CN108047317A - pORF148重组蛋白及其制备方法和应用 - Google Patents
pORF148重组蛋白及其制备方法和应用 Download PDFInfo
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- CN108047317A CN108047317A CN201810128920.XA CN201810128920A CN108047317A CN 108047317 A CN108047317 A CN 108047317A CN 201810128920 A CN201810128920 A CN 201810128920A CN 108047317 A CN108047317 A CN 108047317A
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Abstract
本发明公开了pORF148重组蛋白及其制备方法和应用,所述pORF148重组蛋白依次由SEQ ID NO.13—SEQ ID NO.16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pORF148重组蛋白可以作为包被抗原,能很好地用以检测CyHV‑3pORF148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。
Description
技术领域
本发明涉及抗原抗体领域,具体是涉及pORF148重组蛋白及其制备方法和应用。
背景技术
鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)又称锦鲤疱疹病毒(KoiHerpesvirus,KHV),属于疱疹病毒目(Herpesvirales),异疱疹病毒科(Alloherpesviridae),鲤疱疹病毒属(Cyprinivirus),是一种双链线性DNA病毒,具有囊膜结构。该病毒严重威胁鲤(Cyprinuscarpio)及其观赏品种:锦鲤,致死率高达80%-100%。鲤疱疹病毒3型基因组约295kb,编码156个不同的ORF(Aoki等,2007),目前已经确定其中的14个ORF编码囊膜蛋白(Michel等,2010;Yi等,2014)。囊膜蛋白位于病毒最外层,在病毒与宿主细胞的识别、互作以及入侵过程中发挥着重要作用,也是建立检测方法和防治手段的重要靶分子(Yi等,2014;Fuchs等,2014)。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。
发明内容
本发明的目的之一是提供新的pORF148重组蛋白,其可以用于检测CyHV-3pORF148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。
pORF148重组蛋白,其依次由SEQ ID NO.13—SEQ ID NO.16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pORF148重组蛋白的表达基因,其序列,包括a:其核苷酸序列如SEQID NO.17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pORF148重组蛋白的制备方法,包括以下步骤:
包含上述编码所述pORF148重组蛋白的表达基因序列通过HindIII/XhoI双酶切***pET32a(+)载体,构建重组质粒pET32a-mod pORF148;
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 pORF148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pORF148重组蛋白的表达基因的序列如SEQ ID NO.17。
本发明的另一目的是提供一种检测锦鲤血清中针对CyHV-3 ORF148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pORF148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pORF148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。
本发明经过对CyHV-3的大量研究的基础上,发明人截取了CyHV-3 pORF148全部蛋白序列中的抗原表位优势区段进行了原核表达。表达效果良好。进一步经ELISA显示,该截短表达的蛋白(其优化后的编码DNA为SEQ ID NO.17)可以作为包被抗原,能很好地用以检测CyHV-3 ORF148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 pORF148序列分析:其中,箭头表示信号肽切割位点(SignalP4.1软件预测),粗斜体标出跨膜序列(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,粗体标出最终预测的B细胞表位优势区段。
图2融合PCR电泳图:其中,M:Marker,1、2:mORF148融合PCR扩增。
图3 pORF148的重组表达:其中,M:蛋白分子量标准,1:pet32a(+)未诱导,2:pet32a(+)诱导,3:pet32a(+)-compORF148未诱导,4:pet32a(+)-compORF148诱导,5:pet32a(+)-tORF148未诱导,6:pet32a(+)-tORF148诱导,7:pet32a(+)-mORF148未诱导,8:pet32a(+)-mORF148诱导。
