CN114134165A - 一种新型hpv治疗性核酸疫苗 - Google Patents
一种新型hpv治疗性核酸疫苗 Download PDFInfo
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Abstract
本发明提供的核酸序列，包括1:1的HPV16‑AVLS1序列和HPV16‑AVLC1序列；以及1:1的HPV18‑AVLS1序列和HPV18‑AVLC1序列；序列AVLS1和序列AVLC1分别包括两个串联的E6蛋白、两个L1短肽，两个L2短肽、两个串联的E7蛋白、一个PADRE序列及一个adjuvant序列；序列AVLS1的N端带有小鼠IgK分泌肽序列；序列AVLC1的N端带有泛素序列。本发明提供的核酸序列不仅可以诱导高效价的抗E6/E7抗体，还能诱导高水平的功能性细胞免疫，表现出极佳的预防和治疗HPV相关肿瘤效果。
Description
技术领域
本发明涉及肿瘤的免疫治疗和预防，具体涉及一种新型HPV治疗性核酸疫苗。
背景技术
全世界大约20-25%的癌症病例都是由传染源引起的。其中约15%的人类癌症与病毒感染有关。人类乳头瘤病毒（human papilloma virus，HPV) 引起了约 30% 的所有传染源相关癌症，并且与超过95%的宫颈癌有关。2018年，全球有超过300000 名女性死于宫颈癌。这是高危人乳头瘤病毒 (high risk HPV，hrHPV)持续感染的结果。
目前HPV预防性疫苗已上市，预防性疫苗通过诱导针对主要衣壳蛋白 L1的类型特异性中和抗体来预防持续性宫颈感染。然而它们对预先存在的HPV感染或癌前病变治疗效果不显著。另外，现有的宫颈癌治疗方法均疗效有限，且由于全球范围内的HPV预防接种率不理想，HPV感染和随后发生的HPV相关恶性肿瘤将在未来几十年仍然是公共卫生问题。因此，开发治疗性HPV疫苗和其他癌症疗法是一项紧迫的任务。
已知早期蛋白E6和E7是导致 HPV 相关癌症的恶性进展的原因，E6可以结合和降解肿瘤抑制因子 p53，而 E7 通过抑制 pRb 从而使细胞周期不受控制地进入 S 期。因此，HPV E6和E7 癌蛋白可作为治疗性 HPV 疫苗的理想靶标，目前绝大多数 HPV 治疗性疫苗都靶向这两种抗原。然而，这些靶向E6 和 E7的疫苗对 HPV相关癌症的疗效非常有限。主要原因可能包括以下几点： E6 和 E7 在上皮基底膜细胞中仅适度表达，它可能在恶性细胞中上调之前，成功地逃避对过度表达的 E6 和 E7 的免疫，最终引起持续的病毒感染；E6和E7蛋白较小，包含的抗原表位较少；现有的治疗性疫苗抗原表达水平偏低，递送效率低，结构设计不完善，不能引起足够的免疫反应强度。因此，可能需要更广泛的抗原靶向策略和抗原序列设计来治疗HPV+ 癌症和清除持续的癌前感染。
HPV有两种衣壳蛋白，分别是L1和L2，其中L1是主要的衣壳蛋白，L2可以稳定病毒衣壳结构。目前的HPV预防性疫苗是L1蛋白表达出来后自己会组装成病毒样颗粒（virus-like-particle，VLP），可诱导人体产生中和抗体，表现出很好的预防HPV感染效果。VLP作为亚单位蛋白疫苗刺激细胞免疫反应强度较弱，因此表现出有限的治疗效果。研究结果显示L1和L2的DNA疫苗形式，同时可以引起很强的体液免疫和细胞免疫，体现了作为治疗性疫苗靶标的潜力。
发明内容
本发明通过对HPV16和HPV18 的E6、E7、L1和L2的抗原序列设计以及密码子优化；融合L1/L2辅助T细胞表位，两个全长E6和两个全长E7蛋白，佐剂序列Beta defensin-3和通用Th表位PADRE序列设计出新型HPV治疗性核酸疫苗AVL101核酸序列。AVL101共包括四条核酸序列，每条疫苗序列不仅包含两个全长E6和两个全长E7蛋白，也同时包含了L1/L2的辅助T细胞的免疫表位序列。该序列组合不仅可以诱导高效价的抗E6/E7抗体，同时还能高效诱导T细胞免疫，最终表现出高效抑制表达E6/E7的癌细胞的生长。
具体的，本发明提供一种用于治疗和预防HPV感染疾病的核酸序列，包括：
1:1的序列HPV16-AVLS1和序列HPV16-AVLC1；
以及，1:1的序列HPV18-AVLS1和序列HPV18-AVLC1；
优选为1:1:1:1的HPV16-AVLS1、HPV16-AVLC1、HPV18-AVLS1和HPV18-AVLC1；
其中，序列AVLS1和序列AVLC1分别包括两个串联的蛋白E6分子、两个L1短肽，两个L2的短肽、两个串联的蛋白E7分子、一个PADRE序列及一个adjuvant序列。
所述L1短肽选自SEQ ID No：1~SEQ ID No：4所示序列中的至少一种；
所述L2短肽选自SEQ ID No：5~SEQ ID No：8所示序列中的至少一种；
序列AVLS1的N端带有小鼠IgK分泌肽序列；序列AVLC1的N端带有泛素分子。
本发明通过对HPV16的E6、E7、L1和L2的抗原序列组合设计以及密码子优化提供了用于治疗HPV16或HPV18 或两者共感染的相关疾病的DNA疫苗序列。
本发明AVLS1与AVLC1编码蛋白序列的差别只在N端，其余序列均一样。AVLS2的N端带有一段小鼠IgK分泌肽序列，可以促进蛋白分泌到细胞外被抗原呈递细胞捕获而被呈递，进而促进辅助T细胞免疫响应；而AVLC1 的带有一段泛素(ubiquitin)分子，可以促进蛋白的降解进而增强CD8+ T细胞免疫。其余相同的部分包括两个完整的E6分子，两个L1短肽，两个L2的短肽，两个完整的E7分子，一个PADRE序列及一个adjuvant序列。本发明提供的L1和L2的短肽是助细胞免疫结合表位序列（helper T Cell epitope），将其引入DNA疫苗序列的可以帮助刺激增强对HPV感染细胞的CTL杀伤；
本发明为了克服HLA-2等位基因的高多态性，在构建过程中添加了通用的PADRE(Pan-HLA DR)序列；其中，所述PADRE序列为SEQ ID No：9。
同时，本发明在C末端加了一段佐剂肽Beta-defensin-3，该肽可以作为Toll样受体 (toll like receptor)的激动剂促进先天免疫。其中，所述adjuvant序列为SEQ ID No：10。
其中，所述小鼠IgK分泌肽序列为SEQ ID No：11；
其中，所述泛素分子的序列为SEQ ID No：12。
其中，各个元件之间使用AGA(Ala-Gly-Ala)或AAY (Ala-Ala-Tyr)连接子；佐剂肽使用刚性连接子EAAAK(Glu-Ala-Ala-Ala-Lys)连接；这些连接子将确保最大的免疫原性和抗原表位的呈递。最终疫苗结构图如图1所示。
本发明提供的，HPV16融合蛋白E6序列引入C70G和I135T突变，HPV18融合蛋白E6序列引入C65G和I130T突变，消除它们降解p53的能力；
HPV16的E7蛋白序列引入C24G和E26G突变，HPV18的E7蛋白序列引入C27G和E29G突变，使它们E7蛋白失去恶性转化的能力。
优选的，所述核酸序列为SEQ ID No：13~ SEQ ID No：16所示四条序列之一。
本发明使用HPV16和18两种亚型作为示例，能引起E6和E7特异的体液免疫和细胞免疫应答，而且HPV16疫苗序列表现出高效抑制表达HPV16 E6/E7的TC-1肿瘤的生长。通过比较含有本发明设计的疫苗序列和现在临床上速度最快已经处于临床三期的HPV DNA疫苗序列，本发明序列疫苗表现出了更好的免疫响应效果和抗肿瘤效果。本发明序列设计方法也可以适用于其它HPV亚型相关疾病的治疗性疫苗开发。
本发明提供的疫苗DNA序列可以独立存在，也可以连接到真核表达载体上，也可以转换为mRNA序列，作为疫苗的组分。
即，本发明提供的所述核酸序列转换的mRNA序列，可作为疫苗的组分。
本发明的又一目的在于，提供一种重组载体，包括表达载体和上述任一所述的核酸序列。
其中，序列AVLS1和序列AVLC1的两端通过HindIII和XhoI酶切位点，连入表达载体AVL0318；所述表达载体为无抗生素标记的AVL0318载体。
本发明的又一目的在于，提供一种扩增上述重组载体的制备方法，包括如下步骤：
