CN108017694B - pORF65重组蛋白及其制备方法和应用 - Google Patents
pORF65重组蛋白及其制备方法和应用 Download PDFInfo
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- CN108017694B CN108017694B CN201810079114.8A CN201810079114A CN108017694B CN 108017694 B CN108017694 B CN 108017694B CN 201810079114 A CN201810079114 A CN 201810079114A CN 108017694 B CN108017694 B CN 108017694B
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Abstract
本发明公开了pORF65重组蛋白及其制备方法和应用，所述pORF65重组蛋白依次由SEQ ID NO.17—SEQ ID NO.21所示氨基酸序列通过连接子连接而成，所述连接子为4－7个分子量小的、极性且亲水的氨基酸构成。经ELISA显示，本发明所述pORF65重组蛋白可以作为包被抗原，能很好地用以检测CyHV‑3 pORF65完整编码序列构建的DNA疫苗免疫锦鲤后产生的血清特异性抗体，以及诊断锦鲤是否感染鲤疱疹病毒3型。
Description
技术领域
本发明涉及抗原抗体领域，具体是涉及pORF65重组蛋白及其制备方法和应用。
背景技术
鲤疱疹病毒3型(CyHV-3)又称为锦鲤疱疹病毒(KHV)，是一种双链线性DNA病毒，具有囊膜结构，自上世纪九十年代报道以来已经在世界范围内流行，该病毒引起的锦鲤疱疹病毒病(KHVD)造成巨大经济损失，严重威胁鲤、锦鲤产业安全。在CyHV-3防治研究中基于病毒囊膜蛋白的DNA疫苗成为重要的研究方向，病毒囊膜蛋白参与了病毒与宿主细胞的识别、互作、侵染等过程，可以诱导较强的体液免疫应答，是DNA疫苗研发的重要候选分子。为准确评价DNA疫苗免疫效果、制定免疫方案，需要建立高通量、特异性抗体检测方法作为CyHV-3疫苗研究的技术支撑。因此表达CyHV-3囊膜蛋白抗原与制备抗鲤血清IgM的抗体成为建立检测方法亟待解决的关键问题。
鱼类免疫球蛋白纯化及其单抗、多抗制备是免疫评价、诊断方法建立的基础。Protein A G作为抗体结合蛋白在抗体纯化中得到广泛应用。尽管鲤是一种重要的经济鱼类，但其血清IgM纯化仅见凝胶过滤层析制备的报道，亲和层析作为一种快速制备高纯度抗体的方法尚未应用于鲤血清IgM的纯化。
因此，本发明在密码子优化与预测CyHV-3 pORF65 B细胞表位优势区段的基础上，采用pET32a(+)/BL21系统重组表达pORF65 B细胞表位优势区段；采用rProtein G亲和层析纯化了鲤血清IgM，并制备了鼠抗鲤IgM多克隆抗体；在此基础上初步建立了CyHV-3特异性抗体检测的间接ELISA方法，为进一步开展CyHV-3疫苗研究提供基础。
发明内容
本发明的目的之一是提供新的pORF65重组蛋白，其可以用于检测CyHV-3 pORF65完整编码序列构建的DNA疫苗免疫锦鲤后产生的血清特异性抗体。
实现上述目的的技术方案如下。
pORF65重组蛋白，其依次由SEQ ID NO.17—SEQ ID NO.21所示氨基酸序列通过连接子连接而成，所述连接子为4－7个分子量小的、极性且亲水的氨基酸构成。
所述连接子组成为GGGGS。
一种编码上述pORF65重组蛋白的表达基因，其序列，包括a：其核苷酸序列如SEQID NO.22所示；b：与a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a的核苷酸序列不同的序列；C:对上述a或b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pORF65重组蛋白的制备方法，包括以下步骤：
包含上述编码所述pORF65重组蛋白的表达基因序列通过HindIII/XhoI双酶切插入pET32a(+)载体，构建重组质粒pET32a-mod ORF65；
再转入DH5α，PCR筛选阳性转化菌株，阳性菌株测序鉴定后提取质粒，分别转入BL21(DE3)表达菌株；
挑取转化pET32a-mod ORF65的BL21(DE3)表达菌株，接种于Amp抗性的LB培养基中培养过夜，再接种于LB新鲜培养基中，再加入IPTG诱导表达、纯化，即得。
编码所述pORF65重组蛋白的表达基因的序列如SEQ ID NO.22。
本发明的另一目的是提供一种检测锦鲤血清中针对CyHV-3 pORF65的特异性抗体的试剂盒。
具体技术方案如下：
检测锦鲤的血清特异性抗体的试剂盒，包括有所述pORF65重组蛋白。
