CN110684097B - 卡氏住白细胞虫重组r7蛋白及其制备方法和应用 - Google Patents
卡氏住白细胞虫重组r7蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，公开了一种卡氏住白细胞虫重组R7蛋白及其制备方法；所述卡氏住白细胞虫重组R7蛋白具有的氨基酸序列为MBP‑R7。本发明人工合成了卡氏住白细胞虫重组R7基因及制备卡氏住白细胞虫重组R7蛋白，表达产物经纯化、体外活性检测后发现卡氏住白细胞虫重组R7蛋白具有检测卡氏住白细胞虫抗体的作用，在检测卡氏住白细胞虫病中，用重组R7蛋白代替第二代裂殖体作为包被抗原，检测效果更好，更快速便捷；由于重组R7蛋白更易制备，因此降低了抗原获取的难度和成本。
Description
技术领域
本发明属于生物技术领域，具体涉及一种卡氏住白细胞虫重组R7蛋白及其制备方法。
背景技术
鸡卡氏住白细胞虫病又称“白冠病”，是由卡氏住白细胞虫寄生于鸡的红细胞、白细胞和内脏组织细胞内所引起的一种血孢子虫病。鸡卡氏住白细胞虫病多发生于3～6周龄雏鸡，发病严重，死亡率高；青年鸡感染率比雏鸡高，但是死亡率不高；而成年鸡的感染率最高，但死亡率很低，症状较轻。主要临床症状表现为鸡冠苍白，消瘦，拉水样白色或绿色稀粪，鸡发育受阻，成年鸡产蛋下降甚至停止，会对养鸡行业造成较大经济损失。
根据临床症状、流行病学、病理解剖等诊断方法可对卡氏住白细胞虫病作出初步判断，而确诊一般采用血涂片检查方法，检查外周血液中有无裂殖子或配子体。出现在外周血液中裂殖子体积极小，普通光学显微镜下难以鉴别卡氏住白细胞虫裂殖子与其他血孢子虫的裂殖子，容易误诊。并且卡氏住白细胞虫成熟配子体在外周血液中出现的时间晚且持续的时间短，血涂片检查容易漏检。血涂片检查方法虽然简单易行，但是在检测效率、检出率、虫种鉴别等方面具有很大的局限性，越来越不适应当前防治工作的需要，尤其不能适应大规模的流行病学调查。
而采用抗原—抗体反应来检测鸡卡氏住白细胞虫病是目前探索的方向，抗原—抗体反应的特点主要有四性：即特异性、比例性、可逆性，阶段性。因此，提供一种卡氏住白细胞虫重组R7蛋白来检测重组卡氏住白细胞虫抗体是很有必要的。
发明内容
本发明的目的在于提供一种卡氏住白细胞虫重组R7蛋白及其制备方法，该卡氏住白细胞虫重组R7蛋白代替卡氏住白细胞虫第二代裂殖体作为包被抗原易制备，成本低。
为了实现上述目的，本发明采用的技术方案是：
发明人用原核表达的重组R7蛋白包被酶标板，建立检测抗卡氏住白细胞虫抗体的ELISA方法。R7蛋白是卡氏住白细胞虫第二代裂殖体的一种外膜抗原。基于卡氏住白细胞虫重组R7蛋白的ELISA检测方法比琼脂扩散试验、血涂片检查法等更加灵敏和方便快捷，可以检测出鸡感染卡氏住白细胞虫后产生的抗体和接种重组R7油佐剂疫苗后产生的抗体。为我国广大的养殖场卡氏住白细胞虫病的防控提供帮助。
一种卡氏住白细胞虫重组R7蛋白，所述卡氏住白细胞虫重组R7蛋白具有的氨基酸序列为MBP-R7。
所述MBP-R7：
MGHHHHHHGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGASGLVTFISPNNVQAEIINTHGVRCNQNEEVTHQTHQTHQTHQTHQTHQTHQIHQIHQIHGYMTNQKHEEHGKIINQVKENVKNTVNENVKNNVDENTTSEHEITIPNENDIKTNDENETTHYEREIIYIVDDLPEVNVEESDETEHITYEIDNDIQEEHEKVTHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEKEKEEHEEV。
一种卡氏住白细胞虫重组R7蛋白的制备方法，包括以下步骤：
(1)将卡氏住白细胞虫R7基因进行PCR扩增，得到卡氏住白细胞虫重组R7基因；
(2)用限制性内切酶对psYNO-1质粒进行酶切，将重组卡氏住白细胞虫R7基因与酶切后psYNO-1质粒用连接酶连接，并转化到E.coli TOP10感受态细胞中培养，再进行PCR鉴定阳性克隆，得到PCR鉴定阳性菌液，提取PCR鉴定阳性菌液质粒，经双酶切鉴定得到阳性重组质粒psYNO-R7；
(3)将阳性重组质粒psYNO-R7转化到E.coli BL21表达菌，加入异丙基硫代半乳糖苷进行诱导表达，得到psYNO-R7重组蛋白；
(4)将诱导表达后的psYNO-R7重组蛋白纯化，得到卡氏住白细胞虫重组R7蛋白。
优选地，所述卡氏住白细胞虫重组R7基因的制备方法包括以下步骤：
(1)找到卡氏住白细胞虫R7基因序列，进行密码子优化后再人工合成R7基因序列；
(2)设计一对特异性连接引物的核苷酸序列为：CTGTACTTCCAGGGAGCAAGTGGTCTGGTTACC和GTGGTGGTGCTCGAGTTATCACACTTCTTCATGT，再设计一对鉴定引物的核苷酸序列为GACTAATTCGAGCTCGAACAACAACA和CATGTTCTTCTTTTTCTTTCTCTTCGTG；
(3)以合成的R7基因为模板，PCR扩增R7基因片段，得到卡氏住白细胞虫重组R7基因。
优选地，所述特异性连接引物的5’端含有与表达质粒载体psYNO-1末端同源的15个碱基和3’端含有R7基因特异引物序列。
优选地，步骤(2)中，所述限制性内切酶为BamH Ⅰ和Xho Ⅰ。
优选地，步骤(2)中，所述双酶切鉴定的步骤为：将psYNO-R7、Sac Ⅰ、Xho Ⅰ、10×MBuffer和ddH2O加入到灭菌的1.5mL离心管中混匀，在37℃下恒温水浴2h，得到酶切产物，将酶切产物进行琼脂糖凝胶电泳，将鉴定为阳性的质粒做好标记，并测序，得到阳性重组质粒psYNO-R7，保存于-20℃中。
优选地，步骤(2)中，所述PCR鉴定阳性克隆的步骤为：将菌液进行PCR反应，PCR反应体系的组分为Ex Taq DNA聚合酶、引物、菌液和ddH2O。
更优选地，所述引物为：R7S2-F和R7S2-R；所述Ex Taq DNA聚合酶、引物、菌液和ddH2O的体积分别为：25μL、2μL、4μL和19μL。
更优选地，Ex Taq DNA聚合酶为TaKaRa的产品。
菌液PCR扩增程序为：
反应条件为94℃预变性1min，(94℃变性20s，60℃退火20s，72℃延伸30s)×34循环，72℃延伸5min，4℃结束。