1）将E6/E7蛋白、L1/L2多肽与IgK, Ubquitin，PADRE和adjuvant的氨基酸序列进行拼接合成，合成疫苗序列HPV16-AVLS1、HPV16-AVLC1、HPV18-AVLS1和HPV18-AVLC1四条核酸序列；优选合成为如SEQ ID No：13~ SEQ ID No：16所示序列；
2）将上述四条核酸序列利用HindIII和XhoI酶切位点连入载体PUC57；再把疫苗序列亚克隆到表达载体AVL0318；获得四个重组载体AVL0318-HPV16/18-AVLS1/AVLC1；
3）使用大肠杆菌AVL-DH5α(SacB)对AVL0318-HPV16/18-AVLS1/AVLC1的质粒进行扩增。
其中，所述大肠杆菌（大肠埃希氏菌(Escherichia coli)）AVL-DH5α(SacB)的基因组序列中含有组成型表达的SacB基因，且不含有抗生素筛选基因；
其中，能够表达SacB基因的序列为SEQ ID No：17。
本发明提供的大肠杆菌菌株AVL-DH5α(SacB)的制备方法：在大肠杆菌的attB位点插入SacB基因；所述基因编辑是通过包括下述步骤的方法实现的：
1）分别PCR扩增插入位点上下游同源臂基因序列、p5/6 6/6-SacB基因序列，以上述三个序列为模板重叠延伸PCR扩增长片段SacB-CRISPR核苷酸序列；所述SacB-CRISPR的核苷酸序列如SEQ ID No.18所示；
2）将表达Cas9质粒、SgRNA和长片段SacB-CRISPR核苷酸序列转化到大肠杆菌DH5α感受态细胞中，进行基因编辑和同源重组修复，挑取单克隆培养后PCR测序验证，最后通过温度敏感消除工具质粒和编辑菌的抗性，即得可用于无抗生素筛选目标质粒的AVL-DH5α(SacB)菌株。
本发明进一步提供的，菌株生产无抗生素筛选标记质粒的方法，具体包括如下步骤：
1）将AVL-DH5α(SacB)菌株涂布在6%蔗糖的LB培养基上，37℃培养24h，上述菌株不能生长。
2）制备AVL-DH5α(SacB)感受态细胞，转化带有抑制SacB表达的RNA-out基因片段的AVL0318质粒。涂布在6%蔗糖的LB培养基上，37℃培养24h，上述菌株可以生长。
本发明通过CRISPR-cas9技术将SacB基因（蔗糖致死基因）转入大肠杆菌，SacB编码蔗糖果聚糖酶，该酶能催化蔗糖水解成葡萄糖和果糖，并且将果糖聚合成高分子量的果聚糖，而高分子量果聚糖积累对细胞具有毒性作用，可造成大肠杆菌死亡。本发明构建的质粒系统，带有HPV的目的片段以及抑制SacB表达的RNA-out基因片段，当把质粒系统导入重组的大肠杆菌后，质粒上RNA-out基因会表达一段序列，把SacB基因沉默，即使在有蔗糖的培养基上进行培养，大肠杆菌依然不会死亡。如此能快速将转入HPV疫苗重组载体的菌株与没转入重组载体的菌株进行区分。
本发明提供的一种新型HPV治疗性核酸疫苗，包括上述任一所述的核酸序列，或该核酸序列转化的mRNA序列，或包括所述核酸序列的重组载体，以及上述制备方法制得的重组载体。
本发明联合HPV 治疗性疫苗的设计方法、高效安全的表达载体和高产安全的质粒生产菌株，提供了一种新型适用于不同亚型HPV治疗性核酸疫苗的开发和应用的系统方案。
本发明提供的DNA疫苗序列组合物不仅可以诱导高效价的抗E6 /E7抗体，同时还能高效诱导T细胞免疫，最终高效抑制表达E6/E7癌细胞的生长。该DNA疫苗序列设计方法可同时适用于各亚型HPV，如高危型HPV16，18，58，52，33，这五种亚型占世界所有人群癌前病变和宫颈癌发生的约90%，因此本发明可以用于治疗HPV不同亚型的感染疾病。
附图说明
图1为本发明提供的HPV治疗性核酸疫苗的结构示意图；
图2为AVL0318载体图谱；
图3为本发明提供的疫苗序列表达质粒酶切鉴定图；
图4为本发明提供的疫苗序列质粒在HEK-293T细胞内的表达检测图；
图5a为HPV16 E6和E7特异性细胞免疫检测结果图；
图5b为HPV18 E6和E7特异性细胞免疫检测结果图；
图6a为HPV16 E6和E7特异性抗体检测结果图；
图6b为HPV18 E6和E7特异性抗体检测结果图；
图7为HPV16疫苗对小鼠TC-1同种移植瘤的治疗的效果图；
图8为HPV16疫苗对小鼠TC-1同种移植瘤的预防效果图。
图9为attB上下游同源臂及SacB F3/R1扩增电泳检测图谱；
图10为SacB-CRISPR融合扩增结果图；
图11为AVL-DH5α(SacB)菌落PCR鉴定图谱。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
本发明一种用于治疗HPV感染疾病的核酸序列AVL101包括HPV16的两条核酸序列和HPV18的两条核酸序列；分别为HPV16-AVLS1和HPV16-AVLC1；HPV18-AVLS1和HPV18-AVLC1。
HPV16和18的 AVLS1与AVLC1编码蛋白序列的差别只在N端，其余序列均一样。AVLS1的N端带有一段小鼠IgK分泌肽序列，可以促进蛋白分泌到细胞外被抗原呈递细胞捕获而被呈递，进而促进辅助T细胞免疫响应；而AVLC1 的带有一段泛素(ubiquitin)分子，可以促进蛋白的降解进而增强CD8+T细胞免疫。
其余相同的部分包括两个串联完整的E6分子，两个L1短肽，两个L2的短肽，两个串联完整的E7分子，一个PADRE序列及一个adjuvant序列。L1和L2的短肽是助细胞免疫结合表位序列（helper T Cell epitope），引入我们DNA疫苗序列的原因是可以帮助刺激增强对HPV感染细胞的CTL杀伤；为了克服HLA-2等位基因的高多态性，在构建过程中添加了通用的PADRE (Pan-HLA DR)序列；同时在C末端加了一段佐剂肽Beta-defensin-3，该肽可以作为Toll样受体 (toll like receptor)的激动剂促进先天免疫。各个元件之间使用AGA(Ala-Gly-Ala)或AAY (Ala-Ala-Tyr)连接子；佐剂（adjuvant）肽使用刚性连接子EAAAK(Glu-Ala-Ala-Ala-Lys)；这些连接子将确保最大的免疫原性和抗原表位的呈递；最终疫苗结构如图1所示。
另外，HPV16融合蛋白E6序列引入C70G和I135T突变，HPV18融合蛋白E6序列引入C65G和I130T突变，消除它们降解p53的能力； HPV16的E7蛋白序列引入C24G和E26G突变，HPV18的E7蛋白序列引入C27G和E29G突变，使它们E7蛋白失去恶性转化的能力。
本发明通过融合L1/L2辅助T细胞表位，两个全长E6/E7蛋白，佐剂序列Betadefensin-3和通用Th表位PADRE序列设计出新型HPV治疗性核酸疫苗。本发明使用HPV的两个亚型16和18作为示例，该疫苗能引起E6和E7特异的体液免疫和细胞免疫应答，并且HPV疫苗序列表现出高效的预防和治疗TC-1肿瘤细胞的效果。通过比较含有本发明设计序列的疫苗和现在临床上速度最快已经处于临床三期的HPV DNA疫苗序列，本发明序列相应疫苗表现出了更好的免疫效果和抗肿瘤效果。本发明序列设计方法也可以适用于其它HPV亚型相关疾病的治疗性疫苗开发。
本发明同时提供了作为DNA疫苗使用的表达载体和质粒生产菌株。目前普遍使用的表达载体多采用抗生素抗性如KanR，AmpR等作为选择标记，在发酵培养基中添加相应抗生素维持选择压力可以稳定质粒在菌体中的存在。抗生素作为传统的选择标记，在实验室研究水平和工业生产水平具有广泛的适用性和有效性，是最方便的工具之一。然而，抗生素在医疗中的过度使用已经成为一个严重的问题。许多病原菌在抗生素的泛滥使用环境下已发生变异产生相关抗性，相关抗生素已不能控制其生长和感染。另外，抗性基因通常位于可移动的质粒DNA单元上，它可在不同宿主间进行传递，最终可能导致向环境其它微生物中发生抗性基因的转移。美国食品和药物管理局(FDA)和世界卫生组织(WHO)等监管机构认为质粒骨架中存在抗生素抗性基因是不受欢迎的，需要避免在最终商品化产物例如DNA疫苗中使用抗生素抗性基因。它们认为除了抗生素抗性可能转移到内源性微生物群中外，质粒抗性基因也有一定概率整合到人体染色体中，激活和转录相关癌基因。例如，有关DNA疫苗质粒的监管指南指出：“应避免使用某些选择标记，例如抗生素抗性，这可能会对目标人群的其他临床治疗产生不利影响”。此外，在发酵培养中使用抗生素需要在质粒纯化过程中对抗生素去除进行昂贵的工艺验证。因此考虑以上因素，因此本发明选用无抗生素的质粒筛选策略。
