CN114478776A - 一种抗鸡tlr15蛋白的多克隆抗体及制备方法 - Google Patents
一种抗鸡tlr15蛋白的多克隆抗体及制备方法 Download PDFInfo
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Abstract
本发明提供了一种抗鸡TLR15蛋白多克隆抗体及其制备方法,包括鸡TLR15蛋白的抗原性分析、筛选用于制备抗体的肽段对应基因序列、密码子优化及基因合成、原核表达及免疫。鸡Toll样受体15(chTLR15)是一种禽类特有的细胞表面模式识别受体,介导的先天性免疫反应对禽类抵御细菌以及病毒入侵具有重要作用,但由于其商业化抗体的缺乏,其配体和激活机制不清。本发明通过分析chTLR15蛋白的抗原性,密码子优化及基因合成和原核表达,获得重组蛋白His‑chTLR15,制备了具有高度特异性的抗chTLR15蛋白多克隆抗体,为研究chTLR15在家禽免疫方面作用提供重要支撑。
Description
技术领域
本发明涉及一种抗鸡TLR15蛋白多克隆抗体及其制备技术领域,特别是涉及一种鸡TLR15(Toll-like receptors 15,TLR15)部分基因片段的选择、密码子优化及合成,重组质粒的构建,重组蛋白表达、纯化,多克隆抗体的制备及特异性检测。
背景技术
chTLR15是鸡TLRs家族中特有的先天免疫受体蛋白。chTLR15以同源二聚体形式存在,具有抵抗细菌入侵、激活机体抗病毒能力。chTLR15属于Ⅰ型跨膜糖蛋白,是禽类体内一类重要的模式识别受体,广泛存在于心脏、肝脏、脾脏、肺脏、肾脏、大脑及胸腺等组织中,其中在法氏囊中mRNA转录水平最高,其次是脾脏与盲肠。chTLR15的激活涉及受体胞外域的蛋白水解切割与NF-κB依赖性基因转录的刺激,且chTLR15可识别病毒非甲基化CpG-DNA序列,激活机体抗病毒能力。
目前chTLR15的研究集中在chTLR15介导的先天性免疫反应对禽类抵御细菌以及病毒入侵的研究;通过高效识别病毒肽聚糖,激活并产生IL-1β炎性因子;chTLR15相关信号通路对宿主抗感染免疫应答的研究。
人、大鼠、小鼠、兔子等哺乳动物以及家禽,果蝇的TLRs已鉴定并证实,但TLR15为鸡特有的先天免疫受体蛋白,对chTLR15研究尚不深入,尤其是还未出现商业化鸡TLR15多克隆抗体。
在本次发明中,根据NCBI公布的鸡TLR15序列,分析其抗原性,选定76-681bp基因序列(长606bp)所编码的第25-227 aa(长202aa)作为抗原,对76-681bp基因序列进行密码子优化后,合成基因片段,利用原核表达系统表达pET30-chTRL15,通过纯化浓缩chTRL15重组蛋白制备兔抗TLR15血清多抗。本发明填补了缺乏商品化chTLR15抗体的空白,为TLR15在家禽方面的研究提供了基础资料。
发明内容
本发明克服了抗鸡TLR15蛋白多克隆抗体制备技术的缺陷,提供一种编码鸡TLR15蛋白片段的核苷酸序列优化序列、重组蛋白表达及纯化、多克隆抗体制备及抗体特异性检测方法。
本发明的目的是提供一种抗鸡TLR15蛋白的多克隆抗体,以解决现有技术中针对鸡TLR15蛋白缺少特异性抗体的问题。
为实现上述目的,本发明公开了如下的技术内容:
一种抗鸡TLR15蛋白多克隆抗体,其特征在于,所述多克隆抗体由经重组质粒pET30a-chTLR15编码表达的重组蛋白经生物免疫制得,所述重组质粒pET30a-chTLR15中TLR15基因序列为SEQ ID NO.3所示,由SEQ ID NO.1核苷酸序列经密码子优化而来,该序列长606bp,其编码的氨基酸序列如SEQ ID NO.2所示,该序列编码202个氨基酸(第25-227位),分子量预测为22kDa,等电点为5.74,预测融合His标签后蛋白分子量为27kDa。
本发明进一步公开了抗鸡TLR15蛋白多克隆抗体制备方法包括以下步骤:
步骤一:鸡TLR15抗原性分析、密码子优化及基因片段的合成;