将得到的PCR产物用1％琼脂糖凝胶进行电泳，在135V下电泳25min后，观察并拍照记录结果；PCR鉴定阳性的菌液，取500μL菌液与500μL 50％甘油混匀，做好标记，保存于-20℃。
优选地，步骤(2)中，所述双酶切鉴定的步骤为：将psYNO-R7、Sac Ⅰ、Xho Ⅰ、10×MBuffer和ddH2O加入到灭菌的1.5mL离心管中混匀，在37℃下恒温水浴2h，得到酶切产物，将酶切产物进行琼脂糖凝胶电泳，将鉴定为阳性的质粒做好标记，并测序，得到阳性重组质粒psYNO-R7，保存于-20℃中。
更优选地，所述psYNO-R7、Sac Ⅰ、Xho Ⅰ、10×M Buffer和ddH2O的体积分别为25μL、1μL、1μL、5μL和18μL。
一种卡氏住白细胞虫重组R7蛋白在检测卡氏住白细胞虫病中的应用。
一种采用卡氏住白细胞虫重组R7蛋白的间接ELISA检测方法，包括以下步骤：
(1)包被抗原：将卡氏住白细胞虫重组R7蛋白用包被缓冲液稀释到0.065-1.3μg/mL，在37℃下包被1-2h或4℃包被过夜；
(2)洗涤酶标板：甩干孔内包被液，加入磷酸盐吐温缓冲液洗涤，再甩干残留液体；
(3)封闭：加入封闭液，在37℃下封闭1-2h或4℃封闭过夜；
(4)加入血清：用5％脱脂奶稀释血清，混合静置5-10min，将稀释好的血清加入酶标板，在37℃下孵育0.5-2h；
(5)加入酶标二抗：加入稀释好的HRP标记山羊抗鸡IgG，在37℃下孵育0.5-2h；
(6)显色：加入TMB显色液，在37℃下显色10-30min；
(7)终止：加入终止液终止显色；
(8)读数：用酶标仪检测450nm波长OD值，并读取。
优选地，步骤(1)中，所述包被缓冲液选自碳酸盐缓冲液，所述碳酸盐缓冲液选自pH为9.6的0.2mol/L NaHCO3。
优选的，步骤(2)中，所述磷酸盐吐温缓冲液(PBST)是含0.05％吐温20和pH为7.2的磷酸盐缓冲液。
优选地，步骤(3)之后和步骤(4)之前、步骤(4)之后和步骤(5)之前、步骤(5)之后和步骤(6)之前，还包括甩干孔内液体，加入磷酸盐吐温缓冲液洗涤，再甩干残留液体。
优选地，步骤(3)中，所述封闭液为5％脱脂奶粉；
优选地，步骤(4)中，所述血清与5％脱脂奶的稀释比为1:250-1:2000。更优选地，所述血清与5％脱脂奶的稀释比为1:500。
优选地，步骤(5)中，所述HRP标记山羊抗鸡IgG的稀释比为1:2000-1:12000。更优选地，所述HRP标记山羊抗鸡IgG的稀释比为1:4000。
优选地，步骤(6)中，所述显色液是KPL公司的SureBlue TMB MicrowellSubstrate，底物为3,3',5,5'-四甲基联苯胺，所述显色液的反应时间为10-30min。
优选地，步骤(7)中，所述终止液为2mol/L H2SO4。
本发明的有益技术效果是：
本发明人工合成了卡氏住白细胞虫重组R7基因及制备卡氏住白细胞虫重组R7蛋白，表达产物经纯化、体外活性检测后发现卡氏住白细胞虫重组R7蛋白具有检测卡氏住白细胞虫病的作用，在检测卡氏住白细胞虫病中，用重组R7蛋白代替第二代裂殖体作为包被抗原，检测效果更好，更快速便捷；由于重组R7蛋白更易制备，因此降低了抗原获取的难度和成本。
附图说明
图1：L.caulleryi R7基因PCR扩增结果图；
图2：菌液PCR鉴定结果图；
图3：重组质粒psYNO-R7的双酶切结果图；
图4：诱导时间分析图；
图5：IPTG诱导浓度分析图；
图6：表达产物存在形式的检测结果；
图7：R7蛋白纯化结果；
图8：R7蛋白Western blot检测结果；
图9：重组R7蛋白反应原性分析；
图10：蛋白浓度标准曲线。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明了，下面通过实施例，对本发明进行进一步详细说明。但是应该理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限制本发明的范围。
实施例1
1.基因合成和基因克隆
(1)找到卡氏住白细胞虫R7基因序列，在不改变氨基酸序列的情况下优化密码子，再人工合成R7基因序列；
(2)设计一对连接引物R7S1-F/R7S1-R，根据psYNO-1质粒和R7基因的核苷酸序列，设计一对鉴定引物R7S2-F/R7S2-R，引物序列见表1，连接引物的5’端含有与表达质粒载体psYNO-1末端同源的15个碱基，In-Fusion连接引物的3’端含有R7基因特异引物序列；
(3)以合成的R7基因序列为模板，PCR扩增R7基因片段。
表1 引物设计
50μL的PCR反应体系如下表2：
表2 PCR反应体系
PCR扩增程序为：
反应条件为98℃预变性1min，(98℃变性20s，65℃退火20s，72℃延伸30s)×34循环，72℃延伸5min，4℃结束。
取10μL PCR产物与2μL 6×Loading Buffer混合后，用1％琼脂糖凝胶进行电泳，135V电泳25min后，用凝胶成像系统观察并拍照记录结果；剩余PCR产物电泳后切胶回收目的基因；利用胶回收试剂盒对PCR产物进行回收与纯化，回收产物保存于-20℃。
实施例2
1.卡氏住白细胞虫重组R7蛋白的制备方法与表达
(1)PCR扩增重组基因：找到卡氏住白细胞虫R7基因序列，设计一对特异扩增引物对卡氏住白细胞虫R7基因进行PCR扩增，得到卡氏住白细胞虫重组R7基因，所述一对特异性扩增引物为的核苷酸序列为：CTGTACTTCCAGGGAGCAAGTGGTCTGGTTACC和GTGGTGGTGCTCGAGTTATCACACTTCTTCATGT，另设计一对鉴定引物的核苷酸序列为GACTAATTCGAGCTCGAACAACAACA和CATGTTCTTCTTTTTCTTTCTCTTCGTG；
(2)构建重组表达载体psYNO-R7：用限制性内切酶BamH Ⅰ和Xho Ⅰ对psYNO-1质粒进行酶切，将重组R7基因与酶切后psYNO-1质粒用连接酶连接，并转化到E.coli TOP10感受态细胞中，再进行PCR鉴定阳性克隆，得到PCR鉴定阳性菌液，提取PCR鉴定阳性菌液质粒，经双酶切鉴定得到测序结果正确的阳性质粒psYNO-R7；
(3)诱导表达重组蛋白：将测序正确的阳性重组质粒psYNO-R7转化E.coli BL21表达菌，加入IPTG进行诱导表达，得到卡氏住白细胞虫重组R7蛋白。