本发明以常用的大肠杆菌菌株为基础，利用CRISPR/Cas9技术敲入大肠杆菌DH5α优化后的SacB基因序列制备菌株AVL-DH5α(SacB)，使其适用于无抗生素质粒生产。本发明利用了基于RNA的可选择标记（RNA based selectable marker）对质粒进行筛选和维持。但是不同于Luke等人利用同源重组发明的大肠杆菌SacB表达菌株，需要氯霉素抗性基因来筛选重组子，本发明使用了更方便的CRISPR/Cas9技术，不需要额外的抗性基因对大肠杆菌重组子进行选择，AVL-DH5α(SacB)菌株作为质粒生产的宿主菌安全性会更高。另外相比Luke发明的SacB表达菌株本发明菌株对SacB的密码子进行了优化，使得SacB在大肠杆菌中表达更高，最后筛选到的重组菌株对蔗糖更敏感，筛选无抗质粒的效率更高，而且可以大幅提高质粒的产量。
本发明联合HPV治疗性核酸疫苗的设计方法、高效安全的表达载体和高产安全的质粒生产菌株，提供了一种新型适用于不同亚型HPV治疗性核酸疫苗的开发和应用的系统方案。
实施例1 核酸序列的设计方案以及构建与制备
1、HPV16/18 E6，E7，L1和L2目的片段的获得
从GenBank上下载HPV16毒株NC_001526.4的E6/E7和L1/L2的DNA序列和蛋白序列；
从GenBank上下载HPV18毒株AY262282.1的E6/E7和L1/L2的DNA序列和蛋白序列。
2、序列优化
根据疫苗蛋白结构把所有序列拼接以后，HPV16-AVLS1和HPV16-AVLC1分别包含685和738个氨基酸，HPV18-AVLS1和HPV18-AVLC1分别包含702和755个氨基酸，然后对密码子进行优化，这些优化方式包括但不限于：人类密码子使用偏好性，适度的GC含量，稳定的mRNA二级结构等，消除重复序列和隐蔽剪接位点以及不需要的限制性酶切位点，同时防止细胞中tRNA库的耗尽。
3、序列合成和重组质粒的构建
优化完成后序列直接进行基因合成，利用HindIII和XhoI酶切位点连入载体PUC57，随后把疫苗序列克隆到表达载体AVL0318。
具体的，通过HindIII和XhoI双酶切AVL0318和PUC57- HPV16/18-AVLS1/ AVLC1，琼脂糖凝胶电泳，纯化回收载体AVL0318和目的条带HPV16/18-AVLS1/AVLC1，然后通过T4DNA ligase把HPV16/18-AVLS1/ AVLC1分别连入AVL0318载体，AVL0318载体的图谱如图2所示。
实施例2 重组质粒酶切和表达鉴定
1. 酶切鉴定
使用大肠杆菌AVL-DH5α(SacB)对AVL0318-HPV16/18-AVLS1/AVLC1的质粒分别扩增，然后使用无内毒素质粒大提试剂盒进行质粒纯化。
具体操作方式如下：
1）使用AVL-DH5α(SacB)菌株在6%蔗糖LB培养基上，37℃培养24h。
2）使用质粒大提试剂盒进行质粒提取。
3）使用HindIII和XhoI双酶切验证质粒的正确性，并测序验证序列的正确型，如图3所示。
2.表达鉴定
使用HEK-293T细胞系进行质粒表达鉴定。使用6孔板培养HEK-293T细胞，使用lipofectamine2000把1.5 μg质粒转入细胞中，转染48h后收细胞，进行Western Blo鉴定。四条重组载体均能在HEK-293细胞有效表达；如图4所示。
实施例3 细胞免疫分析
3.1 免疫接种，C57BL/6J小鼠以2周间隔在每个耳朵的耳廓经皮下用30 μg HPV16DNA 疫苗AVL0318-AVLS1和 AVL0318-AVLC1（1：1）或用30μg HPV18 DNA 疫苗AVL0318-AVLS1和 AVL0318-AVLC1（1：1）(质粒溶解在TE缓冲液中，即15 μg (20 μL)/耳)免疫接种3次。每组使用3-5 只小鼠。三次免疫结束后一周对免疫反应进行检测。取小鼠脾细胞ELISPOT检测E6和E7特异性细胞免疫应答。
3.2 ELISPOT检测
3.2.1 脾细胞获取
小鼠取血约500μL于1.5mLEP管，室温静止约1h，取血清分装-80℃储存；小鼠脱颈处死，酒精浸泡5min后，将小鼠转至超净台内，剖腹，取脾。将取好的脾轻轻研磨，期间多次倒入PBS冲洗研磨出的单细胞，离心收获，300 g，5 min离心。
3.2.2 裂红
弃上清，每管加入10 mL红细胞裂解液和约10 mL的PBS，静置约2-3 min，300 g，5min离心，弃上清。
3.2.3 细胞清洗及重悬
使用10 mL的5% FBS的1640混悬，清洗一遍，同等转速离心，弃上清，加入10 mL10%FBS的1640重悬混匀，取20 μL计数。按2x106/mL调节细胞浓度，即2x105/100μL。
3.2.4 孔板清洗及铺板
（1）96孔板润洗：将试剂盒中的96孔板取出，加入PBS 200μL/孔清洗两遍，再加入10%FBS的1640培养基200μL/孔，置于37℃培养箱30min；
（2）细胞铺板：弃培养基，加入已稀释好的细胞100μL；
（3）抗原配置：单肽配制终浓度为20μg/mL，多条肽配制：每条多肽的终浓度为2μg/mL；PMA配制为终浓度50ng/mL；
（4）铺好细胞的96孔板每孔加入相应抗原100μL,Control则补培养基100μL，于37℃5%CO2培养箱孵育24h。
3.2.5 IFN-γ分泌细胞检测（CTL ELISPOT试剂盒）
（1）清洗：取出孵育好的96孔板，弃上清，PBS 200μL/孔清洗2遍，PBST（0.05%tween 20 ）200μL/孔洗2遍；
（2）检测抗体配制：10μL detection抗体加入到10mL Diluent B，每孔80μL，室温孵育2h；
（3）弃上清，使用PBST 200μL/孔洗板3次；
（4）二抗配制：10μL Strep-AP二抗加入到10mL Diluent C，每孔80μL，室温孵育30min；
（5）弃上清，使用200μL/孔PBST清洗两次，再用纯水清洗两次；
（6）准备Blue Developer solution：10mL Diluent Blue内加入160μL S1混匀，再加入160μL S2混匀、再92μL S3混匀，96孔板每孔加入80μL，室温孵育15-30min。
（7）弃上清，纯水200μL/孔洗板3次；
（8）将96孔板每列条孔拆下，在纯水里清洗底部白膜，无需安装回去，每列条孔放于96孔板架上室温过夜使之干燥，读板。
3.3 结果分析
HPV16 DNA疫苗的特异性γ-干扰素分泌如下图5a所示。其中P1和P2分别是国际上进展最快的宫颈癌治疗性DNA疫苗，目前在做临床三期。P1和P2分别是HPV16和HPV18的E6E7融合表达质粒，在此作为阳性对照。结果显示本发明提供的疫苗序列能同时激起靶向E6和E7的细胞免疫，而对照P1只能引起E7的特异性细胞免疫，同时本发明的HPV16 DNA疫苗激起的E7的特异性细胞免疫显著高于P1。
HPV18 DNA疫苗的特异性γ-干扰素分泌如下图5b所示。结果显示本发明疫苗序列和P2均能同时激起靶向E6和E7的细胞免疫，但是本分明疫苗的效果显著高于对照组P2。
实施例4 ELISA检测对E6和E7的抗体应答（体液免疫）
三次免疫结束后一周眼眶取血检测E6和E7特异性体液免疫。
4.1 操作步骤
4.1.1 制备抗原包被板
（1）配制CBS缓冲液（0.05 mol/L、pH 9.6）
碳酸钠 1.59 g
碳酸氢钠 2.93 g
加900 ml超纯水溶解，调节pH到9.6，加超纯水定容到1L。4℃保存。
（2）抗原包被
用（1）中所配CBS将HPV16/18的E6和E7蛋白原液稀释到5 μg/mL，稀释后的抗原溶液包被96孔板（Corning 3590）（100 μL/孔），密封置4°C冰箱包被过夜；
4.1.2 封闭抗原包被板
（1）配制PBST洗液（0.01 mol/L、pH 7.4，PBS，含0.05% Tween 20）