步骤二:构建原核表达载体pET30a-chTLR15;
步骤三:使用重组质粒对感受态大肠杆菌进行转化,制备工程菌;
步骤四:诱导工程菌进行重组蛋白的表达,并进行纯化;
步骤五:使用重组蛋白对家兔进行免疫,制备多克隆抗体。
其中所述步骤一中鸡TLR15抗原性分析及基因片段合成方法如下:
分析NCBI上公布的chTLR15序列,选取抗原性强的片段606bp(76-681 bp),密码子优化后,两端加入EcoRⅠ、XhoI酶切位点,合成基因序列,序列如SEQ ID NO.3所示。
步骤二中构建重组载体的步骤如下:
用EcoRⅠ、XhoI分别酶切pET30载体和chTLR15基因合成片段,切胶回收片段,连接,获得重组质粒pET30a-chTLR15。
步骤三制备工程菌的步骤如下:
将重组质粒pET30a-chTLR15,转化BL21感受态细胞,构建带有重组质粒的工程菌。
步骤四中chTLR15重组蛋白表达纯化的步骤如下:
经IPTG诱导表达,去离子水洗镍柱纯化获得chTLR15重组蛋白。
步骤五中制备多克隆抗体的方法如下:
chTLR15重组蛋白免疫新西兰大白兔,接种方式为背部皮下多点注射,首免使用弗氏完全佐剂,分别于第二周,第四周,第五周使用弗氏不完全佐剂进行一免,二免,三免,于加强免疫后7天取兔血清,得到抗chTLR15蛋白多克隆抗体。
本发明同时也公开了抗鸡TLR15蛋白多克隆抗体在用于特异性检测鸡TLR15蛋白方面的应用;所述的多克隆抗体特异性检测指的是:抗鸡TLR15蛋白抗体识别鸡TLR15蛋白的检测。实验结果显示抗鸡TLR15蛋白多克隆抗体可特异性识别DF-1细胞及鸡法氏囊和胸腺组织中的鸡TLR15蛋白。
本发明还进一步公开了多克隆抗体的特异性检测方法,其特征在于,包括以下步骤:
蛋白样品的获得:DF-1细胞在37℃、5%CO2培养箱内培养至70%左右,提取DF-1细胞蛋白;取鸡法氏囊与胸腺组织100mg左右,加入蛋白裂解液1mL,冷冻研磨仪研磨混匀。
特异性检测:将提取的蛋白经SDS-PAGE凝胶电泳后转至PVDF膜上,转印条件为恒流110V,85min。用快速封闭液室温封闭1h,TBST洗涤2次;β-actin(1:1000)和chTLR15多克隆抗体血清(1:1000)作为一抗,4℃过夜孵育,TBST洗涤3次,每次5min;辣根过氧化酶标记的山羊抗鼠IgG(1:1000)和辣根过氧化酶标记的山羊抗兔IgG(1:1000)作为二抗,常温孵育90min,TBST洗涤3次,每次5min;用发光液曝光。观察特异性结果。
本发明更加详细的描述如下:
(1)一种抗原性强的鸡TLR15蛋白片段,其氨基酸序列如(SEQ ID NO.2)所示。
(2)一种编码上述基因的核苷酸序列如(SEQ ID NO.1)所示。
(3)一种基于如SEQ ID NO.1所示核苷酸序列经密码子优化后更适于原核表达的核苷酸序列如SEQ ID NO.3所示。
(4)一种构建鸡TLR15重组载体的制备方法,包括以下步骤:
分别用EcoRⅠ、XhoI酶切pET30载体和合成的chTLR15部分基因序列,切胶回收目的片段,连接,经菌液PCR鉴定、酶切、测序验证获得质粒pET30a-chTLR15。
(5)一种鸡TLR15重组蛋白的制备方法:
将鸡的TLR15原核表达载体pET30a-chTLR15转入BL21感受态细胞后,经IPTG诱导表达,去离子水洗镍柱纯化获得chTLR15重组蛋白。
如上述制备方法,将诱导表达获得的pET30a-chTLR15重组蛋白纯化浓缩,步骤如下:用去离子水洗镍柱(Ni Sepharose 6 Fast Flow,GE Healthcare),至pH 7.0;挂镍至pH2~3;去离子水洗柱至pH 7.0;用约100 ml的10mM Tris-HCl(pH 8.0)溶液平衡镍柱;稀释样品后上样,后用含0.5 M氯化钠的10 mM Tris-HCl(pH 8.0)溶液洗柱;分别用含15 mM咪唑、60 mM咪唑、300 mM咪唑的10 mM Tris-HCl(pH 8.0)(含0.5 M氯化钠)溶液洗脱,分别收集蛋白峰;最后电泳检测蛋白纯化效果,纯化后利用超滤管/PEG20000进行样品浓缩。