在其中一个实施例中，所述试剂盒为ELISA试剂盒，包括有用所述pORF65重组蛋白包被的酶标板。
在其中一个实施例中，还包括有鼠抗锦鲤IgM的多克隆抗体。
本发明经过发明人截取了CyHV-3 pORF65全部蛋白序列中的抗原表位优势区段进行了原核表达，经ELISA显示，截短表达的蛋白(其优化后的编码DNA为SEQ ID NO.22)可以作为包被抗原，能很好地用以检测CyHV-3 pORF65完整编码序列构建的DNA疫苗免疫锦鲤后产生的血清特异性抗体。
附图说明
图1 pORF65序列分析结果示意图，其中，↓表示信号肽切割位点(SignalP4.1软件预测)，粗体标出跨膜序列(TMHMM软件预测)，波浪线标出DNAstar预测的B细胞表位优势区段，阴影显示ABCpred预测的B细胞表位，方框标出BepiPred预测的B细胞表位，斜体标出最终表达的B细胞表位优势区段(5’-3’依次为A、B、C、D、E)。
图2 ORF65表达结果示意图，其中，M：蛋白Marker；1：pET32a(+)质粒未诱导；2：pET32a(+)质粒诱导；3:pET32a-compORF65质粒未诱导；4：pET32a-compORF65质粒诱导5:pET32a-trunORF65质粒未诱导；6：pET32a-trunORF65质粒诱导7:pET32a-modORF65质粒未诱导；8：pET32a-modORF65质粒诱导。
图3 pORF65抗原表位优势区段融合PCR结果示意图，其中，M:DNA分子标准；1：pORF65抗原表位优势区段。
图4 pORF65Western-blot分析果示意图，其中，M:蛋白质Marker；1:pET32a-modORF65表达蛋白。
图5锦鲤血清IgM纯化结果示意图，其中，M:蛋白Marker，1：未纯化锦鲤血清，2：洗涤穿流液，3：洗脱液。
具体实施方式
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
以下实施例中所使用的试剂或原料，如无特殊说明，均来源于市售。
实施例：
1.方法
1.1引物的设计与合成
根据CyHV419株ORF65(GenBank No.KP004895)序列设计引物(见表1)。引物由上海生工生物技术有限公司合成。
表1 ORF65扩增引物序列
Table 1 Sequence of ORF65 gene amplification
1.2 ORF65基因及蛋白序列分析与密码子优化
将截去信号肽与C端跨膜疏水片段的基因编码序列送金唯智公司进行密码子优化与序列合成。
CyHV-3 419株ORF65全长1785bp，编码594个氨基酸。最终确定5个B细胞表位优势区段，分别是Phe97-Gly172(A),Ala199-Val284(B),Lys307-Ala361(C),Tyr426-Thr445(D),Thr473-Asn546(E)(参见图1)。
图1中↓表示信号肽切割位点，粗体标出跨膜序列(TMHMM软件预测)，波浪线标出DNAstar预测的B细胞表位优势区段，阴影显示ABCpred预测的B细胞表位，方框标出BepiPred预测的B细胞表位，斜体标出最终表达的B细胞表位优势区段(5’-3’依次为A、B、C、D、E),A、B、C、D、E序列之间以连接序列GGGGS连接。
A:FMPSDVQELTSFRLRIYTTSGDNFTYPTTTTGFRPIAKNELFTVGHTPATLGGGVRVKCHVNCSTGLSQFDWFRNG SEQ ID NO.17
B:ASLRSDPVWLGEPVFECPYDVNRWCPEGPITRVVDRIVKPYNEDNLRQMGNVFVCGDPTSTSSKAVMTMGRCPAKYMCSQEAHTV SEQ ID NO.18
C:KYCPNEVAFSRVTVSGVNAIATTDPGRLVVTSGVVKVFCGTEYEIYAPCANATSA SEQ IDNO.19
D:YGVGNSRTMVKTIEVPDINT SEQ ID NO.20
E:TTTTTTTTPTTLPATNATITTAITTNTTTTTTNTTTTNDTATTTTNATTYSNVTLPTTNNTNTNTSPGPASASN.SEQ ID NO.21。
1.3 pET32a-compORF65、pET32a-trunORF65重组质粒的构建与表达