得到的卡氏住白细胞虫重组R7蛋白所具有的氨基酸序列为MBP-R7。
MBP-R7：
MGHHHHHHGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGASGLVTFISPNNVQAEIINTHGVRCNQNEEVTHQTHQTHQTHQTHQTHQTHQIHQIHQIHGYMTNQKHEEHGKIINQVKENVKNTVNENVKNNVDENTTSEHEITIPNENDIKTNDENETTHYEREIIYIVDDLPEVNVEESDETEHITYEIDNDIQEEHEKVTHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEKEKEEHEEV。
2.构建重组表达载体psYNO-R7具体步骤如下：
a.用限制性内切酶BamH Ⅰ和Xho Ⅰ对psYNO-1质粒进行酶切，双酶切体系如下表3。将表3中各组分加入到200μL灭菌PCR管中，轻弹混匀，瞬时离心后，37℃恒温水浴2h，酶切产物在琼脂糖凝胶电泳后进行切胶回收与纯化，回收产物保存于-20℃。
表3 双酶切反应体系
b.将R7基因和限制性内切酶切后的psYNO-1质粒在In-Fusion连接酶的作用下，于50℃恒温水浴20min，得到连接产物。In-Fusion连接体系如下表4：
表4 In-Fusion连接体系
c.重组表达载体的转化：将连接产物加到E.coli TOP10感受态细胞中，混匀，置于冰上35min，在42℃水浴中热应激90s，再立即冰浴2min，冰浴后加到600μL LB液体培养基，在37℃，180rpm摇床中培养45min，得到菌液，取150μL菌液涂布于LK固体培养基(含50μg/mLKan)，将平板正置于37℃生化培养箱中20min，再倒置平板继续培养12～16h，直至长出肉眼可见的单菌落，挑取多个单菌落，分别接种于5mL LK液体培养基(含50μg/mL Kan)中，在37℃下，220rpm培养过夜，甘油保菌。
d.重组表达质粒的PCR扩增：取上述菌液进行PCR扩增，菌液PCR扩增反应体系如下表5。
表5 菌液PCR扩增反应体系
反应条件为94℃预变性3min，(94℃变性30s，60℃退火40s，72℃延伸1min)×34循环，72℃延伸10min，4℃结束，得到PCR产物。
PCR产物用1％琼脂糖凝胶进行电泳，在135V下电泳25min后，用凝胶成像系统观察并拍照记录结果。菌液PCR鉴定阳性的菌液，取500μL菌液与500μL 50％甘油混匀，做好标记，保存于-20℃。
e.抽提PCR鉴定阳性菌液的质粒的步骤如下：
(1)用无菌接种环蘸取PCR鉴定阳性的菌液在LK固体培养基(含50μg/mL Kan)上划线，将平板倒置于37℃生化培养箱中培养12～16h，直至长出肉眼可见的单菌落，挑取单菌落，接种于5mL LK液体培养基(含50μg/mL Kan)中，在37℃和240rpm下培养12～16h；
(2)将菌液在室温下12000rpm离心1min，弃去上清液；
(3)加入250μL Solution I(已加RNase A)，涡旋振荡，使菌体沉淀完全重悬，将悬浮液转移至新的1.5mL灭菌离心管中；
(4)加入250μL Solution II，盖好盖子，轻轻翻转数次，孵育2～3min；
(5)加入350μL Solution III，盖紧盖子，立即翻转几次，充分混匀，直到出现白色絮状沉淀，在室温下13000rpm离心10min；
(6)将DNA Mini Column装在2mL收集管中，将步骤(5)中上清液转移到柱子中，13000rpm离心1min，弃滤液并将柱子重新装在收集管上；
(7)在柱子中加入500μL HBC Buffer(已加异丙醇稀释)，13000rpm离心1min，弃滤液并将柱子装在收集管上；
(8)加入700μL DNA Wash Buffer(已加无水乙醇稀释)，13000rpm离心1min，弃滤液并将柱子装在收集管上，重复一次，
(9)弃滤液并将柱子装回收集管上，13000rpm离心2min，开盖晾2min，
(10)将柱子转移至新的1.5mL灭菌离心管中，加50μL ddH2O到柱子的膜中心，静置2min，13000rpm离心1min，抽提的质粒保存于-20℃备用。
f.重组表达质粒的双酶切鉴定
用抽提的质粒进行双酶切鉴定，双酶切体系如下表6。将表6中各组分加入到灭菌的1.5mL离心管中，轻轻混匀，37℃恒温水浴2h，取酶切产物进行琼脂糖凝胶电泳，将鉴定为阳性的质粒做好标记，并送到华大基因生物技术有限公司测序。测序结果正确的阳性质粒命名为psYNO-R7，保存于-20℃中备用。
表6 双酶切反应体系
3.诱导表达重组蛋白的具体的步骤如下：
a.psYNO-R7重组质粒的转化：抽提阳性重组质粒psYNO-R7，将重组质粒psYNO-R7转化到E.coli BL21表达菌；
b.psYNO-R7重组蛋白的诱导表达：挑取单菌落，分别接种于5mL LK液体培养基中，在37℃和210rpm的条件下培养过夜，甘油保菌，将过夜培养的菌液以1:100的比例接种于200mL LK液体培养基中，在37℃和210rpm的条件下震摇培养2h，至菌液OD600nm＝0.6～0.8，取1mL菌液，在12500rpm的条件下离心5min，弃上清液，沉淀用100μL PBS重悬，加入25μL 5×SDS上样缓冲液，混匀后沸水浴8min，作为未诱导对照组，从剩余菌液中取200mL加入到500mL的灭菌锥形瓶中，加入终浓度为0.1mM的IPTG，在37℃和210rpm的条件下诱导表达；
IPTG诱导浓度的分析：
取甘油保存的菌液在LK固体培养基上划线，在37℃下培养过夜，挑取单菌落，分别接种于5mL LK液体培养基中，在37℃和210rpm条件下培养过夜，将过夜培养的菌液以1:100的比例接种于200mL LK液体培养基中扩大培养，在37℃下，210rpm震摇培养约2h，至菌液OD600nm＝0.6～0.8，取1mL菌液，12500rpm离心5min，弃上清，沉淀用100μL PBS重悬，加入25μL 5×SDS上样缓冲液，混匀后沸水浴8min，作为未诱导对照组。