(6)一种抗鸡TLR15蛋白多克隆抗体的制备,其包括如下步骤:
将chTLR15重组蛋白400μg免疫新西兰大白兔,接种方式为背部皮下多点注射,首免使用弗氏完全佐剂,分别于第二周,第四周,第五周使用弗氏不完全佐剂进行一免,二免,三免,剂量为重组蛋白200μg,于加强免疫后7天取兔血清,得到抗鸡TLR15蛋白多克隆抗体。
(7)一种抗鸡TLR15多克隆抗体特异性检测的方法,多克隆抗体识别鸡源细胞系内和鸡组织内TLR15蛋白的特异性检测方法,包括以下步骤:
S1、DF-1细胞内及鸡组织内蛋白的提取:DF-1细胞在37℃、5%CO2培养箱内培养至70%左右,提取DF-1细胞蛋白;取鸡法氏囊与胸腺组织各100mg左右,加入蛋白裂解液1mL,冷冻研磨仪研磨混匀。
S2、多克隆抗体识别鸡TLR15蛋白的特异性检测:提取蛋白经SDS-PAGE凝胶电泳后转至PVDF膜上,转印条件为恒流110V,85min。用快速封闭液室温封闭1h,TBST洗涤2次;β-actin(1:1000)和chTLR15多克隆抗体血清(1:1000)作为一抗,4℃过夜孵育,TBST洗涤3次,每次5min;辣根过氧化酶标记的山羊抗鼠IgG(1:1000)和辣根过氧化酶标记的山羊抗兔IgG(1:1000)作为二抗,常温孵育90min,TBST洗涤3次,每次5min;用发光液曝光。
本发明主要考察了鸡TLR15多克隆抗体制备技术,重点是解决筛选抗原性强的鸡TLR15蛋白片段、密码子优化及基因合成、重组蛋白表达和纯化、多克隆抗体制备、抗体特异性检测,发明的难点在于筛选适合用于抗体制备的高抗原性蛋白片段、对应基因的合成及重组质粒构建技术、重组蛋白的原核表达技术和纯化技术、多克隆抗体制备技术及特异性检测技术。
本发明公开的抗鸡TLR15多克隆抗体及其制备方法与现有技术相比所具有的积极效果在于:
(1)本发明采用大肠杆菌原核表达系统,构建pET30a-chTLR15质粒载体,并进行蛋白的表达与纯化,不仅能够制备效价较高的多克隆抗体,而且为之后的鸡TLR15相关功能的研究奠定基础,为家禽先天性免疫通路的深入研究提供新思路,为家禽业的可持续发展奠定基础。
(2)本发明填补了抗鸡TLR15蛋白抗体的空白,为研究鸡TLR15基因及蛋白功能提供重要工具。
附图说明
图1为pET-30a-chTLR15重组质粒双酶切结果图:其中泳道1为pET-30a-chTLR15载体大小,泳道2为pET-30a-chTLR15双酶切后产物,MM为蛋白Marke;
图2为pET30a-chTLR15重组质粒蛋白小量表达SDS-PAGE电泳结果图,其中,M为蛋白Marker,泳道1为未诱导对照组,泳道2-4为IPTG诱导组;
图3为pET30a-chTLR15重组质粒蛋白大量表达SDS-PAGE电泳结果图,其中M为蛋白Marker,泳道1为超声后全菌液,泳道2为超声后菌液上清,泳道3为超声后菌液沉淀;
图4为pET30a-chTLR15重组质粒蛋白纯化SDS-PAGE电泳结果图:其中M为蛋白Marker,泳道1为蛋白原样,泳道2为过镍柱后的流出物,泳道3为15mM 咪唑洗脱后蛋白样品,泳道4为60mM 咪唑洗脱后蛋白样品,泳道5-7为300mM 咪唑洗脱后蛋白样品;
图5为pET30a-chTLR15重组质粒蛋白浓缩SDS-PAGE电泳结果图,其中,M为蛋白Marker,泳道1为浓缩后蛋白条带;
图6为ELISA测定血清效价折线图;
图7为兔抗chTLR15蛋白多克隆抗体Western blot特异性检测图,其中检测DF-1细胞、法氏囊与胸腺组织中chTLR15蛋白表达量。
具体实施方式
面通过具体的实施方案叙述本发明。除非特别说明,本发明中所用的技术手段均为本领域技术人员所公知的方法。另外,实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。本发明所用原料及试剂均有市售。