以CyHV-3 HZ419株DNA为模板，用引物65Hind3CF/65XholCR及Prime Star Max高保真酶PCR扩增ORF65全基因，反应程序均采用：94℃5min预变性；94℃30s,59℃30s，72℃90s，35个循环；72℃5min总延伸，4℃保存。采用同样的方法，以金唯智公司优化密码子的合成序列为模板，用引物65Hind3TF/65XholTR扩增去掉信号肽与C端跨膜疏水区段的截短ORF65。将上述扩增产物通过HindIII/XhoI双酶切插入pET32a(+)载体，构建的重组质粒分别命名为pET32a-compORF65、pET32a-trunORF65，转入DH5α，PCR筛选阳性转化菌株，阳性菌株测序鉴定后提取质粒，分别转入BL21(DE3)表达菌株。挑取转入重组质粒的BL21(DE3)表达菌株，接种于Amp抗性的LB培养基中培养过夜，再按1:100比例接种于100mL LB新鲜培养基中，37℃，220rpm·min-1培养至菌液OD值达到0.4-0.5，分别加入0.8mmol·L-1IPTG于28℃诱导表达。参照Novagen PET表达系统手册进行蛋白表达与可溶性分析。
1.4 pET32a-modORF65重组质粒的构建与表达
1)pORF65 B细胞表位优势区段的融合PCR
根据1.2的预测结果，分别扩增5个(A、B、C、D、E)pORF65 B细胞表位优势区段。金唯智公司优化密码子的合成序列为模板，采用Prime Star Max高保真酶，分别利用引物65AFHind3/65AR、65BF/65BR、65CF/65CR、65DF/65DR、65EF/65ERXhol扩增A、B、C、D、E区段(引物序列见表1)，除65D片段外，反应程序均采用：94℃5min预变性；94℃30s,59℃30s，72℃30s，35个循环；72℃2min总延伸，4℃保存。65D片段采用94℃5min预变性；94℃30s,47.5℃30s，72℃30s，35个循环；72℃2min总延伸，PCR产物经琼脂糖凝胶电泳分析后，将扩增的片段采用切胶纯化，5个纯化片段等体积(10μL)混合，进行融合PCR扩增，反应程序为：94℃5min预变性；94℃30s,47.5℃50s，72℃90s，16个循环；72℃2min总延伸。然后以融合后的PCR产物为模板，以65AFHind3/65ERXhol为引物，进行PCR扩增，反应程序：94℃5min预变性；94℃30s,59℃30s，72℃90s，35个循环；72℃2min总延伸，PCR产物进行琼脂糖凝胶电泳分析。
2)pET32a-modORF65重组质粒的构建、表达与纯化
将上述PCR扩增产物切胶回收后通过HindIII/XhoI双酶切插入pET32a(+)载体，构建的重组质粒命名为pET32a-modORF65，后续蛋白表达与可溶性分析同1.3。选取最佳表达条件下的蛋白表达产物，采用Novagen His Bind蛋白纯化试剂盒进行纯化，纯化蛋白经BCA试剂盒定量后-80℃保存。
3)重组pORF65的Western-blot分析
上述纯化蛋白SDS-PAGE电泳后，经100mA，1.5h转至PVDF膜，5％脱脂奶粉封闭液37℃封闭2h后PBST洗膜3次，加入1:1000稀释的HRP标记的抗His标签鼠单克隆抗体37℃孵育2h，PBST洗膜5次后用Bio-Rad生物发光试剂盒检测。
1.5锦鲤血清特异性抗体检测ELISA方法的建立
1)锦鲤血清IgM的纯化
采集锦鲤血清，与4moL·L-1NaCl(pH8.3)等体积混合，取1mL加入2moL·L-1NaCl(pH8.3)平衡后的rProteinG预装柱(北京韦氏博慧色谱科技有限公司)，室温静置15min，2moL·L-1NaCl(pH8.3)洗涤10个柱床，0.05moL·L-1Gly洗脱，收集洗脱液，1:50加入Tris-HCl(pH8.0)调节pH。纯化产物进行SDS-PAGE电泳分析。纯化条带切胶后送美吉生物公司进行LC-MS/MS分析。
2)鼠抗锦鲤IgM的多克隆抗体的制备
以上述纯化的鲤IgM为抗原免疫小鼠(4只)，每只共免疫3次，每次间隔2周，第1次抗原加等体积的弗氏完全佐剂，后2次加等体积的弗氏不完全佐剂，免疫剂量为50μg·只-1，免疫途径为背部和腹部皮下多点注射。采血前3天加强免疫，用不加佐剂抗原腹腔注射，剂量为50μg·只-1。摘除眼球采血，分离血清采用rProtein A(北京韦氏博慧色谱科技有限公司)纯化小鼠IgG，-80℃保存。
3)ORF65 DNA疫苗的免疫
以CyHV-3 HZ419株DNA为模板，用引物ORF65FHind3K/ORF65REcoR1及Prime StarMax高保真酶PCR扩增ORF65全基因，反应程序均采用：94℃5min预变性；94℃30s,59℃30s，72℃90s，35个循环；72℃5min总延伸。然后将PCR扩增产物切胶回收后通过HindIII/EcoRI双酶切插入pEGFP-N1载体，构建的重组质粒命名为pEGFP-ORF65，以pEGFP-ORF65重组质粒(包含ORF65全部编码序列)作为DNA疫苗，以pEGFP-N1空白质粒作为对照，肌肉注射免疫平均体重20g的锦鲤，免疫剂量为3μg·尾-1，免疫3周后采集血清，-80℃保存。
4)间接EILSA方法检测免疫血清抗体效价