图4重组表达pORF148的Western blot鉴定:其中,M:蛋白分子量标准,1:pet32a(+)-mORF148诱导。
图5锦鲤血清IgM纯化: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:
一、材料与方法
1.CyHV-3ORF148全序列原核诱导表达
对ORF148基因(GenBank No.KP004903)进行分析,设计引物(表1)。以CyHV-3-HZ419 DNA为模板,采用Prime Star Max高保真酶,以引物148Hind3CF/148XholCR(表1)扩增ORF148完整编码序列。反应程序采用:94℃5min预变性;94℃30s,59℃30s,72℃90s,35个循环;72℃5min总延伸,4℃保存。
表1 ORF148扩增引物序列(5’-3’)
Table 1 Sequence of ORF148gene amplification
将上述扩增产物通过HindIII/XhoI双酶切***pET32a(+)载体,构建的重组质粒命名为pET32a-compORF148,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,转入BL21(DE3)表达菌株。挑取转化菌株,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100mLLB新鲜培养基中,37℃,220rpm/min培养至菌液OD值达到0.4-0.5,分别加入0.8mM IPTG于28℃诱导表达。
2.pORF148序列分析、密码子优化及重组质粒的构建与表达
对ORF148基因及蛋白序列(GenBank No.KP004903、AJK93609)进行分析,预测抗原表位优势区段。最终,根据发明人的经验,经过多次评估,确定抗原片段(SEQ ID NO.13至SEQ ID NO.16),
以金唯智合成的密码子改造优化的tORF148为模板,以引物148TXho1R/148THind3F(表2)扩增优化的tORF148(SEQ ID NO.17)。扩增条件、载体构建、诱导表达方法同全序列原核诱导表达方法,所构建的重组质粒分别命名为pET32a-tORF148。
表2 ORF148t扩增引物序列(5’-3’)
Table 2Sequence ofORF148t gene amplification(5’-3’)
3.pORF148B细胞表位优势区段融合扩增、重组质粒的构建与表达
3.1.pORF148B细胞表位优势区段融合扩增
根据2中的B细胞表位优势区段预测结果设计引物,将几个B细胞表位优势区段采用Gly-Gly-Gly-Gly-Ser(G4S)柔性区段连接,以金唯智合成的密码子改造优化的tORF148为模板,利用融合PCR技术进行融合扩增,所用引物见表3,反应程序为:94℃5min预变性;94℃30s,59℃30s,72℃90s,35个循环;72℃2min总延伸,4℃保存。获得B细胞表位融合区段mORF148。
表3 ORF148m扩增引物序列(5’-3’)
Table 3Sequence of ORF148m gene amplification
3.2.pORF148B细胞表位优势区段重组质粒的构建与表达
载体构建、诱导表达方法同全序列原核诱导表达方法,所构建的重组质粒命名为
pET32a-mORF148。获得阳性表达产物后,参照Novagen PET表达***手册进行蛋白表达与可溶性分析。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。
4.重组pORF148m的Western-blot分析
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。
5.锦鲤血清特异性抗体检测ELISA方法的建立
5.1.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。
5.2.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。
6.ORF148DNA疫苗的免疫
参照刘振兴(2015)的方法,将ORF148完整编码序列扩增后利用BglⅡ、HindⅢ酶切位点***pEGFP-N1载体,构建的重组质粒pEGFP-ORF148作为DNA疫苗肌肉注射免疫平均体重20g的锦鲤,免疫剂量为3μg/尾,免疫3周后采集血清,-80℃保存。
7.间接EILSA方法检测免疫血清抗体效价
纯化的pORF148m(pET32a-mORF148)用CBS稀释至320μg/mL,100μL/孔包被酶标板,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,25℃孵育1.5h,PBST洗涤5次;加入100μL/孔1:3000稀释鼠抗锦鲤多克隆抗体(4.2制备),37℃孵育2.5h,PBST洗涤5次;加入100μL/孔1:8000稀释HRP标记羊抗小鼠IgG,37℃孵育2h,PBST洗涤5次;加入100μL/孔TMB工作液,37℃孵育30min。加入50μL/孔终止液,检测OD450。
二、结果分析
1.CyHV-3 ORF148全序列原核诱导表达
PAGE电泳结果表明,在本试验所选择的各温度、IPTG浓度、以及诱导时间内,都没有获得CyHV-3ORF148表达产物。
2.pORF148序列分析、密码子优化及重组质粒的构建与表达
CyHV-3 HZ419株ORF148完整编码序列全长1803bp,编码蛋白包含601个氨基酸。该基因包含1个信号肽切割位点以及1个跨膜区段(Ile561-Leu591)。稀有密码子分析显示,CyHV-3 ORF148完整编码序列中低丰度密码子(threshold=10)有134个,为获得表达产物,我们将这些低丰度密码子全部替换为大肠杆菌偏爱密码子,并将信号肽、跨膜区、以及跨膜区紧邻的疏水区去掉后的序列送基因公司合成片段后进行载体构建以及诱导表达。结果显示,进行密码子改造、以及去掉信号肽、跨膜区及疏水结构的后,仍然不能获得表达产物。
