CN113528467A - 一种靶向性双展示噬菌体及其制备方法和应用 - Google Patents
一种靶向性双展示噬菌体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向性双展示噬菌体及其制备方法和应用,属于DNA重组技术领域。本发明公开的一种靶向性双展示噬菌体,利用噬菌体展示技术将编码P53蛋白N端表位多肽SV及DP分别定向克隆到丝状噬菌体的PIII及PVIII基因中,制备出尾部PIII蛋白展示肽SV、背部PVIII蛋白展示肽DP的靶向性双展示噬菌体phage‑SV‑DP。该噬菌体作为一种新型生物制品,具有特异性强、灵敏度高、制备简单、成本低廉等优点,能够应用于肿瘤患者血清P53抗体的检测。
Description
技术领域
本发明涉及DNA重组技术领域,更具体的说是涉及一种靶向性双展示噬菌体及其制备方法和应用。
背景技术
癌症是威胁人类健康的主要杀手,在我国每年癌症发病人数约260万,死亡约180万。导致这一现象的原因很多,其中一个重要的原因是目前人类还不能对肿瘤的发生发展过程进行有效的诊断,尤其是早期诊断。
p53是一种抑癌基因,其蛋白对维持正常的细胞分裂与生长起重要的调节作用,但是一旦该基因发生突变,其编码的突变蛋白半衰期延长,失去抑癌作用,积累于细胞内并刺激免疫系统产生P53抗体。研究表明,血清P53抗体可以作为一种广谱性的肿瘤标志物用于肿瘤早期检测和肿瘤高危人群的筛查。
目前,血清P53抗体的检测主要以重组P53蛋白为基础开展,而蛋白的制备及纯化具有操作繁琐、费时、价格相对昂贵且灵敏度低等缺点。
因此,提供一种靶向性双展示噬菌体及其制备方法和应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种能够用于肿瘤患者血清P53抗体检测的靶向性双展示噬菌体,该噬菌体作为一种新型生物制品具有特异性强、灵敏度高、制备简单、成本低廉等优点。
为了实现上述目的,本发明采用如下技术方案:
一种靶向性双展示噬菌体,利用噬菌体展示技术将编码P53蛋白N端表位多肽SV及DP分别定向克隆到丝状噬菌体的PIII及PVIII基因中,制备出尾部PIII蛋白展示肽SV、背部PVIII蛋白展示肽DP的靶向性双展示噬菌体phage-SV-DP;所述SV的氨基酸序列如SEQ IDNO.2所示;所述DP的氨基酸序列如SEQ ID NO.8所示。
进一步,所述的一种靶向性双展示噬菌体的制备方法,具体步骤如下:
1)构建重组噬菌体载体fADL-le-SV
(1)BglI酶切载体fADL-le,获得酶切后的载体fADL-le;
(2)合成P53蛋白N端表位多肽SV;
(3)将步骤(1)酶切后的载体fADL-le与步骤(2)合成的SV连接,转化,筛选阳性克隆,获得重组噬菌体载体fADL-le-SV;
2)构建重组噬菌体载体fADL-le-SV-DP
以构建好的噬菌体载体fADL-le-SV为模板,利用点突变试剂盒将肽DP定向克隆到噬菌体的PVIII基因中,构建获得重组噬菌体载体fADL-le-SV-DP;
3)制备噬菌体phage-SV-DP
(1)将转化有重组噬菌体载体fADL-le-SV-DP的菌株接种到含有Kar+的LB液体培养基中,37℃剧烈震荡10h;
(2)8000rpm,10min,4℃,留上清;
(3)加入六分之一体积的PEG/NaCl溶液,涡旋,4℃过夜;
(4)12000rpm离心15min,用1ml TBS溶解噬菌体沉淀;
(5)将溶液转移至1.5ml EP管中,14000rpm离心1min,小心将上清转移至1.5ml EP管中,每个EP管中加入150μl PEG/NaCl,混匀后4℃过夜;
(6)14000rpm离心15min,用100μl TBS溶解噬菌体沉淀,4℃冰箱保存。
进一步,所述的靶向性双展示噬菌体在检测血清P53抗体中的应用。
噬菌体展示技术能够将基因型与表现型联系起来,使研究者可以在基因水平上实现对蛋白质构象的体外控制,在体外获得具有良好生物学活性的表达产物。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种靶向性双展示噬菌体及其制备方法和应用,利用噬菌体展示技术得到了一种次要外壳蛋白(PIII)展示p53蛋白表位多肽SV、主要外壳蛋白(PVIII)展示p53蛋白表位多肽DP的靶向性双展示噬菌体phage-SV-DP;该噬菌体作为一种新型生物制品,具有特异性强、灵敏度高、制备简单、成本低廉等优点,能够应用于肿瘤患者血清P53抗体的检测。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明靶向性双展示噬菌体phage-SV-DP制备过程示意图;
图2附图为本发明噬菌体载体fADL-le的图谱;
图3附图为本发明fADL-le载体Bgl I酶切结果;
其中,1,Marker;2,fADL-le载体;3,fADL-le载体BglI酶切后;
图4附图为本发明PCR验证重组载体fADL-le-SV阳性克隆;
其中,1,Marker;2-5,阳性克隆;
图5附图为本发明重组噬菌体载体fADL-le-SV部分测序峰图;第884-919为编码目标多肽的核苷酸序列;
图6附图为本发明重组噬菌体载体fADL-le-SV-DP PVIII位点部分测序峰图;第505-534为编码目标多肽的核苷酸序列;
图7附图为本发明Westernblot分析靶向性双展示噬菌体phage-SV-DP;
其中,1,Marker;2,辅助噬菌体M13K07与P53多克隆抗体杂交(阴性对照);3,phage-DP与P53多克隆抗体杂交;4,phage-SV-DP与P53多克隆抗体杂交;
图8附图为本发明AFM观察phage-SV-DP;
图9附图为本发明检测乳腺癌患者血清P53抗体结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
靶向性双展示噬菌体phage-SV-DP的特征在于P53蛋白两个表位多肽SV及DP分别展示在丝状噬菌体的PIII蛋白及PVIII蛋白,其基因组序列如下:
AACGCTACTACCATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTACATGGAATAAAACTTCCAGACACCGTACTTTAGTTGCATATTTAAAACATGTTGAACTACAGCACCAGATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTGTCTAATCCTGACCTGTTGGAATTTGCTTCCGGTCTGGTTCGCTTTGAGGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATTCGCTTTGCTTCTGACTATAATAGACAGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCTTTTTCTGAACTGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGACGATTCCGCAGTATTGGACGCTATCCAGTCTAAACATTTTACAATTACCCCCTCTGGCAAAACTTCCTTTGCAAAAGCCTCTCGCTATTTTGGTTTCTATCGTCGTCTGGTTAATGAGGGTTATGATAGTGTTGCTCTTACCATGCCTCGTAATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAGTGTGGTATTCCTAAATCTCAATTGATGAATCTTTCCACCTGTAATAATGTTGTTCCGTTAGTTCGTTTTATTAACGTAGATTTTTCCTCCCAACGTCCTGACTGGTATAATGAGCCAGTTCTTAAAATCGCATAAGGTAATTCAAAATGATTAAAGTTGAAATTAAACCATCTCAAGCGCAATTTACTACCCