CN116874578A - 一种var2csa重组蛋白及其制备方法和应用 - Google Patents
一种var2csa重组蛋白及其制备方法和应用 Download PDFInfo
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- CN116874578A CN116874578A CN202310054915.XA CN202310054915A CN116874578A CN 116874578 A CN116874578 A CN 116874578A CN 202310054915 A CN202310054915 A CN 202310054915A CN 116874578 A CN116874578 A CN 116874578A
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Abstract
本申请属于生物医药领域,涉及一种VAR2CSA重组蛋白及其制备方法和应用。本申请提供的VAR2CSA重组蛋白及其制备方法,具有良好的稳定性及较高的产量,同时兼具良好的ofCS及其糖蛋白的检测效能,可用于大规模生产ofCS/ofCSPG的检测蛋白,用于恶性肿瘤的早期筛查、诊断、肿瘤负荷监测及预后预测。
Description
技术领域
本申请属于生物医药领域,涉及一种VAR2CSA重组蛋白及其制备方法和应用。
背景技术
恶性肿瘤细胞中普遍存在着一种特殊结构的硫酸软骨素A(CSA)修饰,该结构是由交替的葡萄糖醛酸和乙酰半乳糖胺形成的二糖单位组成的糖链,并带有不同程度的硫酸基团修饰,称为肿瘤型硫酸软骨素(oncofetal chondroitin sulfate,ofCS),或胎盘样硫酸软骨素(placental chondroitin sulfate,plCS)1。因其与胎盘滋养层细胞表面的硫酸软骨素结构存在相似性,ofCS可被疟原虫表达的VAR2CSA蛋白高亲和力结合2。基于VAR2CSA的ofCS检测技术应用于恶性肿瘤筛查诊断的潜力巨大。
目前,VAR2CSA蛋白已被用于开发靶向硫酸软骨素聚糖异常表达疾病的药物、从肿瘤患者血液中分离循环肿瘤细胞3以及从膀胱癌患者的尿液中检测ofCS修饰的蛋白多糖4。已有研究通过表达一段28个氨基酸的VAR2CSA短肽,构建以VAR2CSA为捕获分子,抗ofCS的抗体为检测分子的ELISA检测体系5(授权公告号:CN 109387627 B)。但是该方法所使用的VAR2CSA短肽对肿瘤型硫酸软骨素的亲和力不详,存在着检测灵敏度低(对早期肿瘤检测效果不详)、尚需要纯化ofCS抗体等缺点。在公开的相关发明中(申请公布号:CN113740521A),还有通过rVAR2检测肾癌和膀胱癌患者尿液中的游离ofCS水平用以诊断癌症。然而该方法所用的ELISA为直接法,同样存在着rVAR2与CSA亲和力未知、灵敏度低等问题。
VAR2CSA是在恶性疟原虫(P.falciparum)感染的红细胞表面表达的大型多结构域跨膜蛋白(350kDa)。其胞外区域包含1个N端片段(N terminal segment,NTS),6个Duffy-Binding-Like(DBL)结构域,和3个结构域间(interdomains,IDs)片段6。现有研究表明VAR2CSA的膜外段存在不同的可与硫酸软骨素A(chondroitin sulfate A,CSA)结合的结构域,且不同的结构域具有不同的亲和力7-9。VAR2CSA可与硫酸软骨素A结合的最小区域为DBL2X及侧翼间区(flanking interdomain,ID)10,11。