CN105254724B - 截短型丙型肝炎病毒hcv ns3抗原及其制备与应用 - Google Patents
截短型丙型肝炎病毒hcv ns3抗原及其制备与应用 Download PDFInfo
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Abstract
本发明公开了一种截短型丙型肝炎病毒HCV NS3抗原,是HCV基因组中NS3全长基因编码的第1252~1526aa片段非结构蛋白氨基酸序列,其氨基酸序列如SEQ ID NO.1所示,对应核苷酸序列如SEQ ID NO.2所示。所述HCV NS3重组抗原蛋白制备方法是:构建用于表达截短型丙型肝炎病毒HCV NS3抗原的大肠杆菌优化密码子,其核苷酸序列如SEQ ID NO.3所示,将所述密码子基因片段经双酶切,再与经同样酶切后的表达载体连接,获得重组表达质粒,以常规方法使抗原表达,经纯化获得。本发明的抗原蛋白灵敏度高、特异性强,用其检测HCV抗体,与丙型肝炎病毒RNA阳性患者的检出符合率高达97%。预示该抗原可以应用于多克隆或单克隆抗体、丙型肝炎病毒检测试剂盒的制备,具有良好的临床应用前景。
Description
技术领域
本发明涉及一种丙型肝炎病毒抗原及其制备与应用,尤其涉及一种截短型丙型肝炎病毒HCV NS3抗原及其制备与应用,属于分子生物学技术领域。
背景技术
丙型肝炎病毒(hepatitis C virus,HCV)感染最显著的临床特征就是引起慢性感染,约有70%的丙肝有可能发展成慢性肝炎,20%发展成肝硬化,12%发展成肝癌,危害十分地严重。全球HCV的感染率为2.8%,约有1.85亿人携带HCV。我国属于HCV低流行区,预计我国HCV感染者1000万,而且每年的新发病例数不断上升。
自1989年HCV基因获得克隆以来,人们一直在致力于HCV相关疫苗的研究工作。HCV基因组长约9.6kb,编码一个长约3000个氨基酸残基的多聚蛋白前体,在宿主信号肽酶和病毒自身编码蛋白酶的作用下生成4种结构蛋白(C、E1、E2、P7)和6种非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A、NS5B)。但因病毒RNA在血液含量较低且非常不稳定,HCV基因组变异率较高,病毒准种多,缺乏理想的体外感染细胞模型,而导致预防性疫苗缺乏保护性抗体。由于目前没有有效治疗丙型肝炎的药物,因此早期发现HCV感染,准确判断病毒亚型及患者感染状况,是有效防控丙肝的重要手段。目前,我国控制该疾病蔓延的手段主要采用血液筛查,这也使得研究开发快速灵敏的丙型肝炎病毒检测试剂盒具有十分重要的临床意义。
已报到的有关HCV检测的ELISA试剂盒主要包含C区、NS3区、NS4区的抗原,有的也包含NS5区的抗原。但对NS5区抗原作用的评价,却褒贬不一。
Core、C33(NS3)抗原在抗原反应性上有很好的互补性。其联合检出率平均值可在99%左右,是研究HCV诊断试剂的重点区段。NS3基因位于HCV全基因序列的3420-5312nt位点,编码含有631aa(1026-1656aa)的NS3蛋白p72,其氨基酸序列相对保守,与黄病毒及瘟病毒相应区段有较高的同源性。NS3蛋白具有多种蛋白酶活性,在病毒多蛋白前体的加工及各蛋白的转化成熟过程中均发挥重要作用,非结构区各蛋白的产生均有赖于该蛋白对病毒多蛋白前体的酶解和修饰。免疫学研究发现,NS3蛋白的抗原性和免疫原性相对较强,是检测HCV感染的主要抗原之一,在诱导机体的抗病毒免疫方面也有一定的意义。感染者对NS3的抗体反应不仅出现早,且滴度高、特异性强,抗体转阳时间一般为8周左右,几乎与抗HCV同步。目前,单片段HCV-NS3抗体的检出率为76.47%-89.47%。