CN106749552B - Wu多瘤病毒重组抗原及其制备方法和应用 - Google Patents
Wu多瘤病毒重组抗原及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种WU多瘤病毒重组抗原，以及WU多瘤病毒重组抗原的制备方法，该方法包括如下步骤：将WU多瘤病毒VP2核酸片段和WU多瘤病毒STAg核酸片段链接，得VP2‑STAg核酸片段，装入PGEX‑20T质粒，得PGEX‑20T‑VP2‑STAg重组质粒然后转入大肠杆菌，并诱导其表达蛋白，蛋白纯化后即得WU多瘤病毒重组抗原。本发明制备方法制得的WU多瘤病毒重组抗原具有良好的抗原性，可用于WUPyV感染患儿抗体的检测，也可用于人群WUPyV感染的流行病学调查研究。
Description
技术领域
本发明涉及生物工程技术领域，具体地涉及WU多瘤病毒重组抗原及其制备方法和应用。
背景技术
WU多瘤病毒(WU polyomavirus,WUPyV)是2007年美国和澳大利亚科学家从1个3岁肺炎患儿的呼吸道标本中发现的一种新的多瘤病毒。研究发现，WUPyV广泛分布于世界各地，在人体多种组织、体液及分泌物中检测到WUPyV的存在，大量的分子生物学和有限的血清学研究结果表明，WUPyV主要经呼吸道感染婴幼儿及年幼儿童，并在部分病例引起严重下呼吸道疾病，如重症肺炎等，由于多数研究未设立无症状对照组且WUPyV感染多呈现多种病毒混合感染的模式，其与人类呼吸道感染间的确切关系有待通过进一步研究予以明确。
1、WUPyV的分子生物学特点：
WUPyV全基因长度为5229bp(或5228bp)，是一闭合环状双链DNA分子，GC含量39％。基因组包括一个早期编码区，编码大T抗原(LTAg)、小T抗原(STAg)，在另一条链上包括一个晚期编码区，编码衣壳蛋白VP1、VP2和VP3，早期编码区和晚期编码区被非编码调控区分开(见图1)。
DNA的合成与转录均起始于调控区，呈相反的两个方向进行，分别控制早期和晚期基因的转录和DNA的复制。
Venter等研究认为WUPyV的基因变异主要在VP1区，VP2及STAg基因较少变异，调节区末端—VP2始端(287-722nt)是WUPyV最大保守区域。本课题组在华南地区率先检出WUPyV，基因组鉴定结果表明，广东株(GenBank序列号GQ926975～926980)与Gaynor、韩国、我国浙江、北京地区检测鉴定的WUPyV株具有高度同源性。
2、研究现状：
迄今为止，尚未分离出完整的WUPyV颗粒，无体内外病毒繁殖的报道，也无相应的细胞系或动物模型可用，根据基因组预测的基因编码开放阅读框能否在病毒感染目标期间进行真实表达有待证实。
绝大多数WUPyV从婴幼儿、低龄儿童呼吸道分泌物中检出，成人呼吸道中检出率非常低。免疫功能低下者体内WUPyV检出率不同报道不同。Neske等发现约89％健康献血人群体内存在WUPyV IgG抗体。
WUPyV检测目前完全依赖分子生物学方法。
WUPyV血清学研究报道较少，目前主要表达了VP1衣壳蛋白，对VP2、VP3和STAg蛋白表达的研究尚未见报道。也就是说，人类对WUPyV的认识极其有限，大多研究限于流行病学研究。已知WUPyV主要感染婴幼儿人群以及免疫功能低下人群，但是该病毒的传播途径、潜伏组织或器官、潜伏期、进入细胞的方式、人体感染病毒后的免疫反应方式、病毒的致病性以及是否象其他多瘤病毒一样引起人体肿瘤等诸多问题尚待明确，特别是其致病性，是首先需要进行明确的。
发明内容
本发明所要解决的技术问题是：提供一种具备高灵敏度和特异性的WU多瘤病毒重组抗原及其制备方法和应用。
为了解决上述技术问题，本发明采用的技术方案为：一种WU多瘤病毒重组抗原，包括SEQ ID NO.1和SEQ ID NO.2所示的氨基酸序列。
上述的WU多瘤病毒重组抗原由核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段和核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段表达得到。
本发明还提供一种WU多瘤病毒重组抗原的制备方法，包括如下步骤：
步骤1、将核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段和核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段链接，得VP2-STAg核酸片段；
步骤2、将VP2-STAg核酸片段装入PGEX-20T质粒，得PGEX-20T-VP2-STAg重组质粒；