VAR2CSA胞外全段可确保其与硫酸软骨素A特异、高亲和力结合12。然而胞外全段的大片段重组蛋白体外表达存在着以下技术缺点:1、表达产量低,2、蛋白易发生错误折叠,不稳定13。加之var2csa基因具有高度的可变性,其遗传多样性也可能会影响与CSA的结合效果。VAR2CSA蛋白的大尺寸和复杂的结构,以及分离株之间的遗传变异,使当前的大规模生产策略复杂化14。开发基于VAR2CSA的肿瘤标志物的重点在于稳定生产可高效检测ofCS的小片段VAR2CSA。
发明内容
为了解决现有可用于ofCS检测的胞外全长VAR2CSA蛋白表达不稳定、产量低的问题,本发明构建了包含最小CSA结合结构域(ID1-DBL2X-ID2a)的不同序列长度、不同虫株来源的VAR2CSA,可用于大规模生产ofCS/ofCSPG的检测蛋白,用于恶性肿瘤的早期筛查、诊断、肿瘤负荷监测及预后预测。
一方面,本申请提供了一种VAR2CSA重组蛋白,所述VAR2CSA重组蛋白包含如SEQID NO:1-4任一所述的氨基酸序列;在一些实施方案中,所述VAR2CSA重组蛋白包含SEQ IDNO:3所述的氨基酸序列。
一方面,本申请提供了一种核酸分子,所述核酸分子编码所述的VAR2CSA重组蛋白;在一些实施方案中,所述核酸分子的序列选自如SEQ ID NO:5-8所述的核苷酸序列;在一些实施方案中,所述核酸分子的序列选自如SEQ ID NO:7所述的核苷酸序列。
一方面,本申请提供了制备如所述的VAR2CSA重组蛋白的方法,包括以下步骤:
S1.以疟原虫VAR2CSA蛋白的氨基酸序列,通过同源重组将所述的核酸分子构建于表达载体中;
S2.取步骤S1制备的测序正确的阳性克隆转化至表达感受态细胞中;
S3.筛选后挑取单克隆再次进行PCR检测;
S4.加入诱导剂诱导重组蛋白表达。
在一些实施方案中,所述疟原虫的虫株选自FCR3株或3D7株;优选地,所述FCR3株的编号为GenBank No:ADG23053.1;优选地,所述3D7株的编号为NCBI:XP_001350415.1;优选地,所述表达载体选自pGEX系列质粒;优选地,所述pGEX系列质粒为pGEX-4T2;优选地,所述pGEX-4T2质粒C端融合表达蛋白酶识别位点及标签蛋白;
优选地,所述表达感受态细胞选自大肠杆菌细胞;优选地,所述S3中的筛选为双抗筛选;优选地,所述双抗包括氨苄西林和/或链霉素;优选地,所述诱导剂选自异丙基硫代半乳糖苷。
在一些实施方案中,所述方法还包括对重组蛋白的纯化。
在一些实施方案中,所述对重组蛋白的纯化包括以下步骤:
1)对表达VAR2CSA重组蛋白的表达感受态细胞重悬后得到的菌液进行破碎;
2)破碎后的菌液进行离心;
3)将离心后的上层清液过滤后,加入到用重悬缓冲液平衡好的可结合C端标签蛋白的层析介质中充分孵育;
4)用洗涤缓冲液洗涤后,用洗脱缓冲液进行VAR2CSA重组蛋白的洗脱;
5)洗脱后的蛋白产物加入到缓冲液平衡好的GST亲和层析介质中充分结合;
6)用缓冲液充分洗涤,随后置换为蛋白酶切缓冲液,酶切过夜;
7)将收集到的酶切产物再次过可结合C端标签蛋白的层析柱,收集蛋白洗脱液,并用脱盐柱进行缓冲液置换或PBS透析,最后进行蛋白浓缩。