在经对丙型肝炎病毒的免疫学信息研究发现,HCV在患者血液中的抗体主要是对以下抗原表位的免疫应答产生,Core(2-120aa),NS3(1026-1457aa),NS4(1694-1735aa),NS4(1859-1931aa),NS5A(2212-2313aa)。但在应用中,存在诊断试剂加入的NS3全长基因表达的蛋白过大,与其它优势抗原表位串联表达后,会因空间结构的遮挡而使部分抗原失去活性的缺陷。基于此,寻找和确立与丙型肝炎病毒RNA阳性患者的检出符合率高、灵敏度高的截短型丙型肝炎病毒HCV NS3抗原,对开发更加有效的早期诊断丙型肝炎病毒HCV的多克隆抗体或单克隆抗体和丙型肝炎病毒抗体检测试剂盒具有重要意义。
发明内容
针对现有技术的不足,本发明的目的在于提供一种截短型丙型肝炎病毒HCV NS3抗原及其制备与应用。
为实现上述目的,本发明人查找阅读国内外HCV NS3文献,并根据HCV sequencesdatabase(http://hepatitis.ibcp.fr.),筛选出6条分属于HCV 6种基因型的NS3抗原序列,通过序列对比、分析,筛选得到NS31252-1526aa截短型HCV NS3氨基酸序列,查找NCBI得到对应核苷酸序列,并进一步选择大肠杆菌的偏好性密码子设计并合成截短型NS3抗原基因核苷酸序列。
本发明所述截短型丙型肝炎病毒HCV NS3抗原,其特征在于:所述抗原是HCV基因组中NS3全长基因编码的第1252~1526aa片段非结构蛋白氨基酸序列,其氨基酸序列如SEQID NO.1所示,对应核苷酸序列如SEQ ID NO.2所示;与SEQ ID NO.2所示的核苷酸序列编码相同序列的蛋白质,但因遗传密码的简并性而与SEQ ID NO.2的核苷酸序列不同的序列;对所述SEQ ID NO.2所示核苷酸序列进行一个或多个碱基的取代、缺失、添加修饰的核苷酸序列。
上述截短型丙型肝炎病毒HCV NS3抗原,优选是HCV基因组中NS3全长基因编码的第1252~1526aa片段非结构蛋白氨基酸序列,其氨基酸序列如SEQ ID NO.1所示,对应核苷酸序列如SEQ ID NO.2所示。
本发明公开了一种用于表达权利要求1所述截短型丙型肝炎病毒HCV NS3抗原的大肠杆菌优化密码子,其特征在于:所述密码子核苷酸序列如SEQ ID NO.3所示,其编码的氨基酸对应SEQ ID NO.2所示的核苷酸序列编码的氨基酸。
本发明所述截短型丙型肝炎病毒HCV NS3重组抗原蛋白制备方法,步骤是:
(1)构建用于表达权利要求1所述截短型丙型肝炎病毒HCV NS3抗原的大肠杆菌优化密码子,其核苷酸序列如SEQ ID NO.3所示;
(2)将所述密码子基因片段经BamHI和XhoI双酶切,酶切产物纯化回收后,与经同样限制性内切酶酶切后的表达载体pET28a连接,获得重组表达质粒(构建的表达载体需用PCR法鉴定,以证明截短型NS3抗原基因片段已获正确***);
(3)通过CaCl2法将制得的重组表达质粒转入大肠杆菌感受态DH5α,涂布于含50μg/ml Kan+的LB琼脂培养基上,筛选得到阳性质粒;
(4)将步骤(3)中得到的阳性质粒,通过CaCl2法转入大肠杆菌E.coli BL21(DE3)感受态细胞,涂布于含Kan+的LB琼脂培养基上,常规培养;挑取单菌落接种至Kan+的LB液体培养基中,培养过夜;次日,转接于新鲜的含Kan+的LB液体培养基中,培养至OD600≈0.6时加入IPTG至终浓度为0.2-0.8mM,37℃诱导培养12-16h,获得含HCV NS3重组抗原蛋白的全菌液(取全菌液进行SDS-PAGE鉴定,证明截短型NS3重组抗原已得到高效表达),经过亲和镍柱层析纯化和离子交换柱纯化,即得到高纯度的截短型HCV NS3重组抗原。