实施例1
抗chTLR15蛋白多克隆抗体及其制备方法
步骤一、chTLR15部分核苷酸序列的优化、基因合成
对NCBI中chTLR15蛋白全序列(NP_001032924.1)进行蛋白序列二级结构分析及亲疏水性、抗原性等分析,选取76-681bp核苷酸序列如SEQ ID NO.1所示编码的25-227aa作为免疫原,对应氨基酸序列如SEQ ID NO.2所示。为提高原核生物中蛋白表达水平,对SEQ IDNO.1所示的76-681bp核苷酸序列进行密码子优化,两端加EcoRⅠ、XhoI酶切位点,序列如SEQID NO.3所示,进行基因片段合成。
其中,SEQ ID NO.1序列为:
cagagaacatctccagtgtcttccttcccattttataactactcttacttaaacctctcttctgtatcacaagcacaagctcccaaaacagcaagagctctcaacttctcatataatgcaattgaaaaaataaccaaaagagactttgaaggttttcatgtactggaagttttggacctttctcacaatcacattaaggacattgaacctggtgcatttgagaacctgctcagccttgtttctgtggatttatcatttaatgataagaatcttctggtatctggtcttgcacctcacctgaaactcataccgaccagcggagcctcggggccttcacagatttatatgtattttcaaaaatcagcagaggctgccctggagccttctgcaccagctgaactgctgccacatttggaagatccacccaatccaggaaatgttaacccaagattcagacaaagaagaacagaggaaaataaaacttcccctccagcagccacgctgaggcctgacttgtgtggagcaccgatcaatgggttgcttgacctatcaaggaccaaactgtctaatgaagagctgacagcaaaattggatgcagacctctgc
SEQ ID NO.2序列为:
QRTSPVSSFPFYNYSYLNLSSVSQAQAPKTARALNFSYNAIEKITKRDFEGFHVLEVLDLSHNHIKDIEPGAFENLLSLVSVDLSFNDKNLLVSGLAPHLKLIPTSGASGPSQIYMYFQKSAEAALEPSAPAELLPHLEDPPNPGNVNPRFRQRRTEENKTSPPAATLRPDLCGAPINGLLDLSRTKLSNEELTAKLDADLC
SEQ ID NO.3序列为:
gaattccaaaggacatctccagtatcaagttttcccttctataactacagctatctgaatttgtcgagcgtgtcgcaagcgcaggctccgaaaactgcgcgtgcactgaacttcagctacaacgcgatcgagaagatcaccaagcgcgattttgaaggttttcatgtcctggaagttctggacttgtcccacaatcatattaaggacatcgagccgggtgccttcgagaacctcttgtccctcgtgtccgtggacctttcttttaacgataagaacctgttggttagcggcctggcaccgcacctgaaattaattccgacctcaggcgctagcggtccgagccaaatttatatgtacttccagaaaagcgctgaagcagcgctggagccgagcgcgccagcagaactgctgccgcacctggaagatccgcccaacccgggtaatgttaatccgcgttttcgccagcgccgtacggaagagaacaaaacctccccgcctgcggcgaccctgcgtccggatctgtgtggtgccccgatcaacggcctgttagacttgtctcgtacgaagctgagtaatgaagagttgaccgcgaaactggacgccgatctgtgcctcgag