步骤3、将PGEX-20T-VP2-STAg重组质粒转入大肠杆菌，并诱导PGEX-20T-VP2-STAg重组质粒表达蛋白，蛋白纯化后即得WU多瘤病毒重组抗原。
本发明还提供所述的WU多瘤病毒重组抗原在制备预防或治疗WU多瘤病毒感染的疫苗或药物上的应用。
本发明的有益效果在于：将WU多瘤病毒VP2核酸片段和WU多瘤病毒STAg核酸片段链接后的嵌合片段构建表达载体，表达出的重组蛋白敏感性好、特异性高，本发明制备方法制得的WU多瘤病毒重组抗原具有良好的抗原性，可用于WUPyV感染患儿抗体的检测，也可用于人群WUPyV感染的流行病学调查。
附图说明
图1为WU多瘤病毒的基因组结构模式图(Gaynor,2007)。
图2为本发明实施例WU多瘤病毒VP2核酸片段引物所得PCR产物图谱。
图3为本发明实施例WU多瘤病毒STAg核酸片段引物所得PCR产物图谱。
图4为本发明实施例PGEX-20T-VP2-STAg重组质粒的鉴定图谱。
图5为本发明实施例GST-Resin纯化柱纯化蛋白抗原SDS-PAGE、免疫印迹鉴定结果。
具体实施方式
为详细说明本发明的技术内容、所实现目的及效果，以下结合实施方式并配合附图予以说明。
本发明最关键的构思在于：将WU多瘤病毒VP2核酸片段和WU多瘤病毒STAg核酸片段链接后的嵌合片段构建表达载体，表达出的重组蛋白敏感性好、特异性高。
本发明提供一种WU多瘤病毒重组抗原，包括SEQ ID NO.1和SEQ ID NO.2所示的氨基酸序列。
上述的WU多瘤病毒重组抗原由核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段和核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段表达得到。
本发明还提供一种重组载体，包含SEQ ID NO.3和SEQ ID NO.4所示的核苷酸序列。
本发明还提供一种WU多瘤病毒重组抗原的制备方法，包括如下步骤：
步骤1、将核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段和核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段链接，得VP2-STAg核酸片段；
步骤2、将VP2-STAg核酸片段装入PGEX-20T质粒，得PGEX-20T-VP2-STAg重组质粒；
步骤3、将PGEX-20T-VP2-STAg重组质粒转入大肠杆菌，并诱导PGEX-20T-VP2-STAg重组质粒表达蛋白，蛋白纯化后即得WU多瘤病毒重组抗原。
其中，步骤1中，核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段由碱基序列如SEQ ID NO.5和SEQ ID NO.6所示的引物组制得，核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段由碱基序列如SEQ ID NO.7和SEQ ID NO.8所示的引物组制得。
上述方法具体包括如下步骤：
步骤1、将碱基序列如SEQ ID NO.5和SEQ ID NO.6所示的引物组进行PCR反应，得WU多瘤病毒VP2核酸片段；将碱基序列如SEQ ID NO.5和SEQ ID NO.6所示的引物组进行PCR反应，得WU多瘤病毒STAg核酸片段；
步骤2、利用T4DNA连接酶将步骤1所得WU多瘤病毒VP2核酸片段和WU多瘤病毒STAg核酸片段进行平端链接，链接后用碱基序列如SEQ ID NO.5和SEQ ID NO.8的引物组进行扩增，得VP2-STAg核酸片段；
步骤3、VP2-STAg核酸片段和PGEX-20T质粒分别用BamHⅠ和Xbal进行双酶切，然后利用T4DNA连接酶进行连接反应，连接产物转化、筛选后得PGEX-20T-VP2-STAg重组质粒；
步骤4、将PGEX-20T-VP2-STAg重组质粒转入E,coli菌株BL21的感受态细胞，IPTG诱导后表达重组蛋白，蛋白纯化后即得WU多瘤病毒重组抗原。
本发明还提供所述的WU多瘤病毒重组抗原在制备预防或治疗WU多瘤病毒感染的疫苗或药物上的应用。
实施例
WUPyV VP2-STAg嵌合蛋白的设计、表达及鉴定
根据本实验室样本GD-WU816克隆的WUPyV基因序列(GQ926979.1)设计VP2核酸片段，目的是要避开VP3的核酸重叠序列，增加VP2抗原的特异性；同样，设计STAg核酸片段，目的是要避开LTAg的核酸重叠序列，增加LTAg抗原的特异性。再将VP2核酸片段和STAg核酸片段链接在一起，得到VP2-STAg嵌合蛋白抗原的核酸片段序列。