取10个灭菌50mL离心管，编号1～10号，按照表9要求在各离心管中加入扩大培养的菌液和IPTG，在37℃下、210rpm的恒温摇床中诱导表达，至SDS-PAGE鉴定的蛋白表达量最高的时间停止诱导，每管取样1mL，12500rpm离心5min，弃去上清液，加入100μL PBS重悬菌体，再加入25μL 5×SDS上样缓冲液，混匀后沸水浴10min，冰浴5min备用，同时设置诱导的空载体菌作为对照，样品处理方法同上。用12％聚丙烯酰胺凝胶进行SDS-PAGE电泳分析。
SDS-PAGE凝胶电泳分析的具体步骤：
(1)制胶：采用常规SDS-PAGE凝胶电泳检测表达产物，分离胶浓度为10％，浓缩胶浓度为5％，按照表7和表8的配方配制SDS-PAGE凝胶，洗干净玻璃板和电泳槽，组装制胶板，用去离子水检漏13min，倒掉去离子水，用滤纸吸干剩余的水渍，将配合好的分离胶缓慢注入玻璃板之间，缓缓覆盖约2mL无水乙醇，室温放置35min，待分离胶凝固，弃掉无水乙醇，用滤纸吸干残留的乙醇，加满浓缩胶，插入梳子，室温放置20min，待胶体凝固。
表7 SDS-PAGE分离胶(10％)
表8 SDS-PAGE浓缩胶(5％)
(2)电泳：将制胶板安装到电泳槽，轻轻拔出梳子，倒入SDS-PAGE电泳缓冲液。在胶孔中加入10～20μL处理好的样品，另加入7μL Protein Marker作为对照，接通电泳槽电源，初始电压70V，约25min后样品到达分离胶，再将电压调至160V，电泳45min左右。
(3)染色：关掉电源，拆下凝胶，将凝胶边缘多余部分切掉，放在考马斯亮蓝染色液中染色50min。
(4)脱色：回收染色液，用清水将胶上多余的染色液洗掉，放入脱色液中脱色。及时更换脱色液，至背景无色为止，在凝胶成像仪中观察并拍照保存。
表9 IPTG诱导浓度的分析
表达产物存在形式的检测：
蘸取甘油保存的菌液在LK固体培养基上划线，37℃培养过夜，挑取单菌落，分别接种于5mL LK液体培养基中，在37℃和210rpm条件下培养过夜，将过夜培养的菌液以1:100的比例接种于500mL LK液体培养基中扩大培养，在37℃和210rpm条件下震摇培养2h，直至菌液OD600nm＝0.6～0.8。取1mL菌液，在12500rpm条件下离心5min，弃去上清液，沉淀物用100μLPBS重悬，加入25μL 5×SDS上样缓冲液，混匀后沸水浴8min，作为未诱导对照组。
剩余菌液中加入IPTG至终浓度为0.1mM，置于37℃下，210rpm恒温摇床中诱导表达，至SDS-PAGE鉴定的蛋白表达量最高的时间停止诱导。将诱导后的菌液置于冰上7min，再于高速冷冻离心机中，在4℃下，12500rpm离心10min，弃去上清液，用25mL PBS重旋菌体。使用功率为200w的超声波细胞粉碎机超声裂解菌体，每间隔6s超声5s，超声时间20min，再在4℃下，12500rpm离心10min，分别取上清液和沉淀，加入5×SDS上样缓冲液，煮沸8min后冰浴5min，12000rpm离心10min，进行SDS-PAGE分析，检测重组蛋白是存在于上清液中还是包涵体中。
实施例3
1.重组蛋白的纯化
卡氏住白细胞虫重组R7蛋白的纯化：将过夜培养的菌液以1:100的比例接种于200mL LK液体培养基中，在37℃下，210rpm震摇培养2h，至菌液OD600nm＝0.6-0.8，在37℃，210rpm和IPTG诱导下，进行卡氏住白细胞虫重组R7蛋白的大量表达，收集诱导表达菌；将诱导后的菌液置于冰上，再于离心机中离心，弃上清液，收集菌体，用MBP(麦芽糖结合蛋白)结合液重悬菌体，使用超声波细胞粉碎机超声裂解菌体，收集上清液，再加入MBP纯化树脂(常州天地人和生物科技有限公司的产品DextrinBeads 6FF)纯化，过滤得到卡氏住白细胞虫重组R7蛋白。
(1)样品准备：将诱导后的菌液置于冰上7min，再于高速冷冻离心机中，在4℃下，10000rpm离心10min，弃去上清液收集菌体，用MBP结合液重悬菌体，使用功率为200w的超声波细胞粉碎机超声裂解菌体，每间隔6s超声5s，超声时间20min，超声结束后，菌体悬液变澄清，悬液在4℃下，10000rpm离心10min；收集上清液，用0.45μm滤器过滤后备用。
(2)装柱：用去离子水冲洗层析柱底筛板与接头，确保柱底筛板上无气泡，关闭柱底出口，并在柱底部留出1～2cm的去离子水，涡旋振荡MBP纯化树脂，使其悬浮起来，将悬浮液小心地倒入层析柱中，静置待填料与酒精保存液分离，再加一块筛板轻轻压实填料，打开柱底出口使液体流出。
(3)平衡：用5倍柱体积的结合液平衡层析柱，使填料与目的蛋白处于相同的缓冲体系下，从而保护蛋白。
(4)上样：将准备好的上清液样品加到平衡好的MBP纯化柱中，堵住柱底出口，样品和填料孵育30min，打开出口，控制流速，使液体缓缓流出，收集流出液。
(5)洗涤：用10倍柱体积的MBP洗杂液清洗柱子，收集洗杂液。
(6)洗脱：将5倍柱体积的MBP洗脱液加到柱子中，孵育20min；打开出口，控制流速，大约1～2秒流出1滴洗脱液，收集洗脱液，即纯化的目的蛋白。
(7)SDS-PAGE：分别取适量上样前样品、流出液、洗杂液和蛋白洗脱液，加5×SDSLoading buffer混匀，沸水浴10min后冰浴5min，进行SDS-PAGE电泳，检测蛋白纯化结果。
2.Western blot检测重组蛋白
(1)电泳：分别取40μL纯化好的蛋白样品和psYNO-1空载体诱导菌处理后加10μL 5×SDS上样缓冲液，煮沸10min，进行SDS-PAGE电泳，结束后将胶切成合适大小。
(2)转膜：将PVDF膜浸泡在甲醇中10s活化，放入预冷的转印缓冲液中与海绵、滤纸、蛋白胶一同浸泡10min，转膜夹从下至上按照负极板、海绵、滤纸、凝胶、PVDF膜、滤纸、海绵、正极板的顺序依次放好，夹好转膜夹，放入转膜槽，接通电源，电压80V，转膜90min。
(3)封闭：转膜结束后，将PVDF膜蛋白面朝下放入盛有封闭液的平皿中，室温摇床孵育2h或4℃过夜，用含吐温-20的磷酸盐缓冲液洗膜3次，每次5～10min。
(4)一抗结合：用封闭液将鼠抗MBP抗体稀释5000倍，将封闭后的PVDF膜蛋白面朝下浸入一抗中，室温摇床孵育1h。含吐温-20的磷酸盐缓冲液洗膜3次，每次5～10min。
(5)二抗结合：用封闭液将山羊抗鼠IgG-HRP稀释10000倍，PVDF膜蛋白面朝下浸入二抗中，室温摇床孵育1h，用含吐温-20的磷酸盐缓冲液洗膜3次，每次5～10min。