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTGCTTGTCAAGATTACTCTCGACGAAGGTCAGCCAGCGTATGCGCCTGGTCTGTACACCGTGCATCTGTCCTCGTTCAAAGTTGGTCAGTTCGGTTCTCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTCGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCCGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGATATTGAACAATGGTTCACTGAAGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGTCGACTAACGAGGGCAAATCATGAAATACCTATTGCCTACGGCGGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAACAACGTTGGCCCGGGAGGCCTGTCTCTAGAAGCCGAAACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCCATTCGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGCGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGTGGCGGTTCCGGTGGCGGCTCCGGTTCCGGTGATTTTGATTATGAAAAAATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCTTTGCCTCAGTCGGTTGAATGTCGCCCTTATGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCGACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAACTTTGTTCGGCTATCTGCTTACTTTCCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCTATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATTAGCGCACAATTACCCTCTGATTTTGTTCAGGGCGTTCAGTTAATTCTCCCGTCTAATGCGCTTCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAAAAATCGTTTCTTATTTGGATTGGGATAAATAAATATGGCTGTTTATTTTGTAACTGGCAAATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGGTGCAAAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAAACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATTTCTGATTTGCTTGCTATTGGTCGTGGTAATGATTCCTACGACGAAAATAAAAACGGTTTGCTTGTTCTTGATGAATGCGGTACTTGGTTTAATACCCGTTCATGGAATGACAAGGAAAGACAGCCGATTATTGATTGGTTTCTTCATGCTCGTAAATTGGGATGGGATATTATTTTTCTTGTTCAGGATTTATCTATTGTTGATAAACAGGCGCGTTCTGCATTAGCTGAACACGTTGTTTATTGTCGCCGTCTGGACAGAATTACTTTACCCTTTGTCGGCACTTTATATTCTCTTGTTACTGGCTCAAAAATGCCTCTGCCTAAATTACATGTTGGTGTTGTTAAATATGGTGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATACTGGTAAGAATTTATATAACGCATATGACACTAAACAGGCTTTTTCCAGTAATTATGATTCAGGTGTTTATTCATATTTAACCCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGATAGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATTTACAGAAGCAAGGTTATTCCATCACATATATTGATTTATGTACTGTTTCAATTAAAAAAGGTAATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTTCTTTTGCTCAAGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTCGTGACTTGGTATTCAAAGCAAACAGGTGAATCTGTTATTGTCTCACCTGATGTTAAAGGTACAGTGACTGTATATTCCTCTGACGTTAAGCCTGAAAATTTACGCAATTTCTTTATCTCTGTTTTACGTGCTAATAATTTTGATATGGTTGGCTCTAATCCTTCCATAATTCAGAAATATAACCCAAATAGTCAGGATTATATTGATGAATTGCCATCATCTGATATTCAGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTCTTTGTTCCGCAAAATGATAATGTTACTCAAACATTTAAAATTAATAACGTTCGCGCAAAGGATTTAATAAGGGTTGTAGAATTGTTTGTTAAATCTAATACATCTAAATCCTCAAATGTATTATCTGTTGATGGTTCTAACTTATTAGTAGTTAGCGCCCCTAAAGATATTTTAGATAACCTTCCGCAATTTCTTTCTACTGTTGATTTGCCAACTGACCAGATATTGATTGAAGGATTAATTTTCGAGGTTCAGCAAGGTGATGCTTTAGATTTTTCCTTTGCTGCTGGCTCTCAGCGCGGCACTGTTGCTGGTGGTGTTAATACTGACCGTCTAACCTCTGTTTTATCTTCTGCGGGTGGTTCGTTCGGTATTTTTAACGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAAAATATTGTCTGTGCCTCGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATTTCTGTTGGCCAGAATGTCCCTTTTATTACTGGTCGTGTAACTGGTGAATCTGCCAATGTAAATAATCCATTTCAGACAATTGAGCGTCAAAATGTTGGTATTTCTATGAGTGTTTTTCCCGTTGCAATGGCTGGCGGTAATATTGTTTTAGATATAACCAGTAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGTCAGACTCTTTTGCTCGGTGGCCTCACTGATTACAAAAACACTTCTCAAGATTCTGGTGTGCCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGTTCTGATTCTAACGAGGAAAGCACGTTGTACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCTCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGATCTCGGGAAAAGCGTTGGTGACCAAAGGTGCCTTTTATCATCACTTTAAAAATAAAAAACAATTACTCAGTGCCTGTTATAAGCAGCAATTAATTATGATTGATGCCTACATCACAACAAAAACTGATTTAACAAATGGTTGGTCTGCCTTAGAAAGTATATTTGAACATTATCTTGATTATATTATTGATAATAATAAAAACCTTATCCCTATCCAAGAAGTGATGCCTATCATTGGTTGGAATGAACTTGAAAAAATTAGCCTTGAATACATTACTGGTAAGGTAAACGCCATTGTCAGCAAATTGATCCAAGAGAACCAACTTAAAGCTTATGATGATGATGTGCTTAAAAACTTACTCAATGGCTGGTTTATGCATATCGCAATACATGCGAAAAACCTAAAAGAGCTTGCCGATAAAAAAGGCCAATTTATTGCTATTTACCGCGGCTTTTTATTGAGCTTGAAAGATAAATAAAATAGATAGGTTTTATTTGAAGCTAAATCTTCTTTATCGTAAAAAATGCCCTCTTGGGTTATCAAGAGGGTCATTATATTTCGCGGAATAAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTTGTTGACAAAGGGAATCATAGATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCTCCAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCACAACTAACCCGGCCTATTCTTTTGATTTATAAGGATTTTTGTCATTTTCTGCTTACTGGTTAAAAAATAAGCTGATTTAACAAATATTTAACGCGAAATTTAACAAAACATTAACGTTTACAATTTAAATATTTGCTTATACAATCATCCTGTTTTTGGGGCTTTTCTGATTATCAATCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTTTCAGGTAATGACCTGATAGCCTTTGTAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGACGGTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTGCCTACTCATTACTCCGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCCTGCGTTGAAATTAAGGCTTCACCAGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAACTCTCTGCCTTGCTTGTACGATTTATTGGATGTT;SEQ ID NO.1。
其中,在SEQ ID NO.1中,第1301-1369位所示核苷酸序列是编码PVIII蛋白的信号肽;第1370-1399位所示核苷酸序列是编码P53蛋白表位多肽DP的核苷酸序列;第1400-1531位所示核苷酸序列是编码PVIII蛋白的核苷酸序列;第1598-1663位所示核苷酸序列是编码PIII蛋白的信号肽;第1664-1699位所示核苷酸序列是编码P53蛋白表位多肽SV的核苷酸序列;第1700-1723位所示核苷酸序列是Linker;第1724-2941位所示核苷酸序列是编码PIII蛋白的核苷酸序列。
靶向性双展示噬菌体phage-SV-DP的制备步骤(示意图见图1)如下:
(一)重组噬菌体载体fADL-le-SV的构建
1)噬菌体载体fADL-le(载体图谱见图2)的提取
利用Axygen的质粒小量提取试剂盒提取商业化的噬菌体载体fADL-le(AntibodyDesign laboratories,Catalog number:PD020),具体步骤如下:
(1)取6ml在LB培养基中过夜培养的转化有载体fADL-le的菌液JM109,12000×g离心1min,弃尽上清;
(2)加250μl Buffer S1,悬浮细菌沉淀,悬浮需均匀,不应留有小的菌块;
(3)加250μl Buffer S2,温和并充分地上下翻转4-6次混合均匀使菌体充分裂解,直至形成透亮的溶液;
(4)加350μl Buffer S3,温和并充分地上下翻转6-8次,12000×g离心10min;
(5)吸取步骤(4)中离心后的上清,并转移到制备管中,12000×g离心1min,弃滤液;
(6)将制备管放回离心管,加500μlBufferW1,12000×g离心1min,弃滤液;
(7)将制备管放回离心管,加700μlBufferW2,12000×g离心1min,弃滤液;以同样的方法再用700μlBufferW2洗涤一次,弃滤液;
(8)将制备管放回2ml离心管,12000×g离心1min;
(9)将制备管移入到新的1.5ml离心管中,在制备管的膜中央加50μlEluent溶液,室温静置1min,12000×g离心1min。
2)酶切fADL-le
BglI酶切载体fADL-le,具体酶切反应体系如下:
酶切反应条件为37℃水浴2h。噬菌体载体fADL-le的PIII基因经Bgl I酶切后,结果见图3。fADL-le载体主要以环状和线状的形式存在,酶切后均变为线性,初步证明酶切成功。
3)酶切载体的回收
对琼脂糖凝胶电泳证明酶切完全的载体进行切胶回收,按照DNA凝胶回收试剂盒(AXYGEN,Cat.No.AP-GX-50)的说明进行操作,具体如下:
(1)在紫外灯下切下含有目的DNA的琼脂糖凝胶,用纸巾吸尽凝胶表面液体并切碎,计算凝胶重量,该重量作为一个凝胶体积;
(2)加入3个凝胶体积的BufferDE-A,混合均匀后于75℃加热,间断混合,直至凝胶完全融化;
(3)加0.5个BufferDE-A体积的BufferDE-B,混合均匀;
(4)吸取步骤(3)中的混合液,转移到DNA制备管中,12000×g离心1min,弃滤液;
(5)将制备管放回2ml离心管,加入500μlBufferW1,12000×g离心1min,弃滤液;
(6)将制备管放回离心管,加700μlBufferW2,12000×g离心1min,弃滤液;以同样的方法再用700μlBufferW2洗涤一次,弃滤液;
(7)将制备管放回2ml离心管,12000×g离心1min;
(8)将制备管移入到新的1.5ml离心管中,在制备管的膜中央加10μlEluent溶液,室温静置1min,12000×g离心1min。
4)噬菌体展示表位的合成
合成编码P53蛋白N端第20-31位氨基酸SDLWKLLPENNV(SEQ ID NO.2;简称SV)的两个互补DNA片段:
5’-CGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAACAACGTTGGCCCGGG-3’;SEQ IDNO.3;
5’-GGGCCAACGTTGTTTTCAGGAAGTAGTTTCCATAGGTCTGATGCCATGGCCGGCT-3’;SEQ IDNO.4;
两种片段溶解后等摩尔混合,94℃变性5min后58℃复性4min,使两条片段互补结合成双链。
5)目的片段与fADL-le酶切载体的连接形成fADL-le-SV
将合成的目的片段与fADL-le酶切后载体连接,16℃反应过夜,连接反应体系如下:
6)重组载体fADL-le-SV转化大肠杆菌JM109感受态细胞
(1)在大肠杆菌JM109感受态细胞中加入10μl的连接产物,混匀,冰浴30min;
(2)42℃,热击90s;
(3)立即放置到冰上,冰浴10min;
(4)加入800μl LB培养基,37℃,100rpm,45min;
(5)将转化产物均匀涂在含有卡那霉素的LB固体培养基上面,完全吸收后,倒置于37℃培养箱中,过夜培养12h;
(6)挑取重组载体fADL-le-SV阳性克隆,进行PCR鉴定。