在一些实施方案中,步骤1)中,加入重悬液对表达感受态细胞进行重悬;优选地,所述重悬液包括10mM Na2HPO4,1.8mM KH2PO4,pH=7.4,140mM NaCl,2.7mM KCl,2.5mMβ-ME,1μM DNase I,1mM PMSF,蛋白酶抑制剂I;优选地,按照表达感受态细胞:重悬液的比例为1g:5ml加入重悬液;优选地,所述步骤1)中为机械破碎;优选地,所述机械破碎的条件设置为:压力800-1200bar,连续破碎2-5次;优选地,所述步骤2)中,离心条件为:30,000×g-50,000×g,3-5℃离心0.5-2小时;优选地,所述步骤3)中,离心后的上层清液经0.45μm和/或0.22μm滤膜过滤;优选地,所述步骤4)中,所述洗涤缓冲液为咪唑浓度为80-150mM的洗涤缓冲液;优选地,所述步骤4)中,所述洗脱缓冲液为咪唑浓度为300-700mM的洗脱缓冲液;优选地,所述步骤6)中,在3-5℃酶切;优选地,所述步骤3)-7)在2-8℃条件下进行。
一方面,本申请提供了一种表达载体,其包含所述的核酸分子;优选地,所述表达载体选自质粒、噬菌体、人工染色体、病毒中的一种或多种;优选地,所述表达载体选自质粒;优选地,所述质粒选自pGEX系列质粒;优选地,所述质粒选自pGEX-4T2。
一方面,本申请提供了一种细胞,其包含所述的表达载体;优选地,所述细胞选自原核细胞和/或真核细胞;优选地,所述原核细胞选自大肠杆菌;优选地,所述大肠杆菌为Shuffle T7 Ecoli。
一方面,本申请提供了一种肿瘤型硫酸软骨素和/或肿瘤型硫酸软骨素修饰的糖蛋白的ELISA试剂,所述ELISA试剂包括所述的VAR2CSA重组蛋白作为检测试剂;优选地,所述ELISA试剂还包括捕获试剂,所述捕获试剂为抗肿瘤型硫酸软骨素和/或肿瘤型硫酸软骨素修饰的糖蛋白的抗体;优选地,所述抗体为单克隆抗体、多克隆抗体、多特异性抗体或抗体片段;优选地,所述捕获试剂为重组VAR2CSA蛋白;优选地,所述检测试剂还包括酶标试剂;优选地,所述的酶标试剂为辣根过氧化物酶、碱性磷酸酶(ALP)、β-半乳糖酶或金胶体,优选地,在使用辣根过氧化物酶的情况下,作为显色底物选自3,3',5,5'-四甲基联苯胺、邻苯二胺;在使用ALP的情况下,作为显色底物选自对硝基苯磷酸酯;在使用β-半乳糖酶时,作为显色底物,选自邻硝基苯基-β-D-吡喃半乳糖苷;优选地,试剂盒还包括封闭液、洗涤液、样品稀释液、显色液、终止液、标准品。优选地,所述封闭液为3-5%BSA或1-5%明胶;优选地,所述封闭液为5%BSA。
一方面,本申请提供了所述的VAR2CSA重组蛋白,或所述的ELISA试剂在制备样本中肿瘤型硫酸软骨素和/或肿瘤型硫酸软骨素修饰的糖蛋白的检测试剂中的应用;优选地,所述肿瘤型硫酸软骨素包括肿瘤型硫酸软骨素糖胺聚糖;优选地,所述肿瘤型硫酸软骨素修饰的糖蛋白选自肿瘤型硫酸软骨素修饰的CD44、肿瘤型硫酸软骨素修饰的CSPG4、肿瘤型硫酸软骨素修饰的SDC1中的一种或多种;优选地,所述肿瘤型硫酸软骨素修饰的糖蛋白选自肿瘤型硫酸软骨素修饰的CD44。
一方面,本申请提供了所述的VAR2CSA重组蛋白,或所述的ELISA试剂在制备检测肿瘤风险的检测试剂中的应用。
在一些实施方案中,所述肿瘤为表达CSA的肿瘤;优选地,所述肿瘤为上皮来源的恶性肿瘤、间叶组织来源的恶性肿瘤、造血系统癌、恶性黑色素瘤、神经上皮组织恶性肿瘤、或神经内分泌癌;优选地,所述上皮来源的恶性肿瘤为:乳腺癌、胰腺癌、卵巢癌、子宫内膜癌、肝细胞癌、肺癌、结直肠癌、前列腺癌、宫颈癌、睾丸癌、基底细胞皮肤癌、肾透明细胞癌、头颈角化鳞状细胞癌、皮肤鳞状细胞癌、外阴角化鳞状细胞癌、外阴基地细胞癌、胃癌、甲状腺癌、肝内胆管癌、口腔癌、鼻咽癌、食管癌、或膀胱癌;优选地,所述间叶组织来源的恶性肿瘤为:脂肪肉瘤、纤维肉瘤、平滑肌肉瘤、横纹肌肉瘤、淋巴管肉瘤或软骨肉瘤;优选地,所述造血系统癌为:淋巴瘤或白血病;优选地,所述神经上皮组织恶性肿瘤为:胶质瘤、弥漫性星形细胞瘤、或神经母细胞瘤。