(6)显色：将ECL的两种发光液按照1:1的比例混合，用移液枪将混合发光液淋在膜的蛋白面，室温避光孵育3min，将膜置于化学发光图像分析系统(Tanon Fine-do X6)发光显色，拍照保存。
3.阴阳性血清制备
购买12只7日龄的无特定病原鸡，在隔离器中养至10日龄备用，免疫前对12只鸡进行翅下采血，分离出血清保存在-20℃，作为阴性血清；免疫组有8只无特定病原鸡，每只S无特定病原鸡免疫35μg重组R7蛋白；剩余4只无特定病原鸡作为未免疫的对照组，做好标记。免疫1周后，给免疫组的鸡加强免疫一次，每只鸡免疫35μg重组R7蛋白。免疫后第3周，对免疫组的鸡进行翅下采血，分离血清保存在-20℃，作为阳性血清。
用Western blot分析重组R7蛋白的反应原性，其中一抗为上述制备的免疫血清，以1:500的比例稀释；二抗为1:5000稀释的山羊抗鸡IgG-HRP。
4.BCA测定蛋白浓度
(1)使用1×PBS将BSA Standard Solution稀释为500μg/mL；
(2)根据样品数，将BCA Solution A和BCA Solution B按50:1的体积比混合，充分混匀后，制得BCA工作液，工作液室温24h内稳定；
(3)按照表10，在96孔板的样品孔中对稀释后的标准液进行配制；
表10 标准液配方
(4)样品按一定比例稀释取20μL加入96孔板的样品孔中；
(5)向样品孔中加入200μL的BCA工作液，37℃放置30～90min；
(6)将96孔板置于562nm波长下检测，如无562nm波长，可以选择在540～595nm波长下测量；
(7)绘制标准曲线，计算待测蛋白样品浓度。
实施例1-3的测试结果如下：
1.结果与分析
R7基因的扩增：以合成的R7基因片段为模板，PCR扩增获得目的基因R7，片段大小与预期一致，约700bp，结果如图1(M：DL 2000DNA Maker；1～3：R7基因PCR扩增产物；4：阴性对照)所示。
2.原核表达载体的构建及鉴定
菌液PCR鉴定：PCR扩增的R7基因和酶切的psYNO-1质粒分别胶回收之后，以适当比例混匀并在In-Fusion连接酶的作用下连接，连接产物转化Top 10感受态细胞，随机挑取单菌落，进行PCR鉴定。结果如图2(M：DL 2000 DNA Maker；1～3：R7基因PCR扩增产物；4：阴性对照)所示，在750bp处有特异性条带，大小与预期相符。
3.重组质粒psYNO-R7的双酶切鉴定
将PCR鉴定阳性的菌液抽提重组质粒，用限制性内切酶Sac Ⅰ和Xho Ⅰ进行双酶切，获得两条带，其中一条为psYNO-1载体，另一条为R7基因，片段大小与预期相符，如图3(M：DL10000 DNA Maker；1～2：psYNO-R7质粒双酶切产物)所示。
4.R7蛋白的表达结果
(1)诱导时间的分析
将构建成功的重组质粒psYNO-R7转化到E.coli BL21，用IPTG在37℃诱导不同的时间。经SDS-PAGE分析，结果如图4(M：170kDa预染蛋白Maker；1：psYNO-1空载体未诱导菌；2：psYNO-1空载体诱导菌；3：psYNO-R7未诱导菌；4～9：psYNO-R7诱导1.5h，3h，4.5h，6h，7.5h，9h后菌液)所示，表达的重组R7蛋白在90kDa处出现明显条带，最佳诱导时间为7.5h。
(2)IPTG诱导浓度分析
分别用0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、0.9mM、1.0mM的IPTG诱导蛋白表达7.5h，经SDS-PAGE分析，可知用0.1mM IPTG诱导表达7.5h的表达量最高，结果可见图5(M：170kDa预染蛋白Maker；1：psYNO-R7未诱导菌；2～11：psYNO-R7分别用0.1mM～1.0mM的IPTG诱导后菌液)。
(3)表达产物存在形式的检测结果
诱导后的菌体超声裂解后，分别取上清液和沉淀进行SDS-PAGE分析，结果显示上清中能看到明显的重组蛋白条带，说明重组蛋白大部分为可溶性表达于上清液中，故选择从上清液中纯化目的蛋白，结果可见图6(M：170kDa预染蛋白Maker；1：未诱导的psYNO-1空载体菌；2：psYNO-1空载体诱导菌；3：未诱导的psYNO-R7菌液；4：psYNO-R7诱导后未裂解菌液；5：37℃诱导7.5h后上清；6：37℃诱导7.5h后沉淀)。
5.R7蛋白纯化结果
重组R7蛋白使用MBP纯化树脂亲和层析，通过含有10mM麦芽糖的洗脱液洗脱下来，纯化结果如图7(M：170kDa预染蛋白Maker；1：psYNO-1诱导后菌液；2：psYNO-R7诱导后上清；3：上样滤过液；4：洗杂液；5：洗脱液)所示，纯化后得到了纯度较高的R7蛋白，几乎没有杂带。
6.R7蛋白Western blot检测结果
用鼠抗MBP单抗对表达的蛋白进行Western Blot检测，鼠抗MBP单抗能特异性识别MBP标签，结果如图8(1：psYNO-1空载体对照组；2：R7蛋白)所示，纯化的目的蛋白可与鼠抗MBP单抗发生反应，约在90kDa大小的位置出现特异性反应条带，表明所获得的蛋白为R7蛋白。
7.阳性血清制备结果
用免疫重组R7蛋白3周后的鸡免疫血清作为一抗，对纯化的重组R7蛋白进行Western Blot检测，结果如图9(1：psYNO-1空载体对照组；2：R7蛋白)所示，能检测到预期大小的目的条带，可证明制备的免疫血清为阳性血清，也表明该R7蛋白具有较好的反应原性。
8.蛋白浓度的测定
按照标准样品测得结果，绘制标准曲线(OD592nm作为横坐标，蛋白浓度作为纵坐标)，见图10，标准蛋白曲线公式为：y＝0.763x－0.099，R2＝0.998。对纯化所得重组R7蛋白液浓度进行测定，用PBS把蛋白原液稀释10倍作为样品测定，其样品OD592nm＝0.300，计算可得稀释液蛋白浓度为0.130mg/mL，即原蛋白液浓度为1.3mg/mL。
本申请根据卡氏住白细胞虫R7基因和psYNO-1表达载体构建了重组表达质粒psYNO-R7。将构建的重组表达质粒psYNO-R7经原核表达分析显示：重组蛋白R7(recombinant R7 protein，R7)在37℃、0.1mM IPTG诱导表达7.5h时可溶性表达量最高，分子量为90kDa。经MBP标签纯化后获得重组R7蛋白，BCA测定纯化重组R7蛋白浓度为1.3mg/mL。
实施例4
一种采用卡氏住白细胞虫重组R7蛋白的间接ELISA检测方法，包括以下步骤：