7)菌液PCR鉴定
挑单克隆,转入5ml含有卡那霉素抗性的LB液体培养基,37℃,200rpm,8h,分别吸取1μl菌液作为模板进行菌液PCR鉴定,引物和扩增条件如下:
上游引物(PF1):5’-ccgtgcatctgtcctcgttcaa-3’;SEQ ID NO.5;
下游引物(PR1):5’-GTTTTCAGGAAGTAGTTTCCATAGGTC-3’;SEQ ID NO.6;
在载体fADL-le的pIII基因插入位点上游大约700bp处设计上游引物PF1,利用插入片段的反义链作为下游引物进行PCR验证,阳性克隆经PCR后会在700bp处出现特异性目的带,阴性克隆经PCR后无特异性条带的出现。
PCR反应体系如下:
PCR反应程序为:94℃预变性8min;94℃30s,55℃30s,72℃30s,35个循环;72℃延伸10min。
反应结束后,通过琼脂糖凝胶电泳,结果见图4;PCR结果显示,在500bp-750bp之间出现一条特异性目的条带,初步证明外源肽SV的编码片段被成功插入到噬菌体的pIII基因中。
将筛选到的阳性克隆送到上海生工生物技术服务有限公司进行测序,重组载体fADL-le-SV测序结果如下:
测序结果表明,目的片段(加粗加下划线部分和图5)被成功克隆到噬菌体载体fADL-le的PIII基因中,且与连接的原始序列完全一致,表明重组载体fADL-le-SV构建成功。
(二)重组噬菌体载体fADL-le-SV-DP的构建
以构建好的噬菌体载体fADL-le-SV为模板,利用点突变试剂盒(Vazyme,货号:C215)将肽DP(编码P53蛋白N端第49-58位氨基酸DIEQWFTEDP,SEQ ID NO.8;简称DP)定向克隆到噬菌体的PVIII基因中,构建出重组噬菌粒fADL-le-SV-DP,具体步骤如下:
1)载体fADL-le-SV的扩增
利用试剂盒自带试剂进行PCR扩增,反应体系如下:
扩增反应所用引物PF2、PR2序列如下:
上游引物(PF2):5’-ttgaacaatggttcactgaagatcccGCAAAAGCGGCC-3’;SEQ IDNO.9;
下游引物(PR2):5’-cagtgaaccattgttcaatatcAGCGAAAGACAGCATCGGAA-3’;SEQ IDNO.10;
其中,小写字母为编码外源肽DP的部分序列,并用于后期同源重组形成环状重组噬菌粒fADL-le-SV-DP,大写字母用于扩增载体fADL-le-SV序列。
2)扩增产物Dpn I消化
因上一步的扩增产物中包含原始模板质粒,为防止其在转化后形成假阳性转化子,PCR扩增反应结束后加入1μlDpn I,37℃消化2h,去除甲基化模板质粒。
3)重组反应形成fADL-le-SV-DP
重组反应体系如下(37℃反应30min):
4)fADL-le-SV-DP转化大肠杆菌JM109细胞
(1)在大肠杆菌JM109感受态细胞中加入10μl的重组产物,混匀,冰浴30min;
(2)42℃,热击90s;
(3)立即放置到冰上,冰浴10min;
(4)加入800μl LB培养基,37℃,100rpm,45min;
(5)将转化产物均匀涂在含有卡那霉素的LB固体培养基上面,完全吸收后,倒置于37℃培养箱中,过夜培养12h;
(6)挑取重组载体fADL-le-SV-DP阳性克隆,将筛选到的阳性克隆送到上海生工生物技术服务有限公司进行测序。
测序结果如下:
测序结果表明,编码肽DP的目的片段(加粗加下划线部分和图6)被成功克隆到噬菌体载体fADL-le-SV的PVIII基因中,且与连接的原始序列完全一致,表明重组噬菌体载体fADL-le-SV-DP构建成功,可用于下一步噬菌体的制备。
(三)噬菌体phage-SV-DP的制备及Westernblot分析验证
1)噬菌体phage-SV-DP的制备
(1)将200μl测序正确的转化fADL-le-SV-DP的JM109接种到含有100ml LB液体培养基(100μg/ml Kar+)的试管中,37℃剧烈震荡10h;
(2)8000rpm,10min,4℃,留上清;
(3)加入六分之一体积的PEG/NaCl溶液,涡旋,4℃过夜;
(4)12000rpm离心15min,用1ml TBS溶解噬菌体沉淀;
(5)将溶液转移至1.5ml EP管中,14000rpm离心1min,小心将上清转移至1.5ml EP管中,每个EP管中加入150μl PEG/NaCl,混匀后4℃过夜;
(6)14000rpm离心15min,用100μl TBS溶解噬菌体沉淀,4℃冰箱保存。
2)Western-blot分析phage-SV-DP
(1)SDS-PAGE后,将分离胶切下,放置于转移缓冲液中;
(2)将PVDF膜和滤纸按照胶的大小进行裁剪,滤纸浸泡在转移缓存液中,PVDF膜用甲醇浸泡1min后再放置到转移缓存液中;
(3)80V恒压电转2h;
(4)将膜放入封闭液中封闭1h;
(5)PBST洗三次,每次间隔5min;
(6)加入按照1:500稀释的P53多克隆抗体(上海生工生物工程股份有限公司,货号:D220082),37℃,1h;
(7)PBST洗三次,每次间隔5min;
(8)羊抗兔IgG二抗稀释至工作浓度后,将膜条放入其中,37℃,45min;
(9)PBST洗三次,每次间隔5min;
(10)将ECL发光液稀释到工作浓度后,滴加在PVDF膜上,曝光30s。
Western-blot分析结果见图7。phage-DP表示噬菌体表面PVIII展示肽DP,故phage-DP与P53多克隆抗体杂交后在PVIII蛋白的位置出现特异性条带;靶向性双展示噬菌体phage-SV-DP能与P53多克隆抗体发生特异性反应,并同时在噬菌体的PIII及PVIII的位置处出现特异性目的带,而辅助噬菌体M13K07与P53多克隆抗体杂交则无任何条带的出现,表明靶向性双展示噬菌体phage-SV-DP制备成功,且能特异性的识别P53抗体。
3)原子力显微镜(AFM)观察phage-SV-DP
将噬菌体phage-SV-DP用PBS缓冲液稀释到107/ml,吸取20μl滴加到云母片上,低速旋涂1min,然后用AFM进行观察。
AFM的结果见图8,靶向性双展示噬菌体phage-SV-DP大约长900nm、宽7nm,具有相对柔性的结构,外源肽的展示对其形态结构无影响。
(四)噬菌体phage-SV-DP检测癌症患者血清P53抗体的结果
(1)phage-SV-DP-ELISA
以phage-SV-DP为检测抗原,利用ELISA方法对60例乳腺癌患者及60例健康人(阴性对照)进行血清P53抗体的检测,具体步骤如下:
1)以phage-SV-DP作为包被抗原(简称phage-SV-DP-ELISA)包被96孔酶标板,浓度为60μg/ml,每孔50μl,放置湿盒中4℃过夜;
2)第二天,先用PBST缓冲液洗涤3次,每次3min,再用PBS溶液洗涤两次,每次3min;
3)每孔加入200μl封闭液封闭,37℃,1h;
4)洗涤后加入按照1:200稀释的乳腺癌患者或健康人血清,每孔50μl,37℃反应1h;
5)重复洗涤后加入1:5000倍稀释的HRP标记的山羊抗人IgG二抗,每孔50μl,37℃温育45min;
6)重复洗涤后加入100μl底物显色液TMB,室温避光反应12min,每孔加入50μl 2M的硫酸终止反应;
7)利用酶标仪检测OD450nm处的吸光值。每个样本的检测均为平行复孔,结果取检查结果的平均值。
(2)P53-ELISA
以重组P53蛋白(购自Abcam,Catalog number:ab82201)作为包被抗原检测血清53抗体的方法简称为P53-ELISA。除了酶标板上面包被的抗原为重组P53蛋白且浓度为5μg/ml外,整个实验流程与phage-SV-DP-ELISA完全相同。
(3)cut-off值的确立
根据之前确立的phage-SV-DP-ELISA及P53-ELISA检测体系,分别检测60例健康人血清,采用平均值+2SD的方法分别确立每种检测方法的cut-off值。