将纯化的pORF65(pET32a-modORF65)用CBS稀释至160μg·mL-1，100μL·孔-1包被酶标板，4℃过夜，PBST洗涤3次；加入封闭液(1％BSA)，37℃封闭1.5h，PBST洗涤3次；加入1:300稀释的锦鲤血清，100μL·孔-1，25℃孵育1.5h，PBST洗涤5次；加入100μL·孔-11:3000稀释鼠抗锦鲤多克隆抗体，37℃孵育2.5h，PBST洗涤5次；加入100μL·孔-11:8000稀释HRP标记羊抗小鼠IgG(北京鼎国昌盛生物技术有限责任公司)，37℃孵育2h，PBST洗涤5次；加入100μL·孔-1TMB工作液，37℃孵育30min。加入50μL·孔-1终止液，检测OD450。
2.结果
2.1 pORF65 B细胞表位优势区段
CyHV-3 419株ORF65全长1785bp，编码594个氨基酸。最终确定5个B细胞表位优势区段，分别是Phe97-Gly172(A),Ala199-Val284(B),Lys307-Ala361(C),Tyr426-Thr445(D),Thr473-Asn546(E)(参见图1)。其中，
pORF65细胞表位优势区段的密码子优化序列
TTTATGCCGAGTGATGTGCAAGAGCTGACCAGTTTTCGCCTGCGCATCTATACCACCAGTGGTGATAATTTTACCTATCCGACCACCACAACAGGCTTTCGCCCGATCGCAAAAAACGAGCTGTTTACCGTTGGCCATACCCCGGCAACACTGGGCGGTGGTGTGCGTGTGAAGTGCCATGTGAACTGCAGTACAGGCCTGAGCCAGTTCGATTGGTTCCGTAACGGCGGCGGCGGCGGCAGCGCAAGCCTGCGCAGTGATCCGGTGTGGTTAGGTGAGCCGGTGTTTGAATGTCCGTACGACGTGAACCGCTGGTGCCCTGAAGGTCCGATTACCCGTGTGGTGGACCGCATTGTGAAACCGTACAACGAAGATAACCTGCGCCAAATGGGCAACGTGTTTGTGTGCGGCGATCCTACCAGCACAAGCAGCAAGGCCGTTATGACCATGGGCCGCTGTCCTGCCAAATACATGTGCAGCCAAGAAGCTCACACAGTTGGCGGCGGCGGCAGCAAGTACTGCCCGAATGAGGTGGCCTTTAGCCGCGTGACCGTGAGCGGTGTGAACGCAATTGCCACCACAGACCCGGGCCGCCTGGTGGTTACCAGCGGCGTTGTGAAGGTTTTCTGCGGCACCGAGTATGAAATTTACGCACCGTGCGCCAATGCCACAAGCGCAGGCGGCGGCGGCAGCTACGGCGTTGGCAACAGCCGCACAATGGTTAAGACCATTGAGGTTCCGGACATCAATACCGGCGGCGGCGGCAGCACCACCACCACCACCACAACCACACCGACAACCCTGCCGGCCACCAATGCCACCATCACCACCGCAATCACAACCAACACCACCACCACCACCACAAATACCACCACCACCAATGACACCGCCACCACCACCACCAACGCAACCACCTACAGCAATGTGACCCTGCCGACCACCAATAACACCAATACCAACACAAGTCCGGGCCCTGCCAGCGCCAGCAACACAGTTACC(SEQ ID NO.22)。
2.2 pORF65的表达
pET32a-compORF65编码全长pORF65，其融合蛋白理论分子量分别为82.3kDa。经PCR鉴定、测序证实上述重组质粒读码框架正确，诱导表达后进行SDS-PAGE分析，结果显示该质粒未见明显表达(图2)，稀有密码子分析显示，CyHV-3ORF65全基因中低丰度密码子(threshold＝10)有99个，严重影响了原核表达，因此本发明构建了密码子优化的pET32a-trunORF65质粒，其编码的融合蛋白理论分子量分别为77.2kDa，该质粒也未见明显表达(图2)。
电泳及测序证实本发明所述融合PCR成功扩增了pORF65B表位优势区段的编码序列(图3)，以此为基础构建了pET32a-modORF65重组质粒，诱导表达获得预期分子量为56.4kDa的目的蛋白(图2)。可溶性分析表明，蛋白以包涵体形式表达。利用Novagen HisBind蛋白纯化试剂盒纯化目的蛋白，经抗His标签的鼠单克隆抗体Western-blot分析，在56.4kDa处杂交到明显的条带(图4)。
2.4鲤血清IgM的纯化与鼠抗鲤多克隆抗体的制备
SDS-PAGE显示在>70kDa附近出现明显条带，推测为锦鲤IgM重链(图5)，美吉公司LC-MS/MS质谱鉴定证实该蛋白为鲤血清IgM重链。以纯化的锦鲤IgM分3次免疫小鼠，加强免疫后3天，摘除眼球采血，ELISA检测表明，制备的鼠抗血清效价>1:160,000。
2.5特异性抗体检测ELISA方法的应用