(1)包被抗原：将卡氏住白细胞虫重组R7蛋白用pH为9.6、0.2mol/L NaHCO3稀释到0.65μg/mL，加入100μL/孔，在37℃下包被2h；
(2)洗涤酶标板：甩干孔内包被液，加入PBST，300μL/孔，洗涤4次，再甩干残留液体；
(3)封闭：加入5％脱脂奶，200μL/孔，在4℃下封闭过夜；
(4)洗涤酶标板：甩干孔内脱脂奶，加入PBST，300μL/孔，洗涤4次，再甩干残留液体；
(5)加入血清：用5％脱脂奶按1:500稀释血清，混合静置6min，将稀释好的血清加入到酶标板，100μL/孔，在37℃下孵育1h；
(6)洗涤酶标板：甩干孔内液体，加入PBST，300μL/孔，洗涤4次，再甩干残留液体；
(7)加入酶标二抗：加入1:4000稀释的HRP标记山羊抗鸡IgG，100μL/孔，在37℃下孵育0.5h；
(8)洗涤酶标板：甩干孔内液体，加入PBST，300μL/孔，洗涤4次，再甩干残留液体；
(9)显色：加入TMB显色液，100μL/孔，37℃显色15min；
(10)终止：加入2mol/L H2SO4终止显色，50μL/孔；
(11)读数：用酶标仪检测450nm波长OD值，并读取。
用建立的间接ELISA方法检测来自广东、广西、福建、江西的临床血清样本，一共1615份鸡血清样品，其中279份为阳性，1336份为阴性，总阳性率为17.28％。具体检测结果如表23，各地区卡氏住白细胞虫阳性率为2.83％～23.44％，其中陆川的阳性率最高，桂林的阳性率最低。对广西省60个鸡场进行抽样检测，共检测了927个血清样本，其中158份为阳性，769份为阴性，阳性率为17.04％。对福建省10个鸡场进行抽样检测，共检测了160个血清样本，其中19份为阳性，141份为阴性，阳性率为11.88％。对广东省28个鸡场进行抽样检测，共检测了448个血清样本，其中89份为阳性，359份为阴性，阳性率为19.87％。对江西省5个鸡场进行抽样检测，共检测了80个血清样本，其中13份为阳性，67份为阴性，阳性率为16.25％。
表23 临床检测结果
序列表
全长的MBP-R7的氨基酸序列：
MGHHHHHHGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGASGLVTFISPNNVQAEIINTHGVRCNQNEEVTHQTHQTHQTHQTHQTHQTHQIHQIHQIHGYMTNQKHEEHGKIINQVKENVKNTVNENVKNNVDENTTSEHEITIPNENDIKTNDENETTHYEREIIYIVDDLPEVNVEESDETEHITYEIDNDIQEEHEKVTHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEKEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEKEKEEHEEVIHEEEKEEVTHEEEEEKVTHEEEEEKVIHEEEKEEVIHEEEKEEVTHEEEKEEVTHEEEKEEVTHEEEKEEVTHEEEKEEVTHEEEEKVTHEEEKEEVTHEEEKEEVTHEEEEKVTHEEEEKVTHEEEEKVTYEEEEEEEEKVTHEEEEKVTHEEEEKVTHEEEEKVIHEEEEKEEDEEEEEEEEEEEEEEEEEDEEEEEEEEEDEEEEEEEENEEEEEEENKEEEEEEKEEHEEEVTHEEEEEKVTHEEEEKVTHEEEENVTYEEEEEKVTHEEEEKVTHEEEEKVTHEEEENVTYEEEEEKVTHEEEEEKVMKKKKIMKYKKKKKKKKKGGGA
SEQUENCE LISTING
<110> 佛山市正典生物技术有限公司
<120> 卡氏住白细胞虫重组R7蛋白及其制备方法
<130> 2019.7
<160> MBP-R7的氨基酸序列
<170> PatentIn version 3.5
<210> 1
<211> 1073
<212> PRT
<213> 人工序列
<400> 1
Met Gly His His His His His His Gly Ser Lys Ile Glu Glu Gly Lys
1 5 10 15
Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
20 25 30
Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
35 40 45
His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
50 55 60
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
65 70 75 80
Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
85 90 95
Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
100 105 110
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
115 120 125
Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
130 135 140
Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
145 150 155 160
Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
165 170 175
Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
180 185 190
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
195 200 205
Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
210 215 220
Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