步骤二、pET30a-chTLR15重组载体的构建和工程菌制备
用EcoRⅠ、XhoI分别双酶切pET-30a载体和合成测定正确的chTLR15基因片段,连接,转化至感受态细胞BL21中。将菌体重悬涂布在LB培养基平板上,在37℃的条件下,倒置培养16h。挑取培养皿上单克隆菌群接种于含有卡那的液体培养基中,过夜培养。利用质粒小型提取试剂盒提取质粒,提取的质粒用EcoRⅠ、XhoI双酶切进行验证,将鉴定的阳性质粒测序。酶切验证结果如图1所示:pET-30a-chTLR15重组质粒载体在约6kb处出现特异性条带,与预期条带大小一致(见泳道1),pET-30a双酶切后产物在约5kb与600bp处出现特异性条带,与载体pET-30a和插入目的基因chTLR15大小一致(见泳道2),说明目的基因插入成功,此结果证实已成功构建pET-30a-chTLR15重组质粒载体。
步骤三、His-chTLR15重组蛋白的表达
1.小量表达:
将测序鉴定正确的pET-30a-chTLR15重组质粒载体转化BL21感受态细胞,挑取挑单克隆菌落到含相应抗性的LB液体培养基中,37℃,200 rpm培养。培养至OD=0.6-0.8,取1ml作为诱导前对照,取剩余的菌液IPTG(0.5 mM)诱导,37℃,200 rpm培养2 h。取1 ml诱导的菌液,12000 rpm/min,离心1 min,收集菌液沉淀,沉淀用50-100 μl 10 mM Tris-HCl(pH8.0)溶液吹散(加入缓冲液的量视菌体量而定),加入与缓冲液等体积的 2×loadingbuffer,100℃煮5 min,经SDS-PAGE电泳检测蛋白是否表达。小量表达结果如图2,IPTG确实可以诱导目的蛋白的表达。
2.大量表达
将100 μl活化的菌液接种到5 ml相应抗性LB液体培养基中37℃培养,将培养的菌液转接到250 ml相应抗性LB液体培养基中,37℃,200 rpm,培养至OD=0.6-0.8,IPTG(0.5mM)16℃诱导过夜,后8000 rpm/min,离心6 min,收集菌液沉淀,菌体用20-30 ml 10 mMTris-HCl(pH 8.0)溶液吹散,超声波破碎(500 W , 180次,每次5 s,间隔5 s)。取100μl超声后的菌悬液,12000 rpm/min,离心10 min,取50 μl上清至另一EP管,上清去除干净后沉淀用50 μl 10 mM Tris-HCl(pH 8.0)溶液吹散,分别用超声后全菌、上清、沉淀SDS-PAGE电泳鉴定目的蛋白表达形式。pET-30a-chTLR25重组蛋白表达形式的鉴定结果如图3,样品pET-30a-chTLR15菌体沉淀(泳道3)在相对分子质量约35KDa处有一条特异性表达带,且比pET-30a-chTLR15全菌、上清中的条带特异性更高(泳道1、2)。说明重组质粒成功表达并主要存在于菌体沉淀中。
步骤四、His-chTLR15重组蛋白纯化浓缩实验
用去离子水洗镍柱(Ni Sepharose 6 Fast Flow,GE Healthcare)至pH 7.0,挂镍至pH 2~3;去离子水洗柱至pH 7.0;用约100 ml的10mM Tris-HCl(pH 8.0)溶液平衡镍柱;稀释样品后上样,后用含0.5 M氯化钠的10 mM Tris-HCl(pH 8.0)溶液洗柱;分别用含15mM咪唑、60 mM咪唑、300 mM咪唑的10 mM Tris-HCl(pH 8.0)(含0.5 M氯化钠)溶液洗脱,分别收集蛋白峰,SDS-PAGE电泳检测蛋白纯化效果,纯化后利用超滤管/PEG20000进行样品浓缩。SDS-PAGE电泳鉴定蛋白纯化效果如图4,由5-7泳道可以看出,经300mM咪唑洗脱的蛋白条带较为单一。蛋白浓缩后特异性条带可见图5。