3.pORF148序列分析与B细胞表位优势区段预测
3.1.pORF148B细胞表位优势区段融合扩增
CyHV-3 HZ419株ORF148完整编码序列全长1803bp,编码蛋白包含601个氨基酸,最终确定pORF148的4个B细胞表位优势区段:Asp157-Vel205,Cys220-Thr241,Vel262-Glu353,Vel412-Ser544(图1)。经融合PCR扩增后,我们获得了4个B细胞表位优势区段串联的片段ORF148m(图2)。
DIGASAYTWYRNGVFADTTSTNEYITVVAGRYTCEPTGSTGTLSDPVWV SEQ ID NO.13
GGGGS
CPNGAITRIHEATSKTYTDIHT SEQ ID NO.14
GGGGS
VSTRGNCPVKLSACNNNEQLTCANQAPIPVLNTCNRVYSYFCPDHYYFSYTAVNSTGASLGLTPVDANEPRRIEMPGTNGTVYRIYCGNDFE SEQ ID NO.15
GGGGS
VKRSAPANPLLVSTGTSELIKVAELSDFTDWECAVFTNPGDSQGIRTRLFNVTQLTTTTATTLTTPSTPPTTPTVITPPTNQSITPTPPTTPTTTPTTPNITTPTTPSTPSTTTPTTPSTPTSTSTSTSTSTS SEQ ID NO.16
pORF148B细胞表位优势区段的密码子优化序列
GATATCGGCGCCAGCGCATATACCTGGTACCGCAATGGCGTGTTCGCCGACACCACCAGCACAAACGAGTATATCACCGTGGTGGCCGGTCGCTATACCTGCGAGCCGACAGGCAGCACCGGTACACTGAGTGATCCTGTGTGGGTTGGCGGCGGCGGCAGCTGCCCGAACGGTGCAATCACCCGTATTCACGAAGCCACCAGCAAAACCTACACCGACATCCATACCGGCGGCGGCGGCAGCGTGAGTACCCGTGGTAACTGCCCGGTGAAACTGAGCGCCTGCAACAACAACGAACAGCTGACCTGCGCCAATCAGGCCCCGATTCCGGTGCTGAATACCTGCAACCGTGTGTACAGCTATTTCTGCCCGGATCACTATTATTTTAGCTACACCGCAGTTAATAGCACCGGCGCCAGCCTGGGTCTGACCCCGGTTGATGCAAATGAGCCGCGTCGCATTGAGATGCCGGGCACCAACGGCACCGTGTATCGTATCTACTGCGGCAACGATTTTGAGGGCGGCGGCGGCAGCGTGAAACGTAGCGCACCGGCCAATCCGCTGCTGGTTAGCACAGGCACCAGCGAACTGATCAAAGTGGCCGAACTGAGTGACTTCACCGATTGGGAATGCGCAGTGTTCACAAACCCGGGCGATAGCCAGGGCATTCGTACCCGCCTGTTCAACGTGACCCAGCTGACCACAACCACAGCCACCACCCTGACCACACCTAGTACCCCGCCGACAACCCCGACCGTGATTACCCCGCCGACCAACCAAAGTATCACCCCGACACCGCCGACCACACCTACCACCACCCCGACCACACCGAATATCACAACACCGACCACCCCGAGTACCCCGAGTACCACCACCCCGACCACACCGAGCACCCCGACCAGTACCAGTACCAGTACCAGCACCAGCACCAGC SEQ ID NO.17。
3.2.pORF148B细胞表位优势区段的表达
pet32a(+)-compORF148、pet32a(+)-tORF148与pet32a(+)-mORF148表达的融合蛋白理论分子量分别为83.5kda、75.6kDa,52.1kDa,采用0.8mM IPTG,28℃诱导4h仅pet32a(+)-modORF148在目的条带附近可见明显表达条带(图3),可溶性分析表明,蛋白以包涵体形式表达。利用Novagen His Bind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52.1kD处杂交到明显的条带(图4)。
3.重组pORF148在ELISA方法中的应用
3.1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。
3.2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pORF148为包被抗原,按照常规方法,建立间接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0.415、0.439、0.466、0.423、0.419,未免疫血清(阴性对照)OD450为0..175,P/N>2.1。重复三次以上,检测结果稳定,该方法可以评价锦鲤特异性抗体水平。
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> pORF148重组蛋白及其制备方法和应用
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cccaagcttg catgataggg tccacgccgc tcctt 35
<210> 2