225 230 235 240
Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
245 250 255
Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
260 265 270
Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
275 280 285
Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
290 295 300
Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
305 310 315 320
Leu Val Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
325 330 335
Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
340 345 350
Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
355 360 365
Glu Ala Leu Lys Asp Ala Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn
370 375 380
Asn Asn Asn Asn Asn Asn Leu Gly Glu Asn Leu Tyr Phe Gln Gly Ala
385 390 395 400
Ser Gly Leu Val Thr Phe Ile Ser Pro Asn Asn Val Gln Ala Glu Ile
405 410 415
Ile Asn Thr His Gly Val Arg Cys Asn Gln Asn Glu Glu Val Thr His
420 425 430
Gln Thr His Gln Thr His Gln Thr His Gln Thr His Gln Thr His Gln
435 440 445
Thr His Gln Ile His Gln Ile His Gln Ile His Gly Tyr Met Thr Asn
450 455 460
Gln Lys His Glu Glu His Gly Lys Ile Ile Asn Gln Val Lys Glu Asn
465 470 475 480
Val Lys Asn Thr Val Asn Glu Asn Val Lys Asn Asn Val Asp Glu Asn
485 490 495
Thr Thr Ser Glu His Glu Ile Thr Ile Pro Asn Glu Asn Asp Ile Lys
500 505 510
Thr Asn Asp Glu Asn Glu Thr Thr His Tyr Glu Arg Glu Ile Ile Tyr
515 520 525
Ile Val Asp Asp Leu Pro Glu Val Asn Val Glu Glu Ser Asp Glu Thr
530 535 540
Glu His Ile Thr Tyr Glu Ile Asp Asn Asp Ile Gln Glu Glu His Glu
545 550 555 560
Lys Val Thr His Glu Glu Glu Lys Glu Glu Val Thr His Glu Glu Ile
565 570 575
Glu Lys Glu Glu His Glu Glu Val Ile His Glu Glu Glu Lys Glu Glu
580 585 590
Val Thr His Glu Glu Ile Glu Lys Glu Glu His Glu Glu Val Ile His
595 600 605
Glu Glu Glu Lys Glu Glu Val Thr His Glu Glu Lys Glu Lys Glu Glu
610 615 620
His Glu Glu Val Ile His Glu Glu Glu Lys Glu Glu Val Thr His Glu
625 630 635 640
Glu Ile Glu Lys Glu Glu His Glu Glu Val Ile His Glu Glu Glu Lys
645 650 655
Glu Glu Val Thr His Glu Glu Ile Glu Lys Glu Glu His Glu Glu Val
660 665 670
Ile His Glu Glu Glu Lys Glu Glu Val Thr His Glu Glu Ile Glu Lys
675 680 685
Glu Glu His Glu Glu Val Ile His Glu Glu Glu Lys Glu Glu Val Thr
690 695 700
His Glu Glu Ile Glu Lys Glu Glu His Glu Glu Val Ile His Glu Glu
705 710 715 720
Glu Lys Glu Glu Val Thr His Glu Glu Lys Glu Lys Glu Glu His Glu
725 730 735
Glu Val Ile His Glu Glu Glu Lys Glu Glu Val Thr His Glu Glu Glu
740 745 750
Glu Glu Lys Val Thr His Glu Glu Glu Glu Glu Lys Val Ile His Glu
755 760 765
Glu Glu Lys Glu Glu Val Ile His Glu Glu Glu Lys Glu Glu Val Thr
770 775 780
His Glu Glu Glu Lys Glu Glu Val Thr His Glu Glu Glu Lys Glu Glu
785 790 795 800
Val Thr His Glu Glu Glu Lys Glu Glu Val Thr His Glu Glu Glu Lys
