封阻2h;
(3)洗板3~5次,用封阻液稀释CypA蛋白(1:1000),100μL/孔,37℃,2h;
(4)洗板3~5次,用封阻液稀释CypA蛋白抗体(1:1000),100μL/孔,37℃,2h;
(5)洗板6次,加入HRP标记的羊抗兔IgG(1:5000),100μL/孔,37℃,1h;
(6)37℃,1h后,TBST洗6~8次;
(7)加入A,B显色剂,37℃避光孵育1h;
(8)加入终止液,用酶标仪450nm处测吸光值A。
结果如下:
利用间接ELISA检测rMgPa与CypA的特异结合,结果表明:与空白对照组相比,rMgPa与CypA孵育组的吸光值大于1.0,如图3所示,阳性对照rMgPa抗体孵育组的吸光值也大于1.0,而空白对照组的吸光度值小于0.5,具有统计学意义(P<0.05),这些结果说明rMgPa能与CypA特异结合。
6、Western blotting鉴定rMgPa与CypA的特异结合
6.1鉴定rMgPa与CypA的直接结合
(1)SDS-PAGE
取20μL的rMgPa加入5μL(5×蛋白电泳上样缓冲液),混匀后煮沸5min,1000g离心1min,每孔加10μL上样进行SDS-PAGE分离。
(2)转膜与封闭
在恒流条件进行转膜:条件为0.03A,30min,其余操作步骤见3.2。
(3)免疫印迹
①CypA蛋白孵育:加入CypA蛋白(1:1000),在4℃孵育6h或过夜,TBST漂洗5次,每次5min。
②孵育一抗:加入稀释1:1000的CypA抗体,37℃孵育2h,TBST漂洗5次,每次5min。
③孵育二抗:加入稀释的HRP标记的羊抗兔IgG(1:5000),37℃孵育1h,TBST漂洗5次,每次5min。
④显影
6.2鉴定rMgPa与细胞膜蛋白的结合
(1)SDS-PAGE
取20μL的rMgPa加入5μL(5×蛋白电泳上样缓冲液),混匀后煮沸5min,1000g离心1min,每孔加10μL上样进行SDS-PAGE分离。
(2)转膜与封闭
在恒流条件进行转膜:条件为0.03A,30min,其余操作步骤见3.2。
(3)免疫印迹
①人尿道上皮细胞膜蛋白孵育:加入膜蛋白,在4℃孵育4h或过夜,TBST漂洗5次,每次5min。
②孵育一抗:加入稀释1:1000的CypA抗体,37℃孵育2h,TBST漂洗5次,每次5min。
③孵育二抗:加入稀释的HRP标记的羊抗兔IgG(1:5000),37℃孵育1h,TBST漂洗5次,每次5min。
④显影。
6.3约17kDa目标蛋白与CypA抗体的作用
(1)SDS-PAGE
取20μL的细胞膜蛋白加入5μL(5×蛋白电泳上样缓冲液),混匀后煮沸5min,1000g离心1min,每孔加10μL上样进行SDS-PAGE分离。
(2)转膜与封闭
在恒压条件进行转膜:条件为15V,30min,其余操作步骤见3.2。
(4)免疫印迹
①孵育一抗:加入稀释1:1000的CypA抗体,37℃孵育2h,TBST漂洗5次,每次5min。
②孵育二抗:加入稀释的HRP标记的羊抗兔IgG(1:5000),37℃孵育1h,TBST漂洗5次,每次5min。
③显影。
鉴定分析结果
rMgPa能与细胞膜蛋白中的CypA特异结合
将rMgPa进行SDS-PAGE并转膜后,用Western blotting检测其能否与细胞膜蛋白中的CypA结合,结果如图4所示,当用膜蛋白与rMgPa孵育后,加入CypA抗体,再加入二抗显色,在约37kDa处有特异性条带出现;而对照组(CypA抗体直接与rMgPa条带孵育)没有条带出现,说明rMgPa能与细胞膜蛋白中的CypA蛋白特异性结合。
用间接免疫荧光试验检测CyPA蛋白在人尿道上皮细胞中的定位,结果如图5所示,由图5中可以看出,人尿道上皮细胞的细胞膜、细胞质和细胞核区域都有红色荧光,细胞核为蓝色;说明在细胞内和细胞膜上都存在CyPA蛋白。
7、CypA对rMgPa和Mg黏附人尿道上皮细胞的抑制作用
(1)取一瓶生长状态良好,铺满细胞培养瓶底部80%~90%的人尿道上皮细胞,细胞洗涤液清洗3次,胰蛋白酶消化,然后加入5mL培养基,吹打均匀;
(2)向24孔板的孔(带细胞爬片)中加入500μL培养液,接着吸取50μL悬浮的人尿道上皮细胞到每个孔中,置于37℃,5%CO2培养箱中培养;
(3)rMgPa(30μg)与CypA单独预孵育,4℃,2h;
(4)100μL的Mg悬浮液(1×107CCU/mL)与CypA单独预孵育,37℃,30min;
(5)PBS(pH7.4)洗涤细胞3次,接着用4%的多聚甲醛固定细胞,4℃,30min;
(6)弃去4%的多聚甲醛,PBS洗涤3次,加入F-12K培养基封闭,37℃,1h;
(7)弃去封闭液后用PBS洗涤3次,加入CypA抗体到孔中,37℃,孵育2h;
(8)分别加入rMgPa蛋白预孵育混合物和100μL Mg悬浮液预孵育混合物到24孔板的孔中,置于37℃,2h;
(9)加入一抗和二抗的步骤及观察样本与rMgPa和Mg的黏附试验相同。
结果如下:
为了进一步验证CypA是rMgPa受体蛋白,将rMgPa与CypA蛋白预孵育后,再检测rMgPa对人尿道上皮细胞的黏附情况。结果如图6所示:与对照组相比,人尿道上皮细胞膜表面的红色荧光明显减少,说明部分rMgPa预孵育时与CypA发生了特异结合,导致rMgPa对人尿道上皮细胞的黏附减少。这些结果说明CypA蛋白能部分抑制rMgPa对人尿道上皮细胞的黏附。