在一些实施方案中,本申请构建了4条包含CSA最小结合结构域的重组VAR2CSA序列。
在一些实施方案中,本申请对表达载体pGEX-4T2质粒进行改造,提高了蛋白的可溶性与表达量,成功表达VAR2CSA蛋白,得到了较高的产率。
4条VAR2CSA序列中,包含结构域片段最多的rVAR2-1号蛋白(DBL1X-ID1-DBL2X-ID2a-ID2b)产量最低,包含结构域片段ID1-DBL2X-ID2a-ID2b的rVAR2-4号蛋白产量稍高,而包含最少结构域片段ID1-DBL2X-ID2a的rVAR2-2号和rVAR2-3号蛋白产量相对更高。在rVAR2-2号和rVAR2-3号中,3D7株来源的rVAR2-3号产量是FCR3来源的rVAR2-2号的近2倍。
进一步将纯化得到的蛋白进行HRP标记后用于ELISA检测,在病例对照队列中,病例组ofCS-CD44水平显著高于对照组(p<0.0001),ROC曲线下面积均达到0.8以上,可用于恶性肿瘤的检测(图5)。
在本申请中,CSA为chondroitin sulfate A的缩写,CSA、硫酸软骨素A、chondroitin sulfateA几者可以互换使用。
在本申请中,rVAR2为recombinant VAR2CSA的缩写;rVAR2、recombinant VAR2、重组VAR2、重组VAR2CSA,几者可互换使用。
在本申请中,ofCSPG为oncofetal chondroitin sulfate proteoglycan的缩写;由于ofCS糖胺聚糖以共价结合的方式附着在多种蛋白上,根据ofCS所结合的具体蛋白类型,ofCSPG有不同的种类,包括但不限于:ofCS-CD44,ofCS-CSPG4,ofCS-SDC1。
本申请中的“检测”同诊断,除了癌症的早期诊断,还包括癌症中期和晚期的诊断,且也包括癌症筛选、风险评估、预后、疾病识别、病症阶段的诊断和治疗性靶标的选择。
附图说明
图1为rVAR21-4的SDS-PAGE考马斯亮蓝染色(A)及Western-Blot(B)鉴定结果图;
图2为rVAR21-4与肿瘤细胞结合的平均荧光强度汇总结果;
图3为rVAR21-4与外周血细胞结合的流式结果(ALL-P1:急性淋巴细胞白血病患者白细胞-1,ALL-P2:急性淋巴细胞白血病患者白细胞-2);
图4为本申请ELISA检测技术原理图;
图5为实施例4中病例对照人群(59个病例、22个对照)中,rVAR21-4检测血浆中ofCS-CD44的效果图,ROC分析ofCS-CD44在病例对照人群中检测恶性肿瘤的效能。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案,具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
制备实施例
大肠杆菌重组载体的克隆和表达
在原质粒限制性酶切位点BamHI的C端融合表达PreScission蛋白酶识别位点、EcoRI与HindIII限制性蛋白酶切位点、V5标签、TEV蛋白酶识别位点及His10标签。以疟原虫FCR3株VAR2CSA蛋白(GenBank No:ADG23053.1),疟原虫3D7株VAR2CSA蛋白(NCBI:XP_001350415.1)的氨基酸序列,参考大肠杆菌的密码子偏好性,对该肽段进行密码子优化后,通过同源重组的方法将目的基因构建于表达载体中,同源重组引物见表1。
表1大肠杆菌表达rVAR2插入片段的重组引物