1、VP2核酸片段引物：(GQ926979.0序列，574-1002nt)429bp
F(SEQ ID NO.5，含有BamHⅠ切点)：
5’-CGCGGATCCCGCATGGGCATATTGCTTGCTGTGCCTGAA-3’；
R(SEQ ID NO.6)：
5’-TCCTGGGTAGGGGGCGTGGAGG-3’
VP2核酸片段的核苷酸序列(SEQ ID NO.3)：
atgggca tattgcttgc tgtgcctgaa ataattgctg catctgtagc tggaggagcagaggcactat caattgctgg atctggagct gcaatagcaa ctggtgaagg tttagctgct cttggtgggcttacagagtc agcagcacta ttaggggaaa ctgttgaaat atctgaagca gctgctactg tactaacaaaagtacctgag cttgtaactg taacacaagg tgtaacagca gctgtacaag ggggtgcagg tcttgtaggtggtatatata cagctttagc agcagatcgc cctggggacc tgcctgcgag taccccaaca ggaagtccaagtggactaca tccccccgca ggatacaatc cccaaggagg tggacttaat atccagtcca tccacaagcccctccacgcc ccctacccag ga
VP2核酸片段所得氨基酸序列(SEQ ID NO.1，143AA)：
MGILLAVPEI IAASVAGGAE ALSIAGSGAA IATGEGLAAL GGLTESAALL GETVEISEAAATVLTKVPEL VTVTQGVTAA VQGGAGLVGG IYTALAADRP GDLPASTPTG SPSGLHPPAG YNPQGGGLNIQSIHKPLHAP YPG
分子量：13564.41Daltons
PCR反应体系及产物获得：PCR反应的总体积为25μl，其中模板1μl，上下游引物各3pmol，10mmol/L dNTP混合物0.5μl，10×High Fidelity PCR buffer with MgCl2 2.5μl，高保真PCR酶混合物0.15μl，用无菌水补足至25μl。PCR反应参数均为94℃预变性3min；94℃变性1min，55℃退火1min，72℃延伸90sec，共循环30次；最后72℃延伸10min。PCR产物用1％琼脂糖凝胶电泳，紫外灯下迅速切下目的产物片段，将凝胶切碎，用AxyPrep DNA凝胶回收试剂盒回收DNA，按照说明书进行操作，方法同前，用30μl灭菌去离子水洗脱，-20℃保存备用。PCR产物图谱见图2。PCR产物经商业化序列测定正确。
2、STAg核酸片段引物：(GQ926979.0序列，4573-5157nt)585bp
F(SEQ ID NO.7)：
5’-ATGGATAAAACTTTGTCCAGAAATGAA-3’
R(SEQ ID NO.8，含有Xbal切点)：
5’-CTAGGTCTAGACTATTACCTGGTTAAGCCAACCCC-3’
STAg核酸片段的核苷酸序列(SEQ ID NO.4，585bp)：
atggataaaactttgtccagaaatgaagcaaaagaacttatgcagctgctgggtcttgatatgacctgctggggaaatttaccactaatgagaacaaaataccttagcaaatgcaaagaatttcatcctgacaaagggggaaatgaggaaaaaatgaaaaagcttaattctttatatttaaaactgcaagagtgtgttagtacagtgcaccaactaaatgaagaagaagatgaagtgtggagctcttcacaggtagaatgcacagaattgtgctgtaactttccccctagaaagtacaggcttgttggagaagtttatggtgatgtttttgaagagtatattttaaaagactgggacatatgcttaaaggggttttattatttgtgtaattgtttttactgctttttagacaagcgccacaagcaaaaatataaaatatttagaaaacctccaatgtggatagagtgttactgctacaggtgctatagagagtggtttggctttgaaattagtgcagaaacatttttttactggaaaaagattatatttcttacaaccatgcaaggggttggcttaaccaggtaa