（2）配制封闭液（5% 脱脂奶粉）——第二天现配
脱脂奶粉 5 g
PBST 100 mL
（3）用PBST洗液洗涤包被板3次，用封闭液于室温封闭2 h（100 μL/孔）；
4.1.3 样品准备和孵育
（1）稀释血清，每孔用封闭液按1：100稀释血清，即每孔加入198μL稀释液+2μL待测血清。
（2）将96孔板盖好盖子后置于37°C振荡（500 rpm）孵育1 h。
4.1.4 二抗孵育
（1）用封闭液按1：8000稀释二抗（HRP anti-mIgG）。
（2）一抗孵育结束用PBST洗液洗板5次；每孔加入50μL二抗稀释液，将96孔板盖好盖子后置于37°C振荡（500 rpm）孵育1 h。
4.1.5 显色
用PBST洗液洗板5次，加TMB显色剂（50 μL/孔），盖好盖子后置于室温下孵育5-20min（依据反应显色情况）；
4.1.6 终止反应
（1）配制终止液（2 M H2SO4）——酸入水
浓硫酸 11.1 mL
超纯水 89.9 mL；
（2）根据反应显色结果，加入终止液，50 μL/孔，终止反应；
4.1.7 检测
酶标仪检测OD450吸光度值，使用GraphPad Prism分析并做图。
4.2 结果分析
HPV16 DNA疫苗的特异性抗体检测结果如下图6a所示。结果显示本发明的疫苗序列能同时激起靶向E6和E7的体液免疫，而且显著高于对照P1体液免疫响应。
HPV18 DNA疫苗的特异性抗体检测结果如下图6b所示。结果显示本发明的疫苗序列能同时激起靶向E6和E7的体液免疫，而且显著高于对照P2体液免疫响应。
实施例5 治疗肿瘤效果
该研究旨在确定是否可以通过标准皮下注射HPV16-AVLS1/AVLC1治疗性给药方式诱导抑制TC-1肿瘤生长或TC-1肿瘤消退。
5.1 TC-1 细胞培养和接种
TC-1 细胞在无菌条件下在含有 20 mM HEPES 缓冲液、10% FCS（Hyclone）、50 μM2-巯基乙醇（Sigma）、1 mM 丙酮酸钠（Gibco）、0.292 mg/mL 谷氨酰胺，100 U/mL青霉素/100 ug/mL 链霉素/0.292 mg/mL谷氨酰胺的 RPMI 培养基中生长在75 cm2平底组织培养瓶中。当贴壁TC-1细胞接近长满时，使用0.25%胰蛋白酶/EDTA消化收集它们，并重新接种到更多的培养瓶中以保持对数生长。
在接种当天，指数期生长的TC-1细胞使用胰蛋白酶/EDTA收获。用上述 RPMI 培养基洗涤细胞，然后用PBS再洗涤两次，然后用台盼蓝染色细胞并使用血细胞计数器计数活细胞。将TC-1 细胞在PBS中调整为1x106个细胞/mL，以准备注射100 ul细胞悬液/小鼠（即1x105 个细胞）。
所有C57BL/6J小鼠购买自维通利华，饲养在无特定病原体的条件下，均为雌性，在实验开始时为 6-10 周龄。使用 BD 1mL 超细胰岛素注射器（0.33mm×12.7 mm）将 TC-1细胞注射到轻度麻醉的小鼠（吸入甲氧基氟醚）的背部髂骨皮下中。所有小鼠均在两小时内完成注射。
5.2 疫苗免疫小鼠
TC-1接种3天、10天和17天后使用BD超细胰岛素注射器将30 μg HPV16-AVLS1/AVLC1 疫苗（溶解在40 μL TE中）皮下递送至双耳耳廓，每次每个耳廓注射20 μL。空载体AVL0318和阳性对照P1同样方式注射相同的质粒量，每组8只小鼠。每天检查和测量肿瘤块记录肿瘤体积，当小鼠肿瘤直径达到2000 mm3伦理处死。每日观测肿瘤大小。
5.3 结果分析
如下图7结果显示HPV16-AVLS1/AVLC1和P1均能抑制肿瘤生长抑制，但HPV16-AVLS1/AVLC1抑制肿瘤生长更明显，整个观察周期都没见到明显的肿瘤生长，而P1在20天才基本达到完全抑制肿瘤生长的效果。
实施例6 预防肿瘤效果
6.1 TC-1 细胞培养和小鼠饲养同实施例5
6.2 共15只C57BL/6J 6-10周雌性小鼠，分成三组，每组五只，每组分别注射对照质粒AVL0318，疫苗序列HPV16-AVLS1/AVLC1和P1，注射三次，分别在第0天，第7天和第14天双耳耳廓注射30 μg质粒。所有小鼠在第21天背部髂骨皮下注射5×105 TC-1肿瘤细胞。在接种当天，将TC-1细胞在 PBS 中调整至 5x106个细胞/mL，每只小鼠注射 100ul 细胞悬液。然后每天检测肿瘤生长情况。
6.3 结果分析
如下图8所示，相比对照组，HPV16-AVLS1/AVLC1和P1组所有小鼠接种TC-1后均无法成瘤，这说明HPV16-AVLS1/AVLC1和P1均有非常明显的预防肿瘤的效果。
实施例7 如上实施例中所使用的能够表达SacB基因的大肠杆菌具体采用如下方式制得：
7.1、设计引物
sacB-F1: ATCAATAATCAGACAACAAGATGAACATCAAAAAGTTTGC
sacB-F2：TGATATAATGGTTTCGCCAAAAATCAATAATCAGACAACAAG
sacB-F3：TAGACACACATCTTGTCATATGATATAATGGTTTCGCCAAAA
sacB-R1: CTCAAGTTAGTATTTATTTGTTAACTGTTAATTGTCCTTG
DH5-attB-down-F：TTAACAGTTAACAAATAAATACTAACTTGAGCGAAACGGGAAG
DH5-attB-UP-R：GACAAGATGTGTGTCTACCAAAAAAGCAGGCTTCAACGGATTCA
DH5 attB-up-F：GAAAGCCCAATCTTCACATCAATC
DH5 attB-down-R：GCATCTGGCGTGGGATGATGTTCCT
SgRNA：TCAAGTTAGTATAAAAAAGC
7.2、 p5/6 6/6-sacB片段获得
以H73 载体为模板，按照如表1所示的如下体系进行：
扩增条件：
94℃ 5min
30Cycle（94℃ 30sec 、55℃ 30sec、68℃30sec）
10℃ 保温
扩增后产物为模板按照如上程序引物换成sacB-F2/sacB-R1,进行第二次扩增，
以扩增引物sacB-F3/sacB-R1依次进行第三次扩增,扩增后获得P5/6 6/6-sacB片段。
7.3、 attB 同源臂扩增
1）、以attB-UP-F/ DH5-attB-UP-R、DH5-attB-down-F/ DH5 attB-down-R引物扩增按照如表2所示的以下体系进行PCR扩增
扩增条件
94℃ 5min
32Cycle（94℃ 30sec 、55℃ 30sec、68℃ 30sec）
10℃ 保温
结果如图9所示；其中，M指DL2000 DNA marker，up指扩增的attB上游同同源片段、SacB指sacB-F3/R1扩增产物（P5/6 6/6-sacB）、down指扩增的attB下游同源臂片段。
2）、重叠延伸PCR扩增修复同源臂获得SacB-CRISPR核酸片段
将图9中的attB上游同源臂片段、sacB F3/R1扩增产物（P5/6 6/6-sacB）和attB下游同源臂片段，3个片段经PCR产物纯化试剂盒回收备用
按照如表3所示的以下体系进行overlapping PCR扩增
扩增条件
94℃ 5min
2 Cycle（94℃ 30sec 、50℃ 30sec、68℃ 1min）
30 Cycle（94℃ 30sec 、55℃ 30sec、68℃ 1min）
10℃ 保温
琼脂糖凝胶电泳检测，结果如图10所示；其中，M指 trans2K plus II DNAmarker，1指SacB-CRISPR核酸片段。
7.4、CRISPR编辑筛选AVL-DH5α(SacB)
7.4.1、制备DH5α电转化感受态
1）菌株平板划线活化培养
2）第二天接种单克隆至5ml LB液体培养基37℃过夜培养
3）次日1%转接至50ml LB 液体培养基中生长至OD600nm约为0.8离心收集菌体
4）用10%甘油洗3次
5) 最后用2ml 10% 甘油重悬菌体即为制备好的电转化感受态细胞
电转化pTcCas9（本实验室改造Tc抗性）质粒，90μl 感受态细胞加入10μl质粒冰上放置5min，2500Kv电转化，加入1ml LB培养基30℃培养1h 涂布Tc 抗性平板，30℃过夜培养。