取测序正确的阳性克隆转化到Shuffle T7 E.coli表达感受态细胞中,氨苄西林与链霉素双抗筛选后挑取单克隆再次进行PCR检测。上述阳性克隆加入含有氨苄西林及链霉素的LB液体培养基中,放至37℃摇床中复苏12小时;活化后的菌种按1:100接种到2L的锥形瓶中在37℃摇床中继续扩大培养2-3小时,调节摇床温度至18℃,待温度降至18℃后加入诱导剂异丙基硫代半乳糖苷(IPTG)诱导重组蛋白表达。
大肠杆菌重组载体表达VAR2CSA的纯化
收集菌体,按每1g菌体加入5mL重悬缓冲液(10mM Na2HPO4,1.8mM KH2PO4,pH=7.4,140mM NaCl,2.7mM KCl,2.5mMβ-ME,1μM DNase I,1mM PMSF,蛋白酶抑制剂I)的比例将菌体充分重悬。在冰浴下对重悬的菌液进行机械破碎,破碎条件设置为:压力1000bar,连续破碎3次。破碎后菌液在40,000×g,4℃离心1小时。为防止重组蛋白发生降解,以下操作均在2-8℃中进行。将离心后的上层清液经0.45μm与0.22μm滤膜双重过滤后,加入到用重悬缓冲液平衡好的Ni-NTA柱中,进行金属螯合。用咪唑浓度为100mM的洗涤缓冲液进行洗涤。充分洗涤后,用咪唑浓度为500mM的洗脱缓冲液进行rVAR2的洗脱。洗脱后的蛋白产物加入到用PBS平衡好的GST亲和层析介质中,放至垂直旋转混合仪上旋转1小时,确保重组蛋白充分结合到介质中。将蛋白与层析介质转至重力层析柱中,待液体流尽后用PBS溶液充分洗涤,随后置换为PreScission Protease酶切缓冲液。加入适量的Prescission Protease,在4℃酶切过夜。第二天将收集到酶切产物再次过Ni-NTA柱,收集咪唑浓度为500mM的蛋白洗脱液,并用脱盐柱进行缓冲液置换,用超滤管进行蛋白的浓缩。BCA法蛋白定量,SDS-PAGE与Western-Blot鉴定(图1)。
表24个重组VAR2CSA蛋白信息
编号 | 包含结构域 | 宿主细胞 | 寄生虫株 | 序列编号 |
rVAR2-1 | DBL1X-ID1-DBL2X-ID2a-ID2b | E.coli | FCR3 | SEQ ID NO:1 |
rVAR2-2 | ID1-DBL2X-ID2a | E.coli | FCR3 | SEQ ID NO:2 |
rVAR2-3 | ID1-DBL2X-ID2a | E.coli | 3D7 | SEQ ID NO:3 |
rVAR2-4 | ID1-DBL2X-ID2a-ID2b | E.coli | FCR3 | SEQ ID NO:4 |
实施例1VAR2CSA的产量
经过大肠杆菌重组载体表达VAR2CSA及纯化后,制备实施例中所制备的4个重组VAR2CSA的产量如表3所示。
表34个重组VAR2CSA蛋白产量
不同的VAR2CSA在表达产量上具有差别,一些特定序列结构的VAR2CSA蛋白更容易稳定表达,表现出较高的产量。rVAR2-2的产量达58.1ug/L;而作为具有与其相当的序列长度的rVAR2-3更是具有高达99.6ug/L的产量。如此高的产量,对于解决现有可用于ofCS检测的VAR2CSA蛋白表达不稳定、产量低的问题具有重要意义。
实施例2VAR2CSA与肿瘤细胞结合的流式检测
细胞封闭后与rVAR2温孵育1小时,洗涤后再次孵育FITC标记的抗V5标签单克隆抗体,室温避光孵育1小时。再次洗涤后用预冷的含5%BSA的PBS溶液进行细胞重悬后,立即使用流式细胞仪对细胞进行分析,记录各组的荧光强度,并以各组平均荧光强度与空白对照的比值作为细胞与rVAR2结合能力的评价指标。流式结果显示4个重组蛋白均可与肺腺癌细胞(A549),结直肠癌细胞(SW480,HCT116,LoVo,HT29,CaCo2,SW620),以及食管鳞癌细胞(KYSE180,KYSE30)结合,平均荧光强度随着孵育的蛋白浓度升高而升高(图2)。