步骤五、抗chTLR15蛋白多克隆抗体的制备
1.新西兰白兔的免疫
首次免疫:选用2.0kg左右的新西兰大白兔,取400ug His-chTLR15重组蛋白作为免疫原,用生理盐水稀释到200-500ul,再加入等体积弗氏佐剂混匀,乳化制成油乳剂,对兔子进行背部皮下注射免疫,打8-10个点。阴性对照组,以同样剂量的弗氏完全佐剂作为对照。同时取健康大白兔少量免疫前血清作为空白对照。免疫周期为34天,共免疫4次,4次免疫后第7天,兔耳缘静脉取血。收集血液置于37℃灭活2h后,4℃过夜凝结释放血清,5000rpm/min,离心10min,两次离心后收集上清,即为血清。
2.ELISA测定血清效价
使用ELISA测定免疫效价。检测双波长(450,630)吸光值,并做图分析,效价检测结果如下表和图6,效价为1/2最大OD值为25600,其所对应的稀释倍数为1.066倍;
步骤六、抗chTLR15蛋白多克隆抗体识别chTLR15蛋白的特异性检测
1. 蛋白样品的获得
DF-1细胞在37℃、5%CO2培养箱内培养至70%左右,提取DF-1细胞蛋白;取鸡法氏囊与胸腺组织100mg左右,加入蛋白裂解液1mL,冷冻研磨仪研磨混匀。
2. 特异性检测
将提取的蛋白经SDS-PAGE凝胶电泳后转至PVDF膜上,转印条件为恒流110V,85min。用快速封闭液室温封闭1h,TBST洗涤2次;β-actin(1:1000)和chTLR15多克隆抗体血清(1:1000)作为一抗,4℃过夜孵育,TBST洗涤3次,每次5min;辣根过氧化酶标记的山羊抗鼠IgG(1:1000)和辣根过氧化酶标记的山羊抗兔IgG(1:1000)作为二抗,常温孵育90min,TBST洗涤3次,每次5min;用发光液曝光。
结果如图7所示,表明免疫大兔阳性血清作为一抗检测出DF-1细胞系、法氏囊和胸腺组织中,出现特异单一条带,与chTLR15蛋白预测大小(98kDa)一致,说明制备的抗体具有特异性。
在详细说明的较佳实施例之后,熟悉该项技术人士可清楚地了解,在不脱离上述申请专利范围与精神下可进行各种变化与修改,凡依据本发明的技术实质对以上实施例所作任何简单修改、等同变化与修饰,均属于本发明技术方案的范围。且本发明亦不受说明书中所举实例实施方式的限制。
SEQUENCE LISTING
<110> 天津农学院
<120> 一种抗鸡TLR15蛋白的多克隆抗体及制备方法
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213> 人工序列
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cagagaacat ctccagtgtc ttccttccca ttttataact actcttactt aaacctctct 60
tctgtatcac aagcacaagc tcccaaaaca gcaagagctc tcaacttctc atataatgca 120
attgaaaaaa taaccaaaag agactttgaa ggttttcatg tactggaagt tttggacctt 180
tctcacaatc acattaagga cattgaacct ggtgcatttg agaacctgct cagccttgtt 240
tctgtggatt tatcatttaa tgataagaat cttctggtat ctggtcttgc acctcacctg 300
aaactcatac cgaccagcgg agcctcgggg ccttcacaga tttatatgta ttttcaaaaa 360
tcagcagagg ctgccctgga gccttctgca ccagctgaac tgctgccaca tttggaagat 420
ccacccaatc caggaaatgt taacccaaga ttcagacaaa gaagaacaga ggaaaataaa 480