一种多克隆抗体或单克隆抗体,其特征在于:所述抗体是能与截短型丙型肝炎病毒HCVNS3抗原特异性结合的多克隆抗体或单克隆抗体。
将制得的截短型HCV NS3重组抗原蛋白免疫小鼠(注射小鼠),经常规单克隆抗体制备方法,纯化分离,即可制备出所述重组抗原特异性结合的多克隆抗体或单克隆抗体。
或者用制得的截短型HCV NS3重组抗原包被酶联板,采用间接酶联免疫法建立抗HCVNS3抗体技术,检测其临床意义。
本发明所述截短型丙型肝炎病毒HCV NS3抗原在制备丙型肝炎病毒HCV抗原或抗体检测试剂盒中的应用。
本发明所述多克隆抗体或单克隆抗体在制备检测丙型肝炎病毒HCV抗原试剂盒中的应用。
本发明所述截短型丙型肝炎病毒HCV NS3抗原和其多克隆抗体或单克隆抗体,可以用于开发丙型肝炎病毒HCV抗原或抗体检测试剂盒或作为其他HCV检测产品的重要原料,对更加有效的开发早期诊断丙型肝炎病毒HCV的多克隆抗体或单克隆抗体和丙型肝炎病毒抗体检测试剂盒提供了支持。
申请人经过分析NS3全长基因片段,将NS3基因分成2段分别表达,1026-1251aa,1252-1526aa,通过基因重组的方法,分别制备出2段重组抗原。经活性检测,1252-1526aa片段重组蛋白具有高灵敏度、高特异性特点。用其检测HCV抗体,与随机丙型肝炎病毒RNA阳性患者的检出符合率高达97%。预示该抗原可以应用于多克隆抗体、单克隆抗体、丙型肝炎病毒检测试剂盒,尤其是丙型肝炎病毒抗体检测试剂盒的制备,具有良好的临床应用前景。
与现有技术比,本发明技术具有以下特点:
1、截短型HCV NS3重组抗原的筛选是通过生物信息学技术实现的
本发明利用生物信息学技术,根据HCV 6种基因型氨基酸序列同源性,对大量HCVNS3序列进行筛选,最终得到1条序列保守的截短型HCV NS3重组片段。
2、截短型HCV NS3重组抗原具有极高的阳性血清覆盖率
本发明将截短型HCV NS3重组序列,通过大肠杆菌偏好性密码子优化后表达,最大限度减小了NS3蛋白分子量,保持了截短型HCV NS3重组抗原的生物活性,进而可以结合不同HCV基因型HCV感染者血清中的HCV NS3抗体。在临床上适用于检测不同HCV基因型HCV感染者筛查工作,不易造成漏检。
3、截短型HCV NS3重组抗原应用于丙型肝炎病毒抗体检测试剂盒的研制
目前,我国主要采取血液筛查来达到控制HCV蔓延的目的。现用的丙型肝炎病毒抗体检测试剂盒主要包被抗原包括C区、NS3区、NS4区的抗原,有的也包含NS5区。但其存在漏检现象,分析其中单个NS3抗原其检出率为76.47%-89.47%。相信通过加入本发明截短型HCVNS3重组融合抗原,可以有效减少丙型肝炎病毒抗体检测试剂盒漏检现象,在临床上具有很大的应用前景和推广价值。
附图说明
图1:纯化后截短型HCV NS3重组抗原SDS-PAGE。
其中:左泳道为样品,右泳道为Mark。
具体实施方式
以下实施例用于进一步阐述本发明,但不用来限制本发明范围。若未特别指明,实施例中所采用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1:截短型HCV NS3重组抗原蛋白序列的获得
发明人根据HCV sequences database(http://hepatitis.ibcp.fr.),筛选出6条分属于HCV 6种基因型的NS3抗原序列,通过序列对比、分析,筛选得到NS31252-1526aa截短型HCV NS3氨基酸序列,查找NCBI得到对应核苷酸序列,其氨基酸序列如SEQ ID NO.1所示,对应核苷酸序列如SEQ ID NO.2所示。并进一步选择大肠杆菌的偏好性密码子,设计并合成截短型NS3抗原基因核苷酸序列,所述密码子核苷酸序列如SEQ ID NO.3所示,其编码的氨基酸对应SEQ ID NO.2所示的核苷酸序列编码的氨基酸。