此外,rVAR2还可以与急性淋巴细胞白血病患者的外周血白细胞结合,却不结合于健康对照的外周血白细胞(图3)。
本申请例从细胞水平(附图2、3)验证了所制备的VAR2CSA蛋白可以特异结合ofCS和ofCSPG。
实施例3“棋盘法”优化夹心法ELISA的实验条件
最佳抗体/rVAR2的包被浓度的确定:设置抗体与rVAR2的包被浓度分别为16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL。最佳包被缓冲液的确定:候选包被缓冲液为常见的0.05M的碳酸氢盐缓冲液(pH=9.6),0.01M Tris缓冲液(pH=8.0),0.01M PBS缓冲液(pH=7.2)。最佳封闭缓冲液的确定:选择用1%明胶,3%明胶,5%BSA溶液,5%牛奶作为候选封闭缓冲液。最佳待检测血浆稀释度的确定:血浆稀释比例由1:25~1:3200共8个梯度进行实验。最佳待测样品作用时间的确定:加入待测血浆样品后分别静置反应30分钟、60分钟、90分钟和120分钟。最佳HRP标记的rVAR2稀释度确定:HRP标记的rVAR2分别设置3.2μg/mL、1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL。最佳HRP标记的rVAR2作用时间的确定:分别设置为15分钟,30分钟,45分钟,60分钟,75分钟,90分钟。最佳TMB作用时间的确定:TMB作用时间分别设置为5分钟,10分钟,15分钟,20分钟,25分钟,30分钟。
每轮实验参照操作流程进行,均使用阳性血浆与阴性血浆,每份血浆样本重复2孔,取其平均值,结果判定P/N值最大时的所对应的实验条件为最佳条件,并用于后续步骤中。最终明确对于rVAR2-5,抗CD44单克隆抗体,抗SDC1单克隆抗体以及抗CSPG4多克隆抗体分别以1-8μg/mL,1-5μg/mL,1-5μg/mL,0.5-5μg/mL的浓度进行包被可获得最佳的反应P/N值。
待测血浆按1:5-1:100进行稀释,HRP标记的rVAR2-3浓度为0.1μg/mL:1:10-1:5000稀释可获得较好反应P/N值。通过梯度反应时间摸索,获得待测血浆样本的反应时间为60-120分钟,HRP标记的rVAR2-3反应时间60-120分钟,TMB的显色时间为5-30分钟。并且,使用pH=9.6的0.05M碳酸氢盐缓冲液作为包被液,5%BSA溶液作为封闭缓冲液,可获得较好的反应P/N值。
实施例4VAR2CSA的病例对照人群中ofCSPG检测效果
检测原理:将抗CD44抗体分别包被在96孔酶标板上,4℃孵育过夜,洗去未结合在孔板上的多余的抗体分子,用含5%BSA封闭,加入稀释的待测血浆样本室温孵育后加入HRP标记的rVAR2,室温孵育后TMB显色(图4)。
反应参数为:抗CD44抗体浓度为1-8μg/mL;待测血浆按1:5-1:100进行稀释,HRP标记的rVAR2浓度为0.1μg/mL:1:10-1:5000稀释;待测血浆样本的反应时间为60-120分钟,HRP标记的rVAR2反应时间60-120分钟,TMB的显色时间为5-30分钟。使用pH=9.6的0.05M碳酸氢盐缓冲液作为包被液,5%BSA作为封闭缓冲液。