结果如图9所示,60例乳腺癌患者中,phage-SV-DP检测到14例P53抗体阳性患者血清,检出率为23.33%,高于重组P53蛋白的检测效率21.67%,两种检测方法的特异性分别为95%(phage-SV-DP-ELISA)、95.00%(P53-ELISA)。该结果表明,phage-SV-DP在检测乳腺癌患者血清P53抗体方面,具有特异性强、灵敏度高且制备简单等优点,可用于乳腺癌患者血清P53抗体的检测应用研究。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 新乡学院
<120> 一种靶向性双展示噬菌体及其制备方法和应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8036
<212> DNA
<213> Artificial Sequence
<400> 1
aacgctacta ccattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60
atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120
cgttcgcaga attgggaatc aactgttaca tggaataaaa cttccagaca ccgtacttta 180
gttgcatatt taaaacatgt tgaactacag caccagattc agcaattaag ctctaagcca 240
tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactgtctaa tcctgacctg 300
ttggaatttg cttccggtct ggttcgcttt gaggctcgaa ttaaaacgcg atatttgaag 360
tctttcgggc ttcctcttaa tctttttgat gcaattcgct ttgcttctga ctataataga 420
cagggtaaag acctgatttt tgatttatgg tcattctctt tttctgaact gtttaaagca 480
tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540
aaacatttta caattacccc ctctggcaaa acttcctttg caaaagcctc tcgctatttt 600
ggtttctatc gtcgtctggt taatgagggt tatgatagtg ttgctcttac catgcctcgt 660
aattcctttt ggcgttatgt atctgcatta gttgagtgtg gtattcctaa atctcaattg 720
atgaatcttt ccacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780
tcctcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840
aaatgattaa agttgaaatt aaaccatctc aagcgcaatt tactacccgt tctggtgttt 900
ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960
aatatccggt gcttgtcaag attactctcg acgaaggtca gccagcgtat gcgcctggtc 1020
tgtacaccgt gcatctgtcc tcgttcaaag ttggtcagtt cggttctctt atgattgacc 1080
gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140
caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200
caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta 1260
gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320
caaagcctcc gtagccgttg ctaccctcgt tccgatgctg tctttcgctg atattgaaca 1380
atggttcact gaagatcccg caaaagcggc ctttaactcc ctgcaagcct cagcgaccga 1440
atatatcggt tatgcgtggg cgatggttgt tgtcattgtc ggcgcaacta tcggtatcaa 1500
gctgtttaag aaattcacct cgaaagcaag ctgataaacc gatacaatta aaggctcctt 1560
ttggagcctt ttttttgtcg actaacgagg gcaaatcatg aaatacctat tgcctacggc 1620
ggccgctgga ttgttattac tcgcggccca gccggccatg gcatcagacc tatggaaact 1680
acttcctgaa aacaacgttg gcccgggagg cctgtctcta gaagccgaaa ctgttgaaag 1740
ttgtttagca aaacctcata cagaaaattc atttactaac gtctggaaag acgacaaaac 1800
tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtggtttg 1860
tactggtgac gaaactcagt gttacggtac atgggttcct attgggcttg ctatccctga 1920
aaatgagggt ggtggctctg agggtggcgg ttctgagggt ggcggttctg agggtggcgg 1980
tactaaacct cctgagtacg gtgatacacc tattccgggc tatacttata tcaaccctct 2040
cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc cttctcttga 2100
ggagtctcag cctcttaata ctttcatgtt tcagaataat aggttccgaa ataggcaggg 2160
tgcattaact gtttatacgg gcactgttac tcaaggcact gaccccgtta aaacttatta 2220
ccagtacact cctgtatcat caaaagccat gtatgacgct tactggaacg gtaaattcag 2280
agactgcgct ttccattctg gctttaatga ggatccattc gtttgtgaat atcaaggcca 2340
atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg gtggttctgg 2400
tggcggctct gagggtggcg gctctgaggg tggcggttct gagggtggcg gctctgaggg 2460
tggcggttcc ggtggcggct ccggttccgg tgattttgat tatgaaaaaa tggcaaacgc 2520
taataagggg gctatgaccg aaaatgccga tgaaaacgcg ctacagtctg acgctaaagg 2580
caaacttgat tctgtcgcta ctgattacgg tgctgctatc gatggtttca ttggtgacgt 2640
ttccggcctt gctaatggta atggtgctac tggtgatttt gctggctcta attcccaaat 2700
ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc aatatttacc 2760