STAg核酸片段所得氨基酸序列(SEQ ID NO.2，195AA)：
MDKTLSRNEAKELMQLLGLDMTCWGNLPLMRTKYLSKCKEFHPDKGGNEEKMKKLNSLYLKLQECVSTVHQLNEEEDEVWSSSQVECTELCCNFPPRKYRLVGEVYGDVFEEYILKDWDICLKGFYYLCNCFYCFLDKRHKQKYKIFRKPPMWIECYCYRCYREWFGFEISAETFFYWKKIIFLTTMQGVGLTR
分子量：23466.41Daltons
PCR反应体系及产物获得：PCR反应的总体积为25μl，其中模板1μl，上下游引物各3pmol，10mmol/L dNTP混合物0.5μl，10×High Fidelity PCR buffer with MgCl2 2.5μl，高保真PCR酶混合物0.15μl，用无菌水补足至25μl。PCR反应参数均为94℃预变性3min；94℃变性1min，55℃退火1min，72℃延伸90sec，共循环30次；最后72℃延伸10min。PCR产物用1％琼脂糖凝胶电泳，紫外灯下迅速切下目的产物片段，将凝胶切碎，用AxyPrep DNA凝胶回收试剂盒回收DNA，按照说明书进行操作，方法同前，用30μl灭菌去离子水洗脱，-20℃保存备用。PCR产物图谱见图3。PCR产物经商业化序列测定正确。
3、VP2核酸片段与STAg核酸片段的链接及表达载体构建：
利用T4DNA连接酶进行常规平端链接，按试剂盒说明书操作。链接后用VP2上游引物(含有BamHⅠ切点)和STAg下游引物(含有Xbal切点)进行VP2-STAg核酸片段扩增，得到1101bp嵌合片段。双酶切后常规方法装入PGEX-20T质粒。
重组质粒的获得
(1)PCR产物和PGEX-20T质粒酶切
目的DNA VP2-STAg核酸片段及质粒DNA分别用BamHⅠ和Xbal进行双酶切，反应体系20μl：10×Buffer MC(MVLTI-CORETM Buffer)2μl，Acetylated BSA(10μg/μl)0.2μl，PCRDNA/质粒DNA 5μl，BamHⅠ0.3μl，Xbal 0.3μl，加入灭菌去离子水12.2μl，37℃，4.5h。反应结束后进行1％的琼脂糖凝胶电泳，紫外灯下观察酶切结果并切下双酶切的条带，用AxyPrepDNA凝胶回收试剂盒回收DNA，方法同前。
(2)PCR产物和质粒DNA连接
利用T4DNA连接酶进行连接反应，VP2-STAg DNA和质粒DNA按照10：1～3：1的比例混合，反应体系10μl：质粒DNA 2.5μl，VP2-STAg DNA 6.0μl，10×T4 DNA Ligase Buffer1.0μl，T4 DNA Ligase 0.5μl，16℃连接过夜。连接物存于-20℃备用。
(3)转化
取5μl连接产物加入冰上预冷的TOP10感受态细胞中，充分混匀，冰上30min，同时用质粒DNA加入感受态细胞中作为阳性对照，感受态细胞作阴性对照。42℃热休克90sec，取出后迅速冰浴2min，加入LB培养液300μl，37℃振荡培养1h。将转化混合物平铺于含100μg/ml氨苄青霉素的LB培养板上，37℃培养过夜。
(4)重组质粒的筛选、检测
挑取LB-氨苄青霉素筛选平板上的单克隆菌落，接种于3ml含100μg/ml氨苄青霉素的LB培养液中，37℃振荡培养过夜，用天根的质粒小提试剂盒说明书进行操作，提取质粒，用60μl灭菌去离子水洗脱，利用BamHⅠ和Xbal内切酶酶切检测，筛选重组质粒。构建的PGEX-20T-VP2-STAg表达质粒酶切鉴定如图4。
4、PGEX-20T-VP2-STAg抗原表达及纯化：
常规方法将PGEX-20T-VP2-STAg重组质粒转入E,coli菌株BL21(DE3)的感受态细胞，IPTG诱导后表达GST-VP2-STAg重组抗原，分子量约为63.5KD(GST约为26.44KD)。
(1)重组蛋白的诱导表达
取1μl筛选阳性的重组质粒加入预冷的25μl表达菌株BL21(DE3)的感受态细胞，混合均匀后冰浴30min，然后42℃热休克90sec，迅速冰上静置2min，加入300μl LB培养液，37℃振荡1h，将转化混合物平铺于含有100μg/ml氨苄青霉素的LB培养板上，37℃培养过夜。挑取3-4个克隆菌落于5ml含有100μg/ml氨苄青霉素的LB培养液，37℃，振荡培养3h，测定OD600在0.6左右，取出2ml菌液作为诱导菌的对照，37℃振摇；取出2ml菌液于灭菌试管中加入2μl 1mol/L IPTG(IPTG终浓度1mmol/L)，37℃振摇3h，离心收菌，弃尽液体，向沉淀中加入50μl无菌去离子水混匀，加入50μl 2×Loading Buffer混匀，沸水中煮5min，12000g离心10min；取10μl上清进行SDS-PAGE(10％分离胶，6％浓缩胶)。考马斯亮兰R-250染色30min；脱色液脱色至背景清晰，利用计算机凝胶扫描仪系统扫描成像。