acttcccctc cagcagccac gctgaggcct gacttgtgtg gagcaccgat caatgggttg 540
cttgacctat caaggaccaa actgtctaat gaagagctga cagcaaaatt ggatgcagac 600
ctctgc 606
<210> 2
<211> 202
<212> PRT
<213> 氨基酸序列
<400> 2
Gln Arg Thr Ser Pro Val Ser Ser Phe Pro Phe Tyr Asn Tyr Ser Tyr
1 5 10 15
Leu Asn Leu Ser Ser Val Ser Gln Ala Gln Ala Pro Lys Thr Ala Arg
20 25 30
Ala Leu Asn Phe Ser Tyr Asn Ala Ile Glu Lys Ile Thr Lys Arg Asp
35 40 45
Phe Glu Gly Phe His Val Leu Glu Val Leu Asp Leu Ser His Asn His
50 55 60
Ile Lys Asp Ile Glu Pro Gly Ala Phe Glu Asn Leu Leu Ser Leu Val
65 70 75 80
Ser Val Asp Leu Ser Phe Asn Asp Lys Asn Leu Leu Val Ser Gly Leu
85 90 95
Ala Pro His Leu Lys Leu Ile Pro Thr Ser Gly Ala Ser Gly Pro Ser
100 105 110
Gln Ile Tyr Met Tyr Phe Gln Lys Ser Ala Glu Ala Ala Leu Glu Pro
115 120 125
Ser Ala Pro Ala Glu Leu Leu Pro His Leu Glu Asp Pro Pro Asn Pro
130 135 140
Gly Asn Val Asn Pro Arg Phe Arg Gln Arg Arg Thr Glu Glu Asn Lys
145 150 155 160
Thr Ser Pro Pro Ala Ala Thr Leu Arg Pro Asp Leu Cys Gly Ala Pro
165 170 175
Ile Asn Gly Leu Leu Asp Leu Ser Arg Thr Lys Leu Ser Asn Glu Glu
180 185 190
Leu Thr Ala Lys Leu Asp Ala Asp Leu Cys
195 200
<210> 3
<211> 618
<212> DNA
<213> 人工序列
<400> 3
gaattccaaa ggacatctcc agtatcaagt tttcccttct ataactacag ctatctgaat 60
ttgtcgagcg tgtcgcaagc gcaggctccg aaaactgcgc gtgcactgaa cttcagctac 120
aacgcgatcg agaagatcac caagcgcgat tttgaaggtt ttcatgtcct ggaagttctg 180
gacttgtccc acaatcatat taaggacatc gagccgggtg ccttcgagaa cctcttgtcc 240
ctcgtgtccg tggacctttc ttttaacgat aagaacctgt tggttagcgg cctggcaccg 300
cacctgaaat taattccgac ctcaggcgct agcggtccga gccaaattta tatgtacttc 360
cagaaaagcg ctgaagcagc gctggagccg agcgcgccag cagaactgct gccgcacctg 420