然后,将Mg与CypA蛋白预孵育后,用间接免疫荧光检测CypA蛋白能否抑制Mg对人尿道上皮细胞的黏附,在荧光显微镜下观察到:与对照组相比,试验组中人尿道上皮细胞膜表面和胞浆内的红色荧光明显减少,说明黏附和侵入人尿道上皮细胞的Mg减少,如图7所示。这些结果表明CypA蛋白能部分抑制Mg对人尿道上皮细胞的黏附与侵入。
<110>:南华大学
<120>:一种与生殖支原体MgPa特异结合的受体蛋白及其分离方法和用途
<160>:1
<210>:1
<211>:165
<212>:PRT
<213>:人工序列
<400>:1
Met Val Asn Pro Thr Val Phe Phe Asp Ile Ala Val Asp Gly Glu Pro
1 5 10 15
Leu Gly Arg Val Ser Phe Glu Leu Phe Ala Asp Lys Val Pro Lys Thr
20 25 30
Ala Glu Asn Phe Arg Ala Leu Ser Thr Gly Glu Lys Gly Phe Gly Tyr
35 40 45
Lys Gly Ser Cys Phe His Arg Ile Ile Pro Gly Phe Met Cys Gln Gly
50 55 60
Gly Asp Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly
65 70 75 80
Glu Lys Phe Glu Asp Glu Asn Phe Ile Leu Lys His Thr Gly Pro Gly
85 90 95
Ile Leu Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe
100 105 110
Phe Ile Cys Thr Ala Lys Thr Glu Trp Leu Asp Gly Lys His Val Val
115 120 125
Phe Gly Lys Val Lys Glu Gly Met Asn Ile Val Glu Ala Met Glu Arg
130 135 140
Phe Gly Ser Arg Asn Gly Lys Thr Ser Lys Lys Ile Thr Ile Ala Asp
145 150 155 160
Cys Gly Gln Leu Glu
165
Claims (3)
1.一种与生殖支原体MgPa特异结合的受体蛋白的分离制备方法,所述受体蛋白分离自人尿道上皮细胞的细胞膜蛋白,命名为亲环蛋白Cyclophinlin A,简称为CypA,其氨基酸序列如SEQ ID NO.1所示,由165个氨基酸组成,其特征在于所述分离制备方法包括以下步骤:
(1)、对生殖支原体的膜蛋白MgPa进行表达、纯化,制得重组蛋白rMgPa;
(2)、采用超声破碎法提取人尿道上皮细胞的膜蛋白;
(3)、采用改进的病毒铺覆蛋白印迹技术筛选步骤(2)中制得的膜蛋白中与重组蛋白rMgPa特异结合的膜蛋白,筛选得到一条约17KDa的蛋白;具体步骤为:
a、SDS-PAGE
将步骤(2)中提取的人尿道上皮细胞膜蛋白,测定浓度后加入蛋白电泳上样缓冲液(5×),混匀后煮沸5min,1000g离心1min,取10μL上样进行SDS-PAGE分离,分离胶为12%,浓缩胶为5%,电泳条件为:浓缩胶80V,分离胶120V;同时设置对照组;
b、转膜与封闭
(i)PVDF膜的处理及凝胶的平衡:裁剪一张比凝胶片段略大的PVDF膜,将其用甲醇浸泡10-15s后再用去离子水漂洗10s,随后将PVDF膜用转膜缓冲液平衡10min,电泳结束后,将目的片段放置于转膜缓冲液中平衡10min;
(ii)在半干转膜仪中,将已经平衡好的滤纸、PVDF膜、凝胶、滤纸从下到上依次叠加,每层均用玻璃棒将气泡排除,在恒压条件进行转膜:条件为15V,30min;
(iii)PVDF膜浸入封闭液中,4℃过夜,或者室温摇床2h,TBST漂洗5次,每次5min;
c、免疫印迹
(i)rMgPa蛋白孵育:加入rMgPa蛋白,在4℃孵育6h,TBST漂洗5次,每次5min;
(ii)孵育一抗:加入稀释1:1000的兔抗rMgPa的多克隆抗体,37℃孵育2h,TBST漂洗5次,每次5min;
(iii)孵育二抗:加入稀释1:5000的HRP标记的羊抗兔IgG,37℃孵育1h,TBST漂洗5次,每次5min;
(iv)显影;
(4)、将步骤(3)中得到的约17KDa蛋白进行质谱分析鉴定,即得到受体蛋白CypA。
2.根据权利要求1所述的受体蛋白的分离制备方法,其特征在于:步骤(2)中,取培养形态良好的人尿道上皮细胞,胰蛋白酶消化5min,加入培养基终止消化,离心6min后收集细胞沉淀;然后加入PBS轻轻吹打细胞沉淀,洗三次;再加入细胞裂解液冰浴20min,加入蛋白酶抑制剂PMSF,超声破碎,10W,5s/次,间歇15s,共计30次,然后2500rpm/min离心10min,取上清离心30min,沉淀用PBS重悬,-80℃保存,即获得人尿道上皮细胞的膜蛋白提取物。
3.权利要求1所述的受体蛋白的用途,其特征在于:利用权利要求1所述的受体蛋白CypA制备治疗或预防生殖支原体感染性疾病的靶向药物中的用途。
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