7.4.2、制备cas9 DH5α感受态细胞
制备方法同上
7.4.3、电转化SacB-CRISPR核酸片段及sgRNA
7.4.4、SacB插入鉴定
挑取10个单克隆进行菌落PCR鉴定，引物如下：
DH5 attB-up-F：GAAAGCCCAATCTTCACATCAATC
DH5 attB-down-R：AGGAACATCATCCCACGCCAGATGC
如图11所示，10个单克隆均为阳性，其中M指DL2000 DNA marker 1-10为平板上随机挑取的10个克隆编号
图中产物送测序，测序引物
attb-JD-F：AATGCCAGCGCCAGACGGGAAAC
attB-JD-R：CTCTGGCAAGCGCCTCGATTACT
测序显示所有单克隆都插入了SacB基因序列，且已准确插入到目标位置。
7.5、编辑菌抗性消除
【AVL-DH5α(SacB)】
将编辑成功的菌接种于无抗LB，37℃培养过夜，第二天稀释涂布于LB无抗培养基平板，37℃培养；
次日长出的单克隆点板在LB（无抗）和LB（TC）、LB（Cm）平板上，在2种抗性上都不长的菌就是消除抗性的菌株，命名为【AVL-DH5α(SacB)】。
实施例8 AVL-DH5α(SacB)蔗糖敏感性和扩增质粒产量的探究
对所提供的大肠杆菌菌株AVL-DH5α(SacB)与Luke等人利用同源重组发明的大肠杆菌SacB表达菌株DH5α attλ::P5/6 6/6-RNA-IN- SacB, catR进行蔗糖敏感性比较。结果显示本发明提供的菌株具有更高的蔗糖敏感性和传代稳定性，具体的，参考表4所示。
进一步对比了常用表达载体pcDNA3.1与本发明的AVL0318载体分别在氨苄抗生素和蔗糖条件下质粒产量进行了比较，结果如表5所示。
结果显示虽然AVL0318在非抗生素条件下使用AVL-DH5α(SacB)进行扩增，AVL0318质粒产量依然高出pcDNA3.1产量22%左右，体现了本发明菌株和表达载体的优越性。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 北京循生生物医学研究有限公司
<120> 一种新型HPV治疗性核酸疫苗
<130> P2021F0462-XS
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ggtaccgagc tcggatccgc cgccaccatg gaaacggaca cgctgctgct gtgggtcctg 60
ctgctgtggg tccccggatc gacgggagac ggatcgatgc accaaaagcg aaccgctatg 120
tttcaggacc cccaggaacg accccgtaaa ctgccccagc tctgcacgga actgcaaacg 180
acgatccatg acatcatcct cgaatgcgtg tactgcaagc aacagctcct gcgacgtgaa 240
gtctacgact ttgcttttcg cgacctgtgc atcgtctaca gagacggaaa cccctacgct 300
gtgggagaca aatgcctgaa gttttactcg aaaatctcgg aataccgcca ctactgctac 360
tcgctgtacg gaaccacgct cgaacagcaa tacaacaaac ccctatgcga cctgctaatc 420
cgctgcatca actgccaaaa gcctctctgc cctgaagaaa agcaacgcca tctcgacaaa 480
aagcaaagat ttcacaacac gcgtggacga tggaccggac gatgcatgtc gtgctgcaga 540
tcgtcacgca cgcgtagaga aacccagctg gctggagcta tgcaccaaaa gcgaaccgct 600
atgtttcagg acccccagga acgaccccgt aaactgcccc agctctgcac ggaactgcaa 660
acgacgatcc atgacatcat cctcgaatgc gtgtactgca agcaacagct cctgcgacgt 720
gaagtctacg actttgcttt tcgcgacctg tgcatcgtct acagagacgg aaacccctac 780
gctgtgggag acaaatgcct gaagttttac tcgaaaatct cggaataccg ccactactgc 840
tactcgctgt acggaaccac gctcgaacag caatacaaca aacccctatg cgacctgcta 900
atccgctgca tcaactgcca aaagcctctc tgccctgaag aaaagcaacg ccatctcgac 960
aaaaagcaaa gatttcacaa cacgcgtgga cgatggaccg gacgatgcat gtcgtgctgc 1020
agatcgtcac gcacgcgtag agaaacccag ctggctgctt acgaagctac cgtctacctc 1080
ccccccgtgc ccgtctcgaa agtggtcgct gcttacgaca cgtacagatt tgtgacctcg 1140
caagctatcg cttgccagaa ggctgcttac cccgactttc tcgacatcgt cgctctgcat 1200
cgccccgctc tgacctcgcg agctgcttac ttttttggag gactgggaat cggaacggga 1260
tcgggaacgg gaggacgtgc tgcttacatg catggagata cgcctacgct ccatgaatat 1320
atgctcgatc tgcaacccga aacgaccgat ctctacggat atggacaact taacgactcg 1380
tcggaagaag aagatgaaat cgatggaccc gctggacaag ctgaacccga ccgtgctcat 1440
tacaacatcg tcacgttttg ttgcaagtgt gactcgacgc tgcgactgtg cgtccaatcg 1500
acccacgtgg acatccgtac gctcgaagac ctgctcatgg gaacgcttgg aatcgtctgc 1560
cccatctgct cgcagaaacc cgctggagct atgcatggag atacgcctac gctccatgaa 1620
tatatgctcg atctgcaacc cgaaacgacc gatctctacg gatatggaca acttaacgac 1680
tcgtcggaag aagaagatga aatcgatgga cccgctggac aagctgaacc cgaccgtgct 1740
cattacaaca tcgtcacgtt ttgttgcaag tgtgactcga cgctgcgact gtgcgtccaa 1800
tcgacccacg tggacatccg tacgctcgaa gacctgctca tgggaacgct tggaatcgtc 1860
tgccccatct gctcgcagaa acccgctgga gctgctaaat ttgtggctgc ttggacgctg 1920
aaggctgctg ctgaagctgc tgctaaagga atcatcaaca cgctgcaaaa gtactactgc 1980
cgtgtccgcg gaggacgatg cgctgtgctc tcgtgcctgc ccaaagaaga acagatcgga 2040
aagtgctcga cgagaggacg taaatgctgc cgccgaaaga aataatga 2088