ttctttgcct cagtcggttg aatgtcgccc ttatgtcttt ggcgctggta aaccatatga 2820
attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt ttcttttata 2880
tgttgccacc tttatgtatg tattttcgac gtttgctaac atactgcgta ataaggagtc 2940
ttaatcatgc cagttctttt gggtattccg ttattattgc gtttcctcgg tttccttctg 3000
gtaactttgt tcggctatct gcttactttc cttaaaaagg gcttcggtaa gatagctatt 3060
gctatttcat tgtttcttgc tcttattatt gggcttaact caattcttgt gggttatctc 3120
tctgatatta gcgcacaatt accctctgat tttgttcagg gcgttcagtt aattctcccg 3180
tctaatgcgc ttccctgttt ttatgttatt ctctctgtaa aggctgctat tttcattttt 3240
gacgttaaac aaaaaatcgt ttcttatttg gattgggata aataaatatg gctgtttatt 3300
ttgtaactgg caaattaggc tctggaaaga cgctcgttag cgttggtaag attcaggata 3360
aaattgtagc tgggtgcaaa atagcaacta atcttgattt aaggcttcaa aacctcccgc 3420
aagtcgggag gttcgctaaa acgcctcgcg ttcttagaat accggataag ccttctattt 3480
ctgatttgct tgctattggt cgtggtaatg attcctacga cgaaaataaa aacggtttgc 3540
ttgttcttga tgaatgcggt acttggttta atacccgttc atggaatgac aaggaaagac 3600
agccgattat tgattggttt cttcatgctc gtaaattggg atgggatatt atttttcttg 3660
ttcaggattt atctattgtt gataaacagg cgcgttctgc attagctgaa cacgttgttt 3720
attgtcgccg tctggacaga attactttac cctttgtcgg cactttatat tctcttgtta 3780
ctggctcaaa aatgcctctg cctaaattac atgttggtgt tgttaaatat ggtgattctc 3840
aattaagccc tactgttgag cgttggcttt atactggtaa gaatttatat aacgcatatg 3900
acactaaaca ggctttttcc agtaattatg attcaggtgt ttattcatat ttaacccctt 3960
atttatcaca cggtcggtat ttcaaaccat taaatttagg tcagaagatg aaattaacta 4020
aaatatattt gaaaaagttt tctcgcgttc tttgtcttgc gataggattt gcatcagcat 4080
ttacatatag ttatataacc caacctaagc cggaggttaa aaaggtagtc tctcagacct 4140
atgattttga taaattcact attgactctt ctcagcgtct taatctaagc tatcgctatg 4200
ttttcaagga ttctaaggga aaattaatta atagcgacga tttacagaag caaggttatt 4260
ccatcacata tattgattta tgtactgttt caattaaaaa aggtaattca aatgaaattg 4320
ttaaatgtaa ttaattttgt tttcttgatg tttgtttcat catcttcttt tgctcaagta 4380
attgaaatga ataattcgcc tctgcgcgat ttcgtgactt ggtattcaaa gcaaacaggt 4440
gaatctgtta ttgtctcacc tgatgttaaa ggtacagtga ctgtatattc ctctgacgtt 4500
aagcctgaaa atttacgcaa tttctttatc tctgttttac gtgctaataa ttttgatatg 4560
gttggctcta atccttccat aattcagaaa tataacccaa atagtcagga ttatattgat 4620
gaattgccat catctgatat tcaggaatat gatgataatt ccgctccttc tggtggtttc 4680
tttgttccgc aaaatgataa tgttactcaa acatttaaaa ttaataacgt tcgcgcaaag 4740
gatttaataa gggttgtaga attgtttgtt aaatctaata catctaaatc ctcaaatgta 4800
ttatctgttg atggttctaa cttattagta gttagcgccc ctaaagatat tttagataac 4860
cttccgcaat ttctttctac tgttgatttg ccaactgacc agatattgat tgaaggatta 4920
attttcgagg ttcagcaagg tgatgcttta gatttttcct ttgctgctgg ctctcagcgc 4980
ggcactgttg ctggtggtgt taatactgac cgtctaacct ctgttttatc ttctgcgggt 5040
ggttcgttcg gtatttttaa cggcgatgtt ttagggctat cagttcgcgc attaaagact 5100
aatagccatt caaaaatatt gtctgtgcct cgtattctta cgctttcagg tcagaagggt 5160
tctatttctg ttggccagaa tgtccctttt attactggtc gtgtaactgg tgaatctgcc 5220
aatgtaaata atccatttca gacaattgag cgtcaaaatg ttggtatttc tatgagtgtt 5280
tttcccgttg caatggctgg cggtaatatt gttttagata taaccagtaa ggccgatagt 5340
ttgagttctt ctactcaggc aagtgatgtt attactaatc aaagaagtat tgcgacaacg 5400
gttaatttgc gtgatggtca gactcttttg ctcggtggcc tcactgatta caaaaacact 5460
tctcaagatt ctggtgtgcc gttcctgtct aaaatccctt taatcggcct cctgtttagc 5520
tcccgttctg attctaacga ggaaagcacg ttgtacgtgc tcgtcaaagc aaccatagta 5580
cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 5640
tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 5700