(2)Western-Blot检测表达产物
将诱导的全菌进行SDS-PAGE；按照Bio-Rad公司的产品说明书，使凝胶靠近电极阴极一侧，聚偏氟乙烯(PVDF)膜靠近阳极一侧，转移缓冲液为480mmol/L Tris-390mmol/L-1mmol/LSDS-甘氨酸-20％甲醇，100mA电转移1h，使蛋白由凝胶转移至PVDF膜，电转结束后，取出PVDF膜用3％脱脂奶室温摇床封闭2h，以封闭非特异性结合位点，加入鼠抗GST单克隆抗体(5μl/ml)，于摇床室温反应1h，用洗涤液室温摇床洗膜4次，每次5min，加入3％脱脂奶稀释的羊抗鼠IgG(Sigma公司生产)(按照1：5000稀释)于摇床室温反应1h，同前洗膜，加入北京宜安泰医疗技术公司生产的试剂2和过氧化脲(100：10)，室温摇床反应5min，用蒸馏水冲洗终止显色反应。经GST-Resin纯化柱纯化蛋白抗原SDS-PAGE、免疫印迹鉴定结果如图5。
5、VP2-STAg重组抗原的初步应用：
将纯化的WUPyV VP2-STAg重组抗原包被板条，采用常规ELISA实验，检测WUPyV感染阳性(PCR核酸检测阳性)患者的血清标本，以及健康的儿童对照血清标本和成人对照血清标本。与VP2、STAg抗原比对结果见表1：WUPyV VP2-STAg、VP2、STAg重组蛋白的IgG抗体检测比对结果。
表1
数据显示，VP2-STAg重组蛋白具有良好的抗原性，可用于WUPyV感染患儿IgG抗体的检测，检出率为34.62％，儿童对照组IgG抗体检出率为52.50％，随着年龄的增长WUPyVIgG抗体检出率成人可达70％以上。提示WUPyV抗体可能具有保护功能，只有当婴幼儿初次感染WUPyV时容易患病。VP2-STAg重组蛋白的敏感性和特异性均高于单独使用VP2、STAg重组蛋白的检测，
综上所述，本发明制备方法制得的WU多瘤病毒重组抗原具有良好的抗原性，可用于WUPyV感染患儿IgG抗体的检测，检出率为34.62％，儿童IgG抗体检出率为52.50％，随着年龄的增长WUPyV IgG抗体检出率成人可达70％以上。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。
SEQUENCE LISTING
<110> 深圳市福田区人民医院
<120> WU多瘤病毒重组抗原及其制备方法和应用
<130> 2016
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 143
<212> PRT
<213> 人工序列
<400> 1
Met Gly Ile Leu Leu Ala Val Pro Glu Ile Ile Ala Ala Ser Val Ala
1 5 10 15
Gly Gly Ala Glu Ala Leu Ser Ile Ala Gly Ser Gly Ala Ala Ile Ala
20 25 30
Thr Gly Glu Gly Leu Ala Ala Leu Gly Gly Leu Thr Glu Ser Ala Ala
35 40 45
Leu Leu Gly Glu Thr Val Glu Ile Ser Glu Ala Ala Ala Thr Val Leu
50 55 60
Thr Lys Val Pro Glu Leu Val Thr Val Thr Gln Gly Val Thr Ala Ala
65 70 75 80
Val Gln Gly Gly Ala Gly Leu Val Gly Gly Ile Tyr Thr Ala Leu Ala
85 90 95
Ala Asp Arg Pro Gly Asp Leu Pro Ala Ser Thr Pro Thr Gly Ser Pro
100 105 110
Ser Gly Leu His Pro Pro Ala Gly Tyr Asn Pro Gln Gly Gly Gly Leu
115 120 125
Asn Ile Gln Ser Ile His Lys Pro Leu His Ala Pro Tyr Pro Gly
130 135 140
<210> 2
<211> 194
<212> PRT
<213> 人工序列
<400> 2