实施例2:截短型HCV NS3重组抗原的克隆与表达
1.截短型HCV NS3重组抗原表达质粒的构建
1.1截短型HCV NS3重组抗原的合成
根据截短型HCV NS3抗原氨基酸序列(其氨基酸序列如SEQ ID NO.1所示),查找NCBI相关核苷酸序列,得到对应核苷酸序列(其核苷酸序列如SEQ ID NO.2所示),利用大肠杆菌密码子偏好性修饰SEQ ID NO.2得到核苷酸序列(如SEQ ID NO.3),委托博尚生物技术有限公司合成型截短型HCV NS3SEQ ID NO.3所示的核苷酸,并分别在5’端和3’端引入BamHI和Xho I两个酶切位点,制得基因产物。
1.2截短型HCV NS3重组抗原表达质粒的构建
1.2.1截短型HCV NS3重组融合抗原基因序列及表达载体pET28a的双酶切
取以上合成基因产物及pET28a表达载体各30ul分别置于1.5ml Eppendorf离心管中,加入10×buffer 5ul、BamHI(10U/ul)和Xho I(10U/ul)各3ul,加入灭菌蒸馏水4ul,置37℃水浴酶切3小时。酶切产物的琼脂糖凝胶电泳纯化和回收:合成基因及pET28a表达载体双酶切后产物按照TaKaRa DNA凝胶回收试剂盒产品说明书进行。
1.2.2连接:于灭菌Eppendorf离心管中加入上述酶切后的载体和目的基因各2ul、10×T4DNA Ligase buffer 2ul、T4DNA Ligase(5U/ul)1ul,加入灭菌蒸馏水至20ul,置16℃过夜。
1.2.3转化:在超净工作台中,用无菌吸头吸取100ul感受态细胞DH5α(感受态细胞按照《分子克隆》(科学出版社,第三版)的方法进行)悬液于Eppendorf中,加入上述连接产物10ul,轻轻涡旋混匀,冰浴30分钟。立即转移到42℃水浴中放置2分钟,每管加入0.5ml LB培养基,37℃温箱振荡培养60分钟,吸取培养液涂布与LB培养基上(含抗生素),置37℃温箱倒置培养过夜。
1.2.4阳性重组子的筛选:各挑取上述培养过夜的平皿中的单克隆菌落,接种于5ml LB液体培养基(含抗生素)中,37℃温箱振荡培养5小时。保存各单克隆菌液并提取质粒(按照《分子克隆》(科学出版社,第三版)的方法进行)。提取后的质粒用BamHI和Xho I酶切,酶切产物用琼脂糖凝胶电泳进行鉴定。
2截短型HCV NS3重组抗原的表达与纯化
2.1表达菌株的培养:取上述阳性表达菌株单克隆菌液20ul接种于100ml LB培养基中,37℃温箱振荡培养过夜。次日,按照1%接种比例转种于LB培养基,37℃温箱振荡培养3小时,当OD600值达到0.6时,加入异丙基硫代半乳糖苷(IPTG)诱导培养,终浓度0.8mmol/L,37℃温箱振荡培养12-16小时。将菌液合并,6000rpm离心20分钟,弃上清,收集沉淀部分。
2.2提取包涵体:将沉淀称湿重,用10倍体积的20mmol/L,pH8.0Tris缓冲液将沉淀悬起,加入溶菌酶,在室温下磁力搅拌10分钟。在冰浴中超声破碎菌体。12000rpm,离心10分钟,其上清,沉淀用1mol/L NaCl洗1次,再用TE洗2次,收集沉淀。沉淀用8M尿素(用20mmol/L,pH8.0Tris配制)溶解,加入1‰DTT,于4℃,12000rpm,离心10分钟,去沉淀取上清。
2.3纯化:将上述包涵体溶解液先用亲和镍柱层析法初步纯化,初纯液过GE-Q柱阶段梯度洗脱,用不同浓度的NaCl(用平衡器配制)洗脱,收集0.25M NaCl洗脱峰,再经Sephadex G50柱脱盐,收集第一个洗脱峰。重组多型别截短型HCV NS3重组融合抗原用SDS-PAGE鉴定。