gaagatccgc ccaacccggg taatgttaat ccgcgttttc gccagcgccg tacggaagag 480
aacaaaacct ccccgcctgc ggcgaccctg cgtccggatc tgtgtggtgc cccgatcaac 540
ggcctgttag acttgtctcg tacgaagctg agtaatgaag agttgaccgc gaaactggac 600
gccgatctgt gcctcgag 618
Claims (9)
1.一种抗鸡TLR15蛋白多克隆抗体,其特征在于,所述多克隆抗体由经重组质粒pET30a-chTLR15编码表达的重组蛋白经生物免疫制得,所述重组质粒pET30a-chTLR15中TLR15基因序列为SEQ ID NO.3所示,由SEQ ID NO.1核苷酸序列经密码子优化而来,编码氨基酸序列如SEQ ID NO.2所示。
2.权利要求1所述抗鸡TLR15蛋白多克隆抗体制备方法包括以下步骤:
步骤一:鸡TLR15抗原性分析、密码子优化及基因片段的合成;
步骤二:构建原核表达载体pET30a-chTLR15;
步骤三:使用重组质粒对感受态大肠杆菌进行转化,制备工程菌;
步骤四:诱导工程菌进行重组蛋白的表达,并进行纯化;
步骤五:使用重组蛋白对家兔进行免疫,制备多克隆抗体。
3.根据权利要求2所述的多克隆抗体,其特征在于,所述步骤一中鸡TLR15抗原性分析及基因片段合成方法如下:
分析NCBI上公布的chTLR15序列,选取抗原性强的片段606bp(76-681 bp),密码子优化后,两端加入EcoRⅠ、XhoI酶切位点,合成基因序列,序列如SEQ ID NO.3所示。
4.根据权利要求2所述的多克隆抗体,其特征在于,所述步骤二中构建重组载体的步骤如下:
用EcoRⅠ、XhoI分别酶切pET30载体和chTLR15基因合成片段,切胶回收片段,连接,获得重组质粒pET30a-chTLR15。
5.根据权利要求2所述的多克隆抗体,其特征在于,在步骤三制备工程菌的步骤如下:
将重组质粒pET30a-chTLR15,转化BL21感受态细胞,构建带有重组质粒的工程菌。
6.根据权利要求2所述的多克隆抗体,其特征在于,所述步骤四中chTLR15重组蛋白表达纯化的步骤如下:
经IPTG诱导表达,去离子水洗镍柱纯化获得chTLR15重组蛋白。
7.根据权利要求2所述的多克隆抗体,其特征在于,所述步骤五中制备多克隆抗体的方法如下:
chTLR15重组蛋白免疫新西兰大白兔,接种方式为背部皮下多点注射,首免使用弗氏完全佐剂,分别于第二周,第四周,第五周使用弗氏不完全佐剂进行一免,二免,三免,于加强免疫后7天取兔血清,得到抗chTLR15蛋白多克隆抗体。
8.权利要求1所述抗鸡TLR15蛋白多克隆抗体在用于特异性检测鸡TLR15蛋白方面的应用;所述的多克隆抗体特异性检测指的是:抗鸡TLR15蛋白抗体识别鸡TLR15蛋白的检测。
9.一种如权利要求8所述抗鸡TLR15蛋白多克隆抗体的特异性检测方法,其特征在于,包括以下步骤:
蛋白样品的获得:DF-1细胞在37℃、5%CO2培养箱内培养至70%左右,提取DF-1细胞蛋白;取鸡法氏囊与胸腺组织100mg左右,加入蛋白裂解液1mL,冷冻研磨仪研磨混匀;特异性检测:将提取的蛋白经SDS-PAGE凝胶电泳后转至PVDF膜上,转印条件为恒流110V,85min;用快速封闭液室温封闭1h,TBST洗涤2次;β-actin(1:1000)和chTLR15多克隆抗体血清(1:1000)作为一抗,4℃过夜孵育,TBST洗涤3次,每次5min;辣根过氧化酶标记的山羊抗鼠IgG(1:1000)和辣根过氧化酶标记的山羊抗兔IgG(1:1000)作为二抗,常温孵育90min,TBST洗涤3次,每次5min;用发光液曝光,观察特异性结果。
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