以本发明制备的鼠抗锦鲤IgM多克隆抗体为检测抗体，以纯化的重组pORF65为包被抗原，建立的间接EILSA方法检测锦鲤免疫血清抗体效价，结果表明5尾免疫锦鲤血清OD450为分别为0.562、0.532、0.501、0.481、0.479，未免疫血清(阴性对照)OD450为0.231～0.21，P/N＞2.1，该方法可以评价特异性抗体水平。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 广东省农业科学院动物卫生研究所
<120> pORF65重组蛋白及其制备方法和应用
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cccaagcttg catggtctcg ccgctcg 27
<210> 2
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccgctcgagc ttgatggtcg cggcggcctt g 31
<210> 3
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cccaagcttg ccaaaccgtg gtgtatacc 29
<210> 4
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgctcgagc agggtaactg tgttgctg 28
<210> 5
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cccaagcttg ctttatgccg agtgatgtg 29
<210> 6
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gccgttacgg aaccaat 17
<210> 7
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
attggttccg taacggcggc ggcggcggca gcgcaagcct gcgcagtg 48
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aactgtgtga gcttcttg 18
<210> 9
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
caagaagctc acacagttgg cggcggcggc agcaagtact gcccgaatga g 51
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tgcgcttgtg gcattggc 18
<210> 11
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gccaatgcca caagcgcagg cggcggcggc agctacggcg ttggcaacag 50
<210> 12
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggtattgatg tccgg 15
<210> 13
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccggacatca ataccggcgg cggcggcagc accaccacca ccaccacaac cacaccg 57
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccgctcgagg gtaactgtgt tgctggc 27
<210> 15
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gggaagcttg ccaccatggt ctcgccgctc g 31
<210> 16
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccggaattcg cttgatggtc gcggcggcct tg 32
<210> 17
<211> 76
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Phe Met Pro Ser Asp Val Gln Glu Leu Thr Ser Phe Arg Leu Arg Ile
1 5 10 15
Tyr Thr Thr Ser Gly Asp Asn Phe Thr Tyr Pro Thr Thr Thr Thr Gly
20 25 30
Phe Arg Pro Ile Ala Lys Asn Glu Leu Phe Thr Val Gly His Thr Pro