3截短型HCV NS3重组抗原活性鉴定
将得到截短型HCV NS3重组抗原多肽作活性鉴定。用pH9.6的碳酸盐缓冲液截短型HCV NS3重组多肽稀释到1.0ug/ml,包被ELISA测定板,经封闭后,对收集的100例HCV阳性血清(经HCV RNA检测确定)进行检测。结果见表1。
表1截短型HCV NS3重组多肽包被测定板检测血清结果
试剂名称 | 阳性血清(例) |
Abbott Architect i2000试剂检测 | 100 |
截短型HCV NS3重组多肽的间接法检测 | 97 |
结果表明,在100例样本中可以检出97例截短型HCV NS3重组抗体阳性标本,还有3例未检出。阳性符合率达到97%,这与之前报道的NS3抗原阳性检出率有提高。但仍有3例漏检样本,这说明我国HCV患者血清中的HCV抗体无法用单一抗原完全检出。
进一步的,我们又采集了200例非HCV疾病类交叉样本,主要包括HBsAg、抗TP、抗HIV、TORCH系列。利用截短型HCV NS3重组抗原检测板检测抗干扰血清,结果见表2。结果显示未出现假阳性病例,说明本发明抗原特异性良好。
表2截短型HCV NS3重组抗原检测板检测抗干扰血清检测结果
抗原活性鉴定结果表明,采用本发明抗原可以具有高特异性、高灵敏度特点,使用该抗原可以提高抗-HCV的检出率,这为多克隆抗体、单克隆抗体、丙型肝炎病毒检测试剂盒,尤其是丙型肝炎病毒抗体检测试剂盒的制备,提供了良好的候选抗原蛋白。
Claims (6)
1.一种截短型丙型肝炎病毒HCV NS3抗原,其特征在于:所述抗原是HCV基因组中NS3全长基因编码的第1252~1526aa片段非结构蛋白氨基酸序列,其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述截短型丙型肝炎病毒HCV NS3抗原的多核苷酸。
3.如权利要求2所述的多核苷酸,其序列如SEQ ID NO.2所示。
4.一种用于在大肠杆菌中表达权利要求1所述截短型丙型肝炎病毒HCV NS3抗原的多核苷酸,其特征在于:其序列如SEQ ID NO.3所示,其编码的氨基酸为SEQ ID NO.2所示的核苷酸序列编码的氨基酸。
5.一种截短型丙型肝炎病毒HCV NS3重组抗原蛋白的制备方法,步骤是:
(1)构建用于在大肠杆菌中表达权利要求1所述截短型丙型肝炎病毒HCV NS3抗原的多核苷酸,其序列如SEQ ID NO.3所示;
(2)将步骤(1)所述多核苷酸片段经BamHI和XhoI双酶切,酶切产物纯化回收后,与经同样限制性内切酶酶切后的表达载体pET28a连接,获得重组表达质粒;
(3)通过CaCl2法将制得的重组质粒载体转入大肠杆菌感受态细胞DH5α,涂布于含50μg/ml卡那霉素的LB琼脂培养基上,筛选得到阳性质粒;
(4)将步骤(3)中得到的阳性质粒,通过CaCl2法转入大肠杆菌E.coli BL21(DE3)感受态细胞,涂布于含卡那霉素的LB琼脂培养基上,常规培养;挑取单菌落接种至卡那霉素的LB液体培养基中,培养过夜;次日,转接于新鲜的含卡那霉素的LB液体培养基中,培养至OD600达到0.6时加入IPTG至终浓度为0.2-0.8mM,37℃诱导培养12-16h,获得含HCV NS3重组抗原蛋白的全菌液,经过亲和镍柱层析纯化和离子交换柱纯化,即得到高纯度的截短型HCV NS3重组抗原。
6.权利要求1所述截短型丙型肝炎病毒HCV NS3抗原在制备丙型肝炎病毒HCV抗体检测试剂盒中的应用。
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