gttctccggc tttccccgtc aagctctaaa tcgggggatc tcgggaaaag cgttggtgac 5760
caaaggtgcc ttttatcatc actttaaaaa taaaaaacaa ttactcagtg cctgttataa 5820
gcagcaatta attatgattg atgcctacat cacaacaaaa actgatttaa caaatggttg 5880
gtctgcctta gaaagtatat ttgaacatta tcttgattat attattgata ataataaaaa 5940
ccttatccct atccaagaag tgatgcctat cattggttgg aatgaacttg aaaaaattag 6000
ccttgaatac attactggta aggtaaacgc cattgtcagc aaattgatcc aagagaacca 6060
acttaaagct tatgatgatg atgtgcttaa aaacttactc aatggctggt ttatgcatat 6120
cgcaatacat gcgaaaaacc taaaagagct tgccgataaa aaaggccaat ttattgctat 6180
ttaccgcggc tttttattga gcttgaaaga taaataaaat agataggttt tatttgaagc 6240
taaatcttct ttatcgtaaa aaatgccctc ttgggttatc aagagggtca ttatatttcg 6300
cggaataaac caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg 6360
caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga 6420
aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat 6480
tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc 6540
aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat 6600
ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc 6660
aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt 6720
aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc 6780
aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg 6840
gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg 6900
aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc 6960
aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg 7020
atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc 7080
agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct 7140
cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat 7200
atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc ttttgttgac 7260
aaagggaatc atagatccct ttagggttcc gatttagtgc tttacggcac ctcgacctcc 7320
aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 7380
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 7440
cactcacaac taacccggcc tattcttttg atttataagg atttttgtca ttttctgctt 7500
actggttaaa aaataagctg atttaacaaa tatttaacgc gaaatttaac aaaacattaa 7560
cgtttacaat ttaaatattt gcttatacaa tcatcctgtt tttggggctt ttctgattat 7620
caatcggggt acatatgatt gacatgctag ttttacgatt accgttcatc gattctcttg 7680
tttgctccag actttcaggt aatgacctga tagcctttgt agacctctca aaaatagcta 7740
ccctctccgg catgaattta tcagctagaa cggttgaata tcatattgac ggtgatttga 7800
ctgtctccgg cctttctcac ccgtttgaat ctttgcctac tcattactcc ggcattgcat 7860
ttaaaatata tgagggttct aaaaattttt atccctgcgt tgaaattaag gcttcaccag 7920
caaaagtatt acagggtcat aatgtttttg gtacaaccga tttagcttta tgctctgagg 7980
ctttattgct taattttgct aactctctgc cttgcttgta cgatttattg gatgtt 8036
<210> 2
<211> 12
<212> PRT
<213> Artificial Sequence
<400> 2
Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val
1 5 10
<210> 3
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 3
cggccatggc atcagaccta tggaaactac ttcctgaaaa caacgttggc ccggg 55
<210> 4
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 4
gggccaacgt tgttttcagg aagtagtttc cataggtctg atgccatggc cggct 55
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 5
ccgtgcatct gtcctcgttc aa 22
<210> 6
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 6
gttttcagga agtagtttcc ataggtc 27
<210> 7
<211> 626
<212> DNA
<213> Artificial Sequence
<400> 7
acaatttatc aggcgatgat acaaatctcc gttgtacttt gtttcgcgct tggtataatc 60
gctgggggtc aaagatgagt gttttagtgt attctttcgc ctctttcgtt ttaggttggt 120
gccttcgtag tggcattacg tattttaccc gtttaatgga aacttcctca tgaaaaagtc 180
tttagtcctc aaagcctccg tagccgttgc taccctcgtt ccgatgctgt ctttcgctgc 240