805 810 815
Glu Glu Val Thr His Glu Glu Glu Glu Lys Val Thr His Glu Glu Glu
820 825 830
Lys Glu Glu Val Thr His Glu Glu Glu Lys Glu Glu Val Thr His Glu
835 840 845
Glu Glu Glu Lys Val Thr His Glu Glu Glu Glu Lys Val Thr His Glu
850 855 860
Glu Glu Glu Lys Val Thr Tyr Glu Glu Glu Glu Glu Glu Glu Glu Lys
865 870 875 880
Val Thr His Glu Glu Glu Glu Lys Val Thr His Glu Glu Glu Glu Lys
885 890 895
Val Thr His Glu Glu Glu Glu Lys Val Ile His Glu Glu Glu Glu Lys
900 905 910
Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
915 920 925
Glu Glu Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu
930 935 940
Glu Glu Glu Glu Glu Glu Glu Asn Glu Glu Glu Glu Glu Glu Glu Asn
945 950 955 960
Lys Glu Glu Glu Glu Glu Glu Lys Glu Glu His Glu Glu Glu Val Thr
965 970 975
His Glu Glu Glu Glu Glu Lys Val Thr His Glu Glu Glu Glu Lys Val
980 985 990
Thr His Glu Glu Glu Glu Asn Val Thr Tyr Glu Glu Glu Glu Glu Lys
995 1000 1005
Val Thr His Glu Glu Glu Glu Lys Val Thr His Glu Glu Glu Glu
1010 1015 1020
Lys Val Thr His Glu Glu Glu Glu Asn Val Thr Tyr Glu Glu Glu
1025 1030 1035
Glu Glu Lys Val Thr His Glu Glu Glu Glu Glu Lys Val Met Lys
1040 1045 1050
Lys Lys Lys Ile Met Lys Tyr Lys Lys Lys Lys Lys Lys Lys Lys
1055 1060 1065
Lys Gly Gly Gly Ala
1070
Claims (7)
1.一种卡氏住白细胞虫重组R7蛋白，其特征在于，所述卡氏住白细胞虫重组R7蛋白的氨基酸序列为MBP-R7，所述MBP-R7的序列为：
MGHHHHHHGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGASGLVTFISPNNVQAEIINTHGVRCNQNEEVTHQTHQTHQTHQTHQTHQTHQIHQIHQIHGYMTNQKHEEHGKIINQVKENVKNTVNENVKNNVDENTTSEHEITIPNENDIKTNDENETTHYEREIIYIVDDLPEVNVEESDETEHITYEIDNDIQEEHEKVTHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEKEKEEHEEV。
2.权利要求1所述的卡氏住白细胞虫重组R7蛋白的制备方法，其特征在于，包括以下步骤：
（1）将卡氏住白细胞虫R7基因进行PCR扩增，得到卡氏住白细胞虫重组R7基因；
（2）用限制性内切酶对psYNO-1质粒进行酶切，将卡氏住白细胞虫重组R7基因与酶切后psYNO-1质粒用连接酶连接，并转化到E.coli TOP10感受态细胞中培养，再进行PCR鉴定阳性克隆，得到PCR鉴定阳性菌液，提取PCR鉴定阳性菌液质粒，经双酶切鉴定得到阳性重组质粒psYNO-R7；
（3）将阳性重组质粒psYNO-R7转化到E.coli BL21表达菌，加入异丙基硫代半乳糖苷进行诱导表达，得到psYNO-R7重组蛋白；
（4）将诱导表达后的 psYNO-R7重组蛋白纯化，得到卡氏住白细胞虫重组R7蛋白。
3.根据权利要求2所述的卡氏住白细胞虫重组R7蛋白的制备方法，其特征在于，所述卡氏住白细胞虫重组R7基因的制备方法包括以下步骤：
（1）找到卡氏住白细胞虫R7核苷酸序列，进行密码子优化后再合成R7基因序列；
（2）设计一对特异性连接引物的核苷酸序列为：CTGTACTTCCAGGGAGCAAGTGGTCTGGTTACC和GTGGTGGTGCTCGAGTTATCACACTTCTTCATGT，再设计一对鉴定引物的核苷酸序列为GACTAATTCGAGCTCGAACAACAACA和CATGTTCTTCTTTTTCTTTCTCTTCGTG；
（3）以合成的R7基因为模板，PCR扩增R7基因片段，得到卡氏住白细胞虫重组R7基因。
4.根据权利要求3所述的卡氏住白细胞虫重组R7蛋白的制备方法，其特征在于，所述特异性连接引物的5’端含有与表达质粒载体psYNO-1末端同源的15个碱基和3’端含有R7基因特异引物序列。
5.根据权利要求2所述的制备方法，其特征在于，步骤（2）中，所述限制性内切酶为BamHⅠ和XhoⅠ。
6.根据权利要求5所述的制备方法，其特征在于，步骤（2）中，所述双酶切鉴定的步骤为：将psYNO-R7、SacⅠ、XhoⅠ、10×M Buffer和ddH2O加入到灭菌的1.5 mL离心管中混匀，在37℃下恒温水浴2 h，得到酶切产物，将酶切产物进行琼脂糖凝胶电泳，将鉴定为阳性的质粒做好标记，并测序，得到阳性重组质粒psYNO-R7，保存于-20℃中。
7.权利要求1所述卡氏住白细胞虫重组R7蛋白在制备检测卡氏住白细胞虫病试剂中的应用。
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