35 40 45
Ala Thr Leu Gly Gly Gly Val Arg Val Lys Cys His Val Asn Cys Ser
50 55 60
Thr Gly Leu Ser Gln Phe Asp Trp Phe Arg Asn Gly
65 70 75
<210> 18
<211> 85
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Ala Ser Leu Arg Ser Asp Pro Val Trp Leu Gly Glu Pro Val Phe Glu
1 5 10 15
Cys Pro Tyr Asp Val Asn Arg Trp Cys Pro Glu Gly Pro Ile Thr Arg
20 25 30
Val Val Asp Arg Ile Val Lys Pro Tyr Asn Glu Asp Asn Leu Arg Gln
35 40 45
Met Gly Asn Val Phe Val Cys Gly Asp Pro Thr Ser Thr Ser Ser Lys
50 55 60
Ala Val Met Thr Met Gly Arg Cys Pro Ala Lys Tyr Met Cys Ser Gln
65 70 75 80
Glu Ala His Thr Val
85
<210> 19
<211> 55
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Lys Tyr Cys Pro Asn Glu Val Ala Phe Ser Arg Val Thr Val Ser Gly
1 5 10 15
Val Asn Ala Ile Ala Thr Thr Asp Pro Gly Arg Leu Val Val Thr Ser
20 25 30
Gly Val Val Lys Val Phe Cys Gly Thr Glu Tyr Glu Ile Tyr Ala Pro
35 40 45
Cys Ala Asn Ala Thr Ser Ala
50 55
<210> 20
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Tyr Gly Val Gly Asn Ser Arg Thr Met Val Lys Thr Ile Glu Val Pro
1 5 10 15
Asp Ile Asn Thr
20
<210> 21
<211> 74
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Thr Thr Thr Thr Thr Thr Thr Thr Pro Thr Thr Leu Pro Ala Thr Asn
1 5 10 15
Ala Thr Ile Thr Thr Ala Ile Thr Thr Asn Thr Thr Thr Thr Thr Thr
20 25 30
Asn Thr Thr Thr Thr Asn Asp Thr Ala Thr Thr Thr Thr Asn Ala Thr
35 40 45
Thr Tyr Ser Asn Val Thr Leu Pro Thr Thr Asn Asn Thr Asn Thr Asn
50 55 60
Thr Ser Pro Gly Pro Ala Ser Ala Ser Asn
65 70
<210> 22
<211> 999
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tttatgccga gtgatgtgca agagctgacc agttttcgcc tgcgcatcta taccaccagt 60
ggtgataatt ttacctatcc gaccaccaca acaggctttc gcccgatcgc aaaaaacgag 120
ctgtttaccg ttggccatac cccggcaaca ctgggcggtg gtgtgcgtgt gaagtgccat 180
gtgaactgca gtacaggcct gagccagttc gattggttcc gtaacggcgg cggcggcggc 240
agcgcaagcc tgcgcagtga tccggtgtgg ttaggtgagc cggtgtttga atgtccgtac 300
gacgtgaacc gctggtgccc tgaaggtccg attacccgtg tggtggaccg cattgtgaaa 360
ccgtacaacg aagataacct gcgccaaatg ggcaacgtgt ttgtgtgcgg cgatcctacc 420