共纳入22例健康对照,来自广东自然人群队列(ChiCTR1800015736);59例肿瘤患者,来自中山大学肿瘤防治中心。通过夹心法ELISA检测,结果提示在所检测的肿瘤患者血浆ofCS修饰的CD44的表达量显著高于健康对照组(450nm处的OD值),将血浆中ofCS修饰的CD44作为自变量进行logistic回归预测其与肿瘤发生的概率,在进行年龄与性别校正后的曲线下面积(AUC)分别为0.864(95%CI=0.7669to 0.9604),Se=0.845,Sp=0.864;0.8258(95%CI=0.7324to 0.9192),Se=0.793,Sp=0.727;0.8429(95%CI=0.748to0.9378),Se=0.879,Sp=0.682;0.8716(95%CI=0.7822to 0.9611),Se=0.810,Sp=0.864。
其中,Se=Sensitivity灵敏性,Sp=Specificity特异性,CI=ConfidenceInternal置信区间。
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Claims (11)
1.一种VAR2CSA重组蛋白,其特征在于,所述VAR2CSA重组蛋白包含如SEQ IDNO:1-4任一所述的氨基酸序列;
优选地,所述VAR2CSA重组蛋白包含SEQ ID NO:3所述的氨基酸序列。
2.一种核酸分子,其特征在于,所述核酸分子编码如权利要求1所述的VAR2CSA重组蛋白;
优选地,所述核酸分子的序列选自如SEQ ID NO:5-8所述的核苷酸序列;
优选地,所述核酸分子的序列选自如SEQ ID NO:7所述的核苷酸序列。
3.一种制备如权利要求1所述的VAR2CSA重组蛋白的方法,其特征在于,包括以下步骤:
S1.以疟原虫VAR2CSA蛋白的氨基酸序列,通过同源重组将如权利要求2所述的核酸分子构建于表达载体中;
S2.取步骤S1制备的测序正确的阳性克隆转化至表达感受态细胞中;
S3.筛选后挑取单克隆再次进行PCR检测;
S4.加入诱导剂诱导重组蛋白表达。
4.如权利要求3所述的方法,其特征在于,所述疟原虫的虫株选自FCR3株或3D7株;
优选地,所述表达载体选自pGEX系列质粒;
优选地,所述pGEX系列质粒为pGEX-4T2;
优选地,所述pGEX-4T2质粒C端融合表达蛋白酶识别位点及标签蛋白;
优选地,所述表达感受态细胞选自大肠杆菌细胞;
优选地,所述S3中的筛选为双抗筛选;
优选地,所述双抗包括氨苄西林和/或链霉素;
优选地,所述诱导剂选自异丙基硫代半乳糖苷。
5.如权利要求3所述的方法,其特征在于,还包括对重组蛋白的纯化;
优选地,所述对重组蛋白的纯化包括以下步骤:
1)对表达VAR2CSA重组蛋白的表达感受态细胞重悬后得到的菌液进行破碎;
2)破碎后的菌液进行离心;
3)将离心后的上层清液过滤后,加入到用重悬缓冲液平衡好的可结合C端标签蛋白的层析柱中充分孵育;
4)用洗涤缓冲液洗涤后,用洗脱缓冲液进行VAR2CSA重组蛋白的洗脱;
5)洗脱后的蛋白产物加入到缓冲液平衡好的GST亲和层析介质中充分结合;
6)用缓冲液充分洗涤,随后置换为蛋白酶切缓冲液,酶切过夜;
7)将收集到的酶切产物再次过可结合C端标签蛋白的层析柱,收集蛋白洗脱液,并用脱盐柱进行缓冲液置换或PBS透析,最后进行蛋白浓缩。
6.如权利要求5所述的纯化方法,其特征在于,步骤1)中,加入重悬液对表达感受态细胞进行重悬;
优选地,所述重悬液包括10mMNa2HPO4,1.8mMKH2PO4,pH=7.4,140mMNaCl,2.7mMKCl,2.5mMβ-ME,1μMDNaseI,1mMPMSF,蛋白酶抑制剂I;
优选地,按照表达感受态细胞:重悬液的比例为1g:5ml加入重悬液;
优选地,所述步骤1)中为机械破碎;
优选地,所述机械破碎的条件设置为:压力800-1200bar,连续破碎2-5次;