<210> 14
<211> 2247
<212> DNA
<213> Artificial Sequence
<400> 14
ggtaccgagc tcggatccgc cgccaccatg caaatctttg tgaagacgct gacgggaaag 60
accatcacgc tcgaagtgga accctcggac acgatcgaaa acgtgaaagc taagatccag 120
gacaaggaag gaatcccccc cgaccagcag agactgatct ttgctggaaa gcagctcgaa 180
gacggacgca cgctgtcgga ctacaacatc cagaaagaat cgacgctcca cctggtcctg 240
agactccgcg gagctatgca ccaaaagcga accgctatgt ttcaggaccc ccaggaacga 300
ccccgtaaac tgccccagct ctgcacggaa ctgcaaacga cgatccatga catcatcctc 360
gaatgcgtgt actgcaagca acagctcctg cgacgtgaag tctacgactt tgcttttcgc 420
gacctgtgca tcgtctacag agacggaaac ccctacgctg tgggagacaa atgcctgaag 480
ttttactcga aaatctcgga ataccgccac tactgctact cgctgtacgg aaccacgctc 540
gaacagcaat acaacaaacc cctatgcgac ctgctaatcc gctgcatcaa ctgccaaaag 600
cctctctgcc ctgaagaaaa gcaacgccat ctcgacaaaa agcaaagatt tcacaacacg 660
cgtggacgat ggaccggacg atgcatgtcg tgctgcagat cgtcacgcac gcgtagagaa 720
acccagctgg ctggagctat gcaccaaaag cgaaccgcta tgtttcagga cccccaggaa 780
cgaccccgta aactgcccca gctctgcacg gaactgcaaa cgacgatcca tgacatcatc 840
ctcgaatgcg tgtactgcaa gcaacagctc ctgcgacgtg aagtctacga ctttgctttt 900
cgcgacctgt gcatcgtcta cagagacgga aacccctacg ctgtgggaga caaatgcctg 960
aagttttact cgaaaatctc ggaataccgc cactactgct actcgctgta cggaaccacg 1020
ctcgaacagc aatacaacaa acccctatgc gacctgctaa tccgctgcat caactgccaa 1080
aagcctctct gccctgaaga aaagcaacgc catctcgaca aaaagcaaag atttcacaac 1140
acgcgtggac gatggaccgg acgatgcatg tcgtgctgca gatcgtcacg cacgcgtaga 1200
gaaacccagc tggctgctta cgaagctacc gtctacctcc cccccgtgcc cgtctcgaaa 1260
gtggtcgctg cttacgacac gtacagattt gtgacctcgc aagctatcgc ttgccagaag 1320
gctgcttacc ccgactttct cgacatcgtc gctctgcatc gccccgctct gacctcgcga 1380
gctgcttact tttttggagg actgggaatc ggaacgggat cgggaacggg aggacgtgct 1440
gcttacatgc atggagatac gcctacgctc catgaatata tgctcgatct gcaacccgaa 1500
acgaccgatc tctacggata tggacaactt aacgactcgt cggaagaaga agatgaaatc 1560
gatggacccg ctggacaagc tgaacccgac cgtgctcatt acaacatcgt cacgttttgt 1620
tgcaagtgtg actcgacgct gcgactgtgc gtccaatcga cccacgtgga catccgtacg 1680
ctcgaagacc tgctcatggg aacgcttgga atcgtctgcc ccatctgctc gcagaaaccc 1740
gctggagcta tgcatggaga tacgcctacg ctccatgaat atatgctcga tctgcaaccc 1800
gaaacgaccg atctctacgg atatggacaa cttaacgact cgtcggaaga agaagatgaa 1860
atcgatggac ccgctggaca agctgaaccc gaccgtgctc attacaacat cgtcacgttt 1920
tgttgcaagt gtgactcgac gctgcgactg tgcgtccaat cgacccacgt ggacatccgt 1980
acgctcgaag acctgctcat gggaacgctt ggaatcgtct gccccatctg ctcgcagaaa 2040
cccgctggag ctgctaaatt tgtggctgct tggacgctga aggctgctgc tgaagctgct 2100
gctaaaggaa tcatcaacac gctgcaaaag tactactgcc gtgtccgcgg aggacgatgc 2160
gctgtgctct cgtgcctgcc caaagaagaa cagatcggaa agtgctcgac gagaggacgt 2220
aaatgctgcc gccgaaagaa ataatga 2247
<210> 15
<211> 2139
<212> DNA
<213> Artificial Sequence
<400> 15
ggtaccgagc tcggatccgc cgccaccatg gaaacggaca cgctgctgct gtgggtcctg 60
ctgctgtggg tccccggatc gacgggagac ggatcgatgg ctcgctttga agacccaacg 120
cgccgaccct acaagctccc cgacctgtgc acggaactga acacgtcgct gcaagacatc 180
gaaatcacct gcgtgtactg caagacggtc ctggaactca cggaagtgtt tgaatttgct 240
tttaaagacc tctttgtggt ctacagagac tcgatcccgc atgctgctgg acataaatgc 300
atcgactttt actcgcgcat cagagaactc agacattact ctgactcggt ctacggagac 360
acgctggaaa agctgacgaa cacgggactc tacaacctcc tgatccgttg cctgcgttgc 420
cagaaaccgc tcaacccagc tgaaaagctg cgccacctca acgaaaaacg acgttttcac 480
aacacggctg gacactacag aggacagtgc cattcgtgct gcaaccgagc tcgtcaggaa 540
cgactccaac gacgcagaga aacgcaagtg gctggagcta tggctcgctt tgaagaccca 600