Met Asp Lys Thr Leu Ser Arg Asn Glu Ala Lys Glu Leu Met Gln Leu
1 5 10 15
Leu Gly Leu Asp Met Thr Cys Trp Gly Asn Leu Pro Leu Met Arg Thr
20 25 30
Lys Tyr Leu Ser Lys Cys Lys Glu Phe His Pro Asp Lys Gly Gly Asn
35 40 45
Glu Glu Lys Met Lys Lys Leu Asn Ser Leu Tyr Leu Lys Leu Gln Glu
50 55 60
Cys Val Ser Thr Val His Gln Leu Asn Glu Glu Glu Asp Glu Val Trp
65 70 75 80
Ser Ser Ser Gln Val Glu Cys Thr Glu Leu Cys Cys Asn Phe Pro Pro
85 90 95
Arg Lys Tyr Arg Leu Val Gly Glu Val Tyr Gly Asp Val Phe Glu Glu
100 105 110
Tyr Ile Leu Lys Asp Trp Asp Ile Cys Leu Lys Gly Phe Tyr Tyr Leu
115 120 125
Cys Asn Cys Phe Tyr Cys Phe Leu Asp Lys Arg His Lys Gln Lys Tyr
130 135 140
Lys Ile Phe Arg Lys Pro Pro Met Trp Ile Glu Cys Tyr Cys Tyr Arg
145 150 155 160
Cys Tyr Arg Glu Trp Phe Gly Phe Glu Ile Ser Ala Glu Thr Phe Phe
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Tyr Trp Lys Lys Ile Ile Phe Leu Thr Thr Met Gln Gly Val Gly Leu
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Thr Arg
<210> 3
<211> 429
<212> DNA
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atgggcatat tgcttgctgt gcctgaaata attgctgcat ctgtagctgg aggagcagag 60
gcactatcaa ttgctggatc tggagctgca atagcaactg gtgaaggttt agctgctctt 120
ggtgggctta cagagtcagc agcactatta ggggaaactg ttgaaatatc tgaagcagct 180
gctactgtac taacaaaagt acctgagctt gtaactgtaa cacaaggtgt aacagcagct 240
gtacaagggg gtgcaggtct tgtaggtggt atatatacag ctttagcagc agatcgccct 300
ggggacctgc ctgcgagtac cccaacagga agtccaagtg gactacatcc ccccgcagga 360
tacaatcccc aaggaggtgg acttaatatc cagtccatcc acaagcccct ccacgccccc 420
tacccagga 429
<210> 4
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atggataaaa ctttgtccag aaatgaagca aaagaactta tgcagctgct gggtcttgat 60
atgacctgct ggggaaattt accactaatg agaacaaaat accttagcaa atgcaaagaa 120
tttcatcctg acaaaggggg aaatgaggaa aaaatgaaaa agcttaattc tttatattta 180
aaactgcaag agtgtgttag tacagtgcac caactaaatg aagaagaaga tgaagtgtgg 240
agctcttcac aggtagaatg cacagaattg tgctgtaact ttccccctag aaagtacagg 300