agcacaagca gcaaggccgt tatgaccatg ggccgctgtc ctgccaaata catgtgcagc 480
caagaagctc acacagttgg cggcggcggc agcaagtact gcccgaatga ggtggccttt 540
agccgcgtga ccgtgagcgg tgtgaacgca attgccacca cagacccggg ccgcctggtg 600
gttaccagcg gcgttgtgaa ggttttctgc ggcaccgagt atgaaattta cgcaccgtgc 660
gccaatgcca caagcgcagg cggcggcggc agctacggcg ttggcaacag ccgcacaatg 720
gttaagacca ttgaggttcc ggacatcaat accggcggcg gcggcagcac caccaccacc 780
accacaacca caccgacaac cctgccggcc accaatgcca ccatcaccac cgcaatcaca 840
accaacacca ccaccaccac cacaaatacc accaccacca atgacaccgc caccaccacc 900
accaacgcaa ccacctacag caatgtgacc ctgccgacca ccaataacac caataccaac 960
acaagtccgg gccctgccag cgccagcaac acagttacc 999
Claims (9)
1.一种编码pORF65重组蛋白的表达基因，其特征是，其序列为a：其核苷酸序列如SEQID NO.22所示；或b：与a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a的核苷酸序列不同的序列。
2.pORF65重组蛋白，其特征是，所述pORF65重组蛋白的制备方法包括以下步骤：
包含权利要求1所述编码pORF65重组蛋白的表达基因序列通过HindIII/XhoI双酶切插入pET32a(+)载体，构建重组质粒pET32a-mod pORF65；
再转入DH5α，PCR筛选阳性转化菌株，阳性菌株测序鉴定后提取质粒，分别转入BL21(DE3)表达菌株；
挑取转化pET32a-mod pORF65的BL21(DE3)表达菌株，接种于Amp抗性的LB培养基中培养过夜，再接种于LB新鲜培养基中，再加入IPTG诱导表达、纯化，即得。
3.权利要求2所述的pORF65重组蛋白的制备方法，其特征是，包括以下步骤：
包含权利要求1所述编码pORF65重组蛋白的表达基因序列通过HindIII/XhoI双酶切插入pET32a(+)载体，构建重组质粒pET32a-mod pORF65；
再转入DH5α，PCR筛选阳性转化菌株，阳性菌株测序鉴定后提取质粒，分别转入BL21(DE3)表达菌株；
挑取转化pET32a-mod pORF65的BL21(DE3)表达菌株，接种于Amp抗性的LB培养基中培养过夜，再接种于LB新鲜培养基中，再加入IPTG诱导表达、纯化，即得。
4.根据权利要求3所述的制备方法，其特征是，所述编码所述pORF65重组蛋白的表达基因的序列如SEQ ID NO.22所示。
5.根据权利要求4所述的制备方法，其特征是，如SEQ ID NO.22所示的编码所述pORF65重组蛋白的表达基因其是通过SEQ ID NO.5和SEQ ID NO.14扩增得到的。
6.权利要求2所述的pORF65重组蛋白在制备检测锦鲤的血清特异性抗体的试剂盒中的应用。
7.检测锦鲤的血清特异性抗体的试剂盒，其特征是，包括有权利要求2所述的pORF65重组蛋白。
8.根据权利要求7所述的检测锦鲤的血清特异性抗体的试剂盒，其特征是，所述试剂盒为ELISA试剂盒，包括有用权利要求2所述pORF65重组蛋白包被的酶标板。
9.根据权利要求7或8所述的检测锦鲤的血清特异性抗体的试剂盒，其特征是，还包括有鼠抗锦鲤IgM的多克隆抗体。
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| CN107056898A (zh) * | 2017-02-13 | 2017-08-18 | 中国水产科学研究院珠江水产研究所 | 鲤疱疹病毒3型1301株orf136基因重组表达蛋白、抗体及其应用 |
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| CN107056898A (zh) * | 2017-02-13 | 2017-08-18 | 中国水产科学研究院珠江水产研究所 | 鲤疱疹病毒3型1301株orf136基因重组表达蛋白、抗体及其应用 |
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