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccgctcgagt ttgaagttct tgtagggcac g 31
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cccaagcttg ccaagtgaca tatagccgtg 30
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgctcgagt ttaaaatttt tatacgg 27
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cccaagcttg cgatatcggc gccagcgc 28
<210> 6
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aacccacaca ggatcac 17
<210> 7
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtgatcctgt gtgggttggc ggcggcggca gctgcccgaa cggtgcaat 49
<210> 8
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggtatggatg tcggtgt 17
<210> 9
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acaccgacat ccataccggc ggcggcggca gcgtgagtac ccgtggtaac 50
<210> 10
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctcaaaatcg ttgccgc 17
<210> 11
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gcggcaacga ttttgagggc ggcggcggca gcgtgaaacg tagcgcacc 49
<210> 12
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccgctcgagg ctggtgctgg tgctggtact ggtactggta ctggt 45
<210> 13
<211> 49
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Ile Gly Ala Ser Ala Tyr Thr Trp Tyr Arg Asn Gly Val Phe Ala
1 5 10 15
Asp Thr Thr Ser Thr Asn Glu Tyr Ile Thr Val Val Ala Gly Arg Tyr
20 25 30
Thr Cys Glu Pro Thr Gly Ser Thr Gly Thr Leu Ser Asp Pro Val Trp
35 40 45
Val
<210> 14
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Cys Pro Asn Gly Ala Ile Thr Arg Ile His Glu Ala Thr Ser Lys Thr
1 5 10 15
Tyr Thr Asp Ile His Thr
20
<210> 15
<211> 92
<212> PRT
<213> 人工序列(Artificial Sequence)
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Val Ser Thr Arg Gly Asn Cys Pro Val Lys Leu Ser Ala Cys Asn Asn
1 5 10 15
Asn Glu Gln Leu Thr Cys Ala Asn Gln Ala Pro Ile Pro Val Leu Asn
20 25 30
Thr Cys Asn Arg Val Tyr Ser Tyr Phe Cys Pro Asp His Tyr Tyr Phe
35 40 45
Ser Tyr Thr Ala Val Asn Ser Thr Gly Ala Ser Leu Gly Leu Thr Pro
50 55 60
Val Asp Ala Asn Glu Pro Arg Arg Ile Glu Met Pro Gly Thr Asn Gly
65 70 75 80
Thr Val Tyr Arg Ile Tyr Cys Gly Asn Asp Phe Glu
85 90
<210> 16
<211> 133
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Val Lys Arg Ser Ala Pro Ala Asn Pro Leu Leu Val Ser Thr Gly Thr
1 5 10 15
Ser Glu Leu Ile Lys Val Ala Glu Leu Ser Asp Phe Thr Asp Trp Glu
20 25 30
Cys Ala Val Phe Thr Asn Pro Gly Asp Ser Gln Gly Ile Arg Thr Arg
35 40 45
Leu Phe Asn Val Thr Gln Leu Thr Thr Thr Thr Ala Thr Thr Leu Thr
50 55 60
Thr Pro Ser Thr Pro Pro Thr Thr Pro Thr Val Ile Thr Pro Pro Thr