优选地,所述步骤2)中,离心条件为:30,000×g-50,000×g,3-5℃离心0.5-2小时;
优选地,所述步骤3)中,离心后的上层清液经0.45μm和/或0.22μm滤膜过滤;
优选地,所述步骤4)中,所述洗涤缓冲液为咪唑浓度为80-150mM的洗涤缓冲液;
优选地,所述步骤4)中,所述洗脱缓冲液为咪唑浓度为300-700mM的洗脱缓冲液;
优选地,所述步骤6)中,在3-5℃酶切;
优选地,所述步骤3)-7)在2-8℃条件下进行。
7.一种表达载体,其包含如权利要求2所述的核酸分子;
优选地,所述表达载体选自质粒、噬菌体、人工染色体、病毒中的一种或多种;
优选地,所述表达载体选自质粒;
优选地,所述质粒选自pGEX系列质粒;
优选地,所述质粒选自pGEX-4T2。
8.一种细胞,其包含如权利要求7所述的表达载体;
优选地,所述细胞选自原核细胞和/或真核细胞;
优选地,所述原核细胞选自大肠杆菌;
优选地,所述大肠杆菌为ShuffleT7Ecoli。
9.一种肿瘤型硫酸软骨素和/或肿瘤型硫酸软骨素修饰的糖蛋白的ELISA试剂,其特征在于,所述ELISA试剂包括如权利要求1所述的VAR2CSA重组蛋白作为检测试剂;
优选地,所述ELISA试剂还包括捕获试剂,所述捕获试剂为抗肿瘤型硫酸软骨素和/或肿瘤型硫酸软骨素修饰的糖蛋白的抗体;
优选地,所述抗体为单克隆抗体、多克隆抗体、多特异性抗体或抗体片段;
优选地,所述捕获试剂为重组VAR2CSA蛋白;
优选地,所述检测试剂还包括酶标试剂;
优选地,所述的酶标试剂为辣根过氧化物酶、碱性磷酸酶(ALP)、β-半乳糖酶或金胶体,
优选地,在使用辣根过氧化物酶的情况下,作为显色底物选自3,3',5,5'-四甲基联苯胺、邻苯二胺;在使用ALP的情况下,作为显色底物选自对硝基苯磷酸酯;在使用β-半乳糖酶时,作为显色底物,选自邻硝基苯基-β-D-吡喃半乳糖苷;
优选地,试剂还包括封闭液、洗涤液、样品稀释液、显色液、终止液、标准品;
优选地,所述封闭液为3-5%BSA或1-5%明胶;
优选地,所述封闭液为5%BSA。
10.如权利要求1所述的VAR2CSA重组蛋白,或权利要求9所述的ELISA试剂在制备样本中肿瘤型硫酸软骨素和/或肿瘤型硫酸软骨素修饰的糖蛋白的检测试剂中的应用;
优选地,所述肿瘤型硫酸软骨素包括肿瘤型硫酸软骨素糖胺聚糖;
优选地,所述肿瘤型硫酸软骨素修饰的糖蛋白选自肿瘤型硫酸软骨素修饰的CD44、肿瘤型硫酸软骨素修饰的CSPG4、肿瘤型硫酸软骨素修饰的SDC1中的一种或多种;
优选地,所述肿瘤型硫酸软骨素修饰的糖蛋白选自肿瘤型硫酸软骨素修饰的CD44。
11.如权利要求1所述的VAR2CSA重组蛋白,或权利要求9所述的ELISA试剂在制备检测肿瘤风险的检测试剂中的应用;
优选地,所述肿瘤为表达CSA的肿瘤;
优选地,所述肿瘤为上皮来源的恶性肿瘤、间叶组织来源的恶性肿瘤、造血系统癌、恶性黑色素瘤、神经上皮组织恶性肿瘤、或神经内分泌癌;
优选地,所述上皮来源的恶性肿瘤为:乳腺癌、胰腺癌、卵巢癌、子宫内膜癌、肝细胞癌、肺癌、结直肠癌、前列腺癌、宫颈癌、睾丸癌、基底细胞皮肤癌、肾透明细胞癌、头颈角化鳞状细胞癌、皮肤鳞状细胞癌、外阴角化鳞状细胞癌、外阴基地细胞癌、胃癌、甲状腺癌、肝内胆管癌、口腔癌、鼻咽癌、食管癌、或膀胱癌;
优选地,所述间叶组织来源的恶性肿瘤为:脂肪肉瘤、纤维肉瘤、平滑肌肉瘤、横纹肌肉瘤、淋巴管肉瘤或软骨肉瘤;
优选地,所述造血系统癌为:淋巴瘤或白血病;
优选地,所述神经上皮组织恶性肿瘤为:胶质瘤、弥漫性星形细胞瘤、或神经母细胞瘤。
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