acgcgccgac cctacaagct ccccgacctg tgcacggaac tgaacacgtc gctgcaagac 660
atcgaaatca cctgcgtgta ctgcaagacg gtcctggaac tcacggaagt gtttgaattt 720
gcttttaaag acctctttgt ggtctacaga gactcgatcc cgcatgctgc tggacataaa 780
tgcatcgact tttactcgcg catcagagaa ctcagacatt actctgactc ggtctacgga 840
gacacgctgg aaaagctgac gaacacggga ctctacaacc tcctgatccg ttgcctgcgt 900
tgccagaaac cgctcaaccc agctgaaaag ctgcgccacc tcaacgaaaa acgacgtttt 960
cacaacacgg ctggacacta cagaggacag tgccattcgt gctgcaaccg agctcgtcag 1020
gaacgactcc aacgacgcag agaaacgcaa gtggctgctt acgacaacac ggtgtacctc 1080
ccccccccct cggtcgctag agtggtcgct gcttacgaca cgtaccgttt tgtgcaatcg 1140
gtcgctatca cctgccagaa agctgcttac gacccctcga tcgtgacgct gatcgaagac 1200
tcgtcggtgg tcacgtcggg agctcccgct gcttactcgg actttatgga catcatccgc 1260
ctccaccgac ccgctctgac gtcgagagct gcttacatgc atggacccaa agctacgctg 1320
caagacatcg tgctccatct cgaaccccaa aacgaaatcc cggtcgacct cctcggacac 1380
ggacaactct cggactcgga agaagaaaac gacgaaatcg acggagtgaa ccatcaacac 1440
ctcccagctc gacgcgctga accacaacgt cacacgatgc tgtgcatgtg ctgcaagtgc 1500
gaagctagaa tcgaactggt cgtggaatcg tctgctgacg acctgcgagc ttttcagcag 1560
ctgtttctga acaccctgtc gtttgtctgc ccgtggtgcg cttcgcagca ggctggagct 1620
atgcatggac ccaaagctac gctgcaagac atcgtgctcc atctcgaacc ccaaaacgaa 1680
atcccggtcg acctcctcgg acacggacaa ctctcggact cggaagaaga aaacgacgaa 1740
atcgacggag tgaaccatca acacctccca gctcgacgcg ctgaaccaca acgtcacacg 1800
atgctgtgca tgtgctgcaa gtgcgaagct agaatcgaac tggtcgtgga atcgtctgct 1860
gacgacctgc gagcttttca gcagctgttt ctgaacaccc tgtcgtttgt ctgcccgtgg 1920
tgcgcttcgc agcaggctgg agctgctaaa tttgtggctg cttggacgct gaaggctgct 1980
gctgaagctg ctgctaaagg aatcatcaac acgctgcaaa agtactactg ccgtgtccgc 2040
ggaggacgat gcgctgtgct ctcgtgcctg cccaaagaag aacagatcgg aaagtgctcg 2100
acgagaggac gtaaatgctg ccgccgaaag aaataatga 2139
<210> 16
<211> 2298
<212> DNA
<213> Artificial Sequence
<400> 16
ggtaccgagc tcggatccgc cgccaccatg caaatctttg tgaagacgct gacgggaaag 60
accatcacgc tcgaagtgga accctcggac acgatcgaaa acgtgaaagc taagatccag 120
gacaaggaag gaatcccccc cgaccagcag agactgatct ttgctggaaa gcagctcgaa 180
gacggacgca cgctgtcgga ctacaacatc cagaaagaat cgacgctcca cctggtcctg 240
agactccgcg gagctatggc tcgctttgaa gacccaacgc gccgacccta caagctcccc 300
gacctgtgca cggaactgaa cacgtcgctg caagacatcg aaatcacctg cgtgtactgc 360
aagacggtcc tggaactcac ggaagtgttt gaatttgctt ttaaagacct ctttgtggtc 420
tacagagact cgatcccgca tgctgctgga cataaatgca tcgactttta ctcgcgcatc 480
agagaactca gacattactc tgactcggtc tacggagaca cgctggaaaa gctgacgaac 540
acgggactct acaacctcct gatccgttgc ctgcgttgcc agaaaccgct caacccagct 600
gaaaagctgc gccacctcaa cgaaaaacga cgttttcaca acacggctgg acactacaga 660
ggacagtgcc attcgtgctg caaccgagct cgtcaggaac gactccaacg acgcagagaa 720
acgcaagtgg ctggagctat ggctcgcttt gaagacccaa cgcgccgacc ctacaagctc 780
cccgacctgt gcacggaact gaacacgtcg ctgcaagaca tcgaaatcac ctgcgtgtac 840
tgcaagacgg tcctggaact cacggaagtg tttgaatttg cttttaaaga cctctttgtg 900
gtctacagag actcgatccc gcatgctgct ggacataaat gcatcgactt ttactcgcgc 960
atcagagaac tcagacatta ctctgactcg gtctacggag acacgctgga aaagctgacg 1020
aacacgggac tctacaacct cctgatccgt tgcctgcgtt gccagaaacc gctcaaccca 1080
gctgaaaagc tgcgccacct caacgaaaaa cgacgttttc acaacacggc tggacactac 1140
agaggacagt gccattcgtg ctgcaaccga gctcgtcagg aacgactcca acgacgcaga 1200
gaaacgcaag tggctgctta cgacaacacg gtgtacctcc cccccccctc ggtcgctaga 1260
gtggtcgctg cttacgacac gtaccgtttt gtgcaatcgg tcgctatcac ctgccagaaa 1320
gctgcttacg acccctcgat cgtgacgctg atcgaagact cgtcggtggt cacgtcggga 1380