65 70 75 80
Asn Gln Ser Ile Thr Pro Thr Pro Pro Thr Thr Pro Thr Thr Thr Pro
85 90 95
Thr Thr Pro Asn Ile Thr Thr Pro Thr Thr Pro Ser Thr Pro Ser Thr
100 105 110
Thr Thr Pro Thr Thr Pro Ser Thr Pro Thr Ser Thr Ser Thr Ser Thr
115 120 125
Ser Thr Ser Thr Ser
130
<210> 17
<211> 933
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gatatcggcg ccagcgcata tacctggtac cgcaatggcg tgttcgccga caccaccagc 60
acaaacgagt atatcaccgt ggtggccggt cgctatacct gcgagccgac aggcagcacc 120
ggtacactga gtgatcctgt gtgggttggc ggcggcggca gctgcccgaa cggtgcaatc 180
acccgtattc acgaagccac cagcaaaacc tacaccgaca tccataccgg cggcggcggc 240
agcgtgagta cccgtggtaa ctgcccggtg aaactgagcg cctgcaacaa caacgaacag 300
ctgacctgcg ccaatcaggc cccgattccg gtgctgaata cctgcaaccg tgtgtacagc 360
tatttctgcc cggatcacta ttattttagc tacaccgcag ttaatagcac cggcgccagc 420
ctgggtctga ccccggttga tgcaaatgag ccgcgtcgca ttgagatgcc gggcaccaac 480
ggcaccgtgt atcgtatcta ctgcggcaac gattttgagg gcggcggcgg cagcgtgaaa 540
cgtagcgcac cggccaatcc gctgctggtt agcacaggca ccagcgaact gatcaaagtg 600
gccgaactga gtgacttcac cgattgggaa tgcgcagtgt tcacaaaccc gggcgatagc 660
cagggcattc gtacccgcct gttcaacgtg acccagctga ccacaaccac agccaccacc 720
ctgaccacac ctagtacccc gccgacaacc ccgaccgtga ttaccccgcc gaccaaccaa 780
agtatcaccc cgacaccgcc gaccacacct accaccaccc cgaccacacc gaatatcaca 840
acaccgacca ccccgagtac cccgagtacc accaccccga ccacaccgag caccccgacc 900
agtaccagta ccagtaccag caccagcacc agc 933
Claims (10)
1.pORF148重组蛋白,其特征是,其依次由SEQ ID NO.13—SEQ ID NO.16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。
2.根据权利要求1所述的重组蛋白,其特征是,所述连接子组成为Gly-Gly-Gly-Gly-Ser。
3.一种编码权利要求1所述的pORF148重组蛋白的表达基因,其特征是,其序列,包括a:其核苷酸序列如SEQ ID NO.17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。
4.权利要求1所述的pORF148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3所述编码所述pORF148重组蛋白的表达基因序列通过HindIII/XhoI双酶切***pET32a(+)载体,构建重组质粒pET32a-mod pORF148;
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 pORF148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。
5.根据权利要求4所述的制备方法,其特征是,所述编码所述pORF148重组蛋白的表达基因的序列如SEQ ID NO.17所示。
6.根据权利要求5所述的制备方法,其特征是,如SEQ ID NO.22所示的编码所述pORF148重组蛋白的表达基因其是通过SEQ ID NO.3和SEQ ID NO.4扩增得到的。
7.权利要求1-2任一项所述的pORF148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。
8.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1或2所述的pORF148重组蛋白。
9.根据权利要求8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1或2所述pORF148重组蛋白包被的酶标板。
10.根据权利要求8或9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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