tgagggtgac gatcccgcaa aagcggcctt taactccctg caagcctcag cgaccgaata 300
tatcggttat gcgtgggcga tggttgttgt cattgtcggc gcaactatcg gtatcaagct 360
gtttaagaaa ttcacctcga aagcaagctg ataaaccgat acaattaaag gctccttttg 420
gagccttttt tttgtcgact aacgagggca aatcatgaaa tacctattgc ctacggcggc 480
cgctggattg ttattactcg cggcccagcc ggccatggca tcagacctat ggaaactact 540
tcctgaaaac aacgttggcc cgggaggcct gtctctagaa gccgaaactg ttgaaagttg 600
tttagcaaaa cctcatacag aaaatc 626
<210> 8
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 8
Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro
1 5 10
<210> 9
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 9
ttgaacaatg gttcactgaa gatcccgcaa aagcggcc 38
<210> 10
<211> 42
<212> DNA
<213> Artificial Sequence
<400> 10
cagtgaacca ttgttcaata tcagcgaaag acagcatcgg aa 42
<210> 11
<211> 606
<212> DNA
<213> Artificial Sequence
<400> 11
gagcaggtcg cggatttcga cacaatttat caggcgatga tacaaatctc cgttgtactt 60
tgtttcgcgc ttggtataat cgctgggggt caaagatgag tgttttagtg tattctttcg 120
cctctttcgt tttaggttgg tgccttcgta gtggcattac gtattttacc cgtttaatgg 180
aaacttcctc atgaaaaagt ctttagtcct caaagcctcc gtagccgttg ctaccctcgt 240
tccgatgctg tctttcgctg atattgaaca atggttcact gaagatcccg caaaagcggc 300
ctttaactcc ctgcaagcct cagcgaccga atatatcggt tatgcgtggg cgatggttgt 360
tgtcattgtc ggcgcaacta tcggtatcaa gctgtttaag aaattcacct cgaaagcaag 420
ctgataaacc gatacaatta aaggctcctt ttggagcctt ttttttgtcg actaacgagg 480
gcaaatcatg aaatacctat tgcctacggc ggccgctgga ttgttattac tcgcggccca 540
gccggccatg gcatcagacc tatggaaact acttcctgaa aacaacgttg gcccgggagg 600
cctgtc 606
Claims (3)
1.一种靶向性双展示噬菌体,其特征在于,利用噬菌体展示技术将编码P53蛋白N端表位多肽SV及DP分别定向克隆到丝状噬菌体的PIII及PVIII基因中,制备出尾部PIII蛋白展示肽SV、背部PVIII蛋白展示肽DP的靶向性双展示噬菌体phage-SV-DP;所述SV的氨基酸序列如SEQ ID NO.2所示;所述DP的氨基酸序列如SEQ ID NO.8所示。
2.根据权利要求1所述的一种靶向性双展示噬菌体的制备方法,其特征在于,具体步骤如下:
1)构建重组噬菌体载体fADL-le-SV
(1)BglI酶切载体fADL-le,获得酶切后的载体fADL-le;
(2)合成P53蛋白N端表位多肽SV;
(3)将步骤(1)酶切后的载体fADL-le与步骤(2)合成的SV连接,转化,筛选阳性克隆,获得重组噬菌体载体fADL-le-SV;
2)构建重组噬菌体载体fADL-le-SV-DP
以构建好的噬菌体载体fADL-le-SV为模板,利用点突变试剂盒将肽DP定向克隆到噬菌体的PVIII基因中,构建获得重组噬菌体载体fADL-le-SV-DP;
3)制备噬菌体phage-SV-DP
(1)将转化有重组噬菌体载体fADL-le-SV-DP的菌株接种到含有Kar+的LB液体培养基中,37℃剧烈震荡10h;
(2)8000rpm,10min,4℃,留上清;
(3)加入六分之一体积的PEG/NaCl溶液,涡旋,4℃过夜;
(4)12000rpm离心15min,用1ml TBS溶解噬菌体沉淀;
(5)将溶液转移至1.5ml EP管中,14000rpm离心1min,小心将上清转移至1.5ml EP管中,每个EP管中加入150μl PEG/NaCl,混匀后4℃过夜;
(6)14000rpm离心15min,用100μl TBS溶解噬菌体沉淀,4℃冰箱保存。
3.权利要求1或2所述的靶向性双展示噬菌体在检测血清P53抗体中的应用。
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WO2001068801A2 (en) * | 2000-03-16 | 2001-09-20 | Ramot At Tel-Aviv University Ltd. | Single chain antibody against mutant p53 |
CN1508254A (zh) * | 2002-12-19 | 2004-06-30 | 中国人民解放军第二军医大学 | 新型噬菌粒展示载体pCANTAB5L |
CN105018507A (zh) * | 2014-04-17 | 2015-11-04 | 东北师范大学 | 一种用于双表位展示的噬菌体载体及其构建方法 |
CN106906187A (zh) * | 2017-03-17 | 2017-06-30 | 浙江大学 | 一种双功能性噬菌体及用途 |
CN108752425A (zh) * | 2018-06-07 | 2018-11-06 | 南方医科大学 | 利用噬菌体展示技术构建细胞穿透肽表达文库的方法 |
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WO2001068801A2 (en) * | 2000-03-16 | 2001-09-20 | Ramot At Tel-Aviv University Ltd. | Single chain antibody against mutant p53 |
US20030022244A1 (en) * | 2000-03-16 | 2003-01-30 | Ramot University Authority For Applied Research & Industrial Development Ltd. | Single chain antibody against mutant P53 |
CN1508254A (zh) * | 2002-12-19 | 2004-06-30 | 中国人民解放军第二军医大学 | 新型噬菌粒展示载体pCANTAB5L |
CN105018507A (zh) * | 2014-04-17 | 2015-11-04 | 东北师范大学 | 一种用于双表位展示的噬菌体载体及其构建方法 |
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