cttgttggag aagtttatgg tgatgttttt gaagagtata ttttaaaaga ctgggacata 360
tgcttaaagg ggttttatta tttgtgtaat tgtttttact gctttttaga caagcgccac 420
aagcaaaaat ataaaatatt tagaaaacct ccaatgtgga tagagtgtta ctgctacagg 480
tgctatagag agtggtttgg ctttgaaatt agtgcagaaa cattttttta ctggaaaaag 540
attatatttc ttacaaccat gcaaggggtt ggcttaacca ggtaa 585
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cgcggatccc gcatgggcat attgcttgct gtgcctgaa 39
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tcctgggtag ggggcgtgga gg 22
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atggataaaa ctttgtccag aaatgaa 27
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<212> DNA
<213> 人工序列
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ctaggtctag actattacct ggttaagcca acccc 35
Claims (6)
1.一种WU多瘤病毒重组抗原，其特征在于：由SEQ ID NO.1和SEQ ID NO.2所示的氨基酸序列顺序连接得到。
2.根据权利要求1所述的WU多瘤病毒重组抗原，其特征在于：由核苷酸序列如SEQ IDNO.3所示的核酸片段和核苷酸序列如SEQ ID NO.4所示的核酸片段表达得到。
3.一种WU多瘤病毒重组抗原的制备方法，其特征在于，包括如下步骤：
步骤1、将核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段和核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段链接，得VP2-STAg核酸片段；
步骤2、将VP2-STAg核酸片段装入PGEX-20T质粒，得PGEX-20T-VP2-STAg重组质粒；
步骤3、将PGEX-20T-VP2-STAg重组质粒转入大肠杆菌，并诱导PGEX-20T-VP2-STAg重组质粒表达蛋白，蛋白纯化后即得WU多瘤病毒重组抗原。
4.根据权利要求3所述的WU多瘤病毒重组抗原的制备方法，其特征在于，步骤1中，核苷酸序列如SEQ ID NO.3所示的WU多瘤病毒VP2核酸片段由碱基序列如SEQ ID NO.5和SEQ IDNO.6所示的引物组制得，核苷酸序列如SEQ ID NO.4所示的WU多瘤病毒STAg核酸片段由碱基序列如SEQ ID NO.7和SEQ ID NO.8所示的引物组制得。
5.根据权利要求3所述的WU多瘤病毒重组抗原的制备方法，其特征在于，包括如下步骤：
步骤1、将碱基序列如SEQ ID NO.5和SEQ ID NO.6所示的引物组进行PCR反应，得WU多瘤病毒VP2核酸片段；将碱基序列如SEQ ID NO.7和SEQ ID NO.8所示的引物组进行PCR反应，得WU多瘤病毒STAg核酸片段；
步骤2、利用T4DNA连接酶将步骤1所得WU多瘤病毒VP2核酸片段和WU多瘤病毒STAg核酸片段进行平端链接，链接后用碱基序列如SEQ ID NO.5和SEQ ID NO.8的引物组进行扩增，得VP2-STAg核酸片段；
步骤3、VP2-STAg核酸片段和PGEX-20T质粒分别用BamHⅠ和Xbal进行双酶切，然后利用T4DNA连接酶进行连接反应，连接产物转化、筛选后得PGEX-20T-VP2-STAg重组质粒；
步骤4、将PGEX-20T-VP2-STAg重组质粒转入E,coli菌株BL21的感受态细胞，IPTG诱导后表达重组蛋白，蛋白纯化后即得WU多瘤病毒重组抗原。
6.权利要求1～5任一项所述的WU多瘤病毒重组抗原的应用，其特征在于：用于制备预防或治疗WU多瘤病毒感染的疫苗或药物。
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