gctcccgctg cttactcgga ctttatggac atcatccgcc tccaccgacc cgctctgacg 1440
tcgagagctg cttacatgca tggacccaaa gctacgctgc aagacatcgt gctccatctc 1500
gaaccccaaa acgaaatccc ggtcgacctc ctcggacacg gacaactctc ggactcggaa 1560
gaagaaaacg acgaaatcga cggagtgaac catcaacacc tcccagctcg acgcgctgaa 1620
ccacaacgtc acacgatgct gtgcatgtgc tgcaagtgcg aagctagaat cgaactggtc 1680
gtggaatcgt ctgctgacga cctgcgagct tttcagcagc tgtttctgaa caccctgtcg 1740
tttgtctgcc cgtggtgcgc ttcgcagcag gctggagcta tgcatggacc caaagctacg 1800
ctgcaagaca tcgtgctcca tctcgaaccc caaaacgaaa tcccggtcga cctcctcgga 1860
cacggacaac tctcggactc ggaagaagaa aacgacgaaa tcgacggagt gaaccatcaa 1920
cacctcccag ctcgacgcgc tgaaccacaa cgtcacacga tgctgtgcat gtgctgcaag 1980
tgcgaagcta gaatcgaact ggtcgtggaa tcgtctgctg acgacctgcg agcttttcag 2040
cagctgtttc tgaacaccct gtcgtttgtc tgcccgtggt gcgcttcgca gcaggctgga 2100
gctgctaaat ttgtggctgc ttggacgctg aaggctgctg ctgaagctgc tgctaaagga 2160
atcatcaaca cgctgcaaaa gtactactgc cgtgtccgcg gaggacgatg cgctgtgctc 2220
tcgtgcctgc ccaaagaaga acagatcgga aagtgctcga cgagaggacg taaatgctgc 2280
cgccgaaaga aataatga 2298
<210> 17
<211> 1422
<212> DNA
<213> Artificial Sequence
<400> 17
atgaacatta aaaaattcgc gaaacaggcg accgttctga ccttcaccac cgcgctgctg 60
gcgggcggtg cgacccaggc gttcgctaaa gaaaccaacc agaaaccgta taaagaaacc 120
tacggtattt ctcacattac ccgccacgat atgctgcaga tcccggaaca gcagaaaaac 180
gaaaaatacc aggttccgga attcgacagc tccaccatca aaaacatcag cagcgcgaaa 240
ggcctggatg tttgggattc ttggccgctg cagaacgctg atggcaccgt cgcgaactac 300
cacggctatc atattgtgtt cgcgctggcg ggcgacccga aaaacgcgga tgacacctct 360
atctacatgt tctaccagaa agttggtgaa accagtatcg acagctggaa aaacgcaggc 420
cgtgttttca aagatagcga taaattcgat gcgaacgata gcatcctgaa agatcagact 480
caggaatgga gcggtagcgc taccttcacc tccgacggca aaattcgtct gttctacacc 540
gatttcagcg gcaaacacta tggtaaacag accctgacca ccgcgcaggt gaacgtgtcc 600
gcgtccgact cctctctgaa catcaacggc gttgaagatt acaaatctat cttcgatggt 660
gacggcaaaa cttatcagaa cgttcagcag ttcatcgatg aaggcaacta ctcttctggc 720
gataaccaca ccctgcgtga tccgcactac gttgaagata aaggccacaa atacctggtt 780
ttcgaagcta acaccggcac cgaagatggc taccagggtg aagaaagcct gttcaacaaa 840
gcgtattacg gtaaatctac cagctttttc cgtcaggaat cccagaaact gctgcagagc 900
gataaaaaac gtaccgcgga actggcgaac ggcgcgctgg gcatgatcga actgaacgat 960
gattacaccc tgaaaaaagt tatgaaaccg ctgatcgcta gcaacaccgt taccgatgaa 1020
atcgaacgtg cgaacgtttt caaaatgaac ggcaaatggt acctgttcac cgactctcgt 1080
ggtagcaaaa tgaccatcga tggtatcacc tctaacgaca tctacatgct gggttacgtt 1140
agcaacagcc tgaccggtcc gtacaaaccg ctgaacaaaa ccggtctggt tctgaaaatg 1200
gacctggatc cgaacgacgt taccttcacc tatagccatt tcgcggtgcc gcaggcgaaa 1260
ggtaacaacg tggtgatcac ctcctacatg accaaccgtg gtttctacgc tgataaacag 1320
agcaccttcg cgccgagctt cctgctgaac atcaaaggta agaaaaccag cgtcgttaaa 1380
gacagcatcc tggaacaggg ccagctgacc gttaacaaat aa 1422
<210> 18
<211> 2474
<212> DNA
<213> Artificial Sequence
<400> 18
gaaagcccaa tcttcacatc aatcggtttt tcacctgtac cgtacagagt aattccaccc 60
ggagcggcag ggacatatac cgttccctga tactcaccag gcatcacggc aatatactgg 120
cgcttgttgg tacgcttgat aattgccgca tctaccgccg cctgaatcgt ggtatgcgtt 180
acaccttgag tgcccgccgg gccgacaaca aagtcaggtt gcgcaggcag ggtaatcggg 240
gaaggattcc acgctgcagc acctggtgtc agggatgcaa aatagtgttg agcatcgaaa 300
ttctgcgctt cttttgccga cagaatcggg cgagaagagg taccaggcgc ggtttgatca 360
gaaggacgtt gatcgggcgg ggttgagcta caggcggtca gcgtcacgcc aaaagccaat 420
gccagcgcca gacgggaaac tgaaaatgtg ttcacaggtt gctccgggct atgaaataga 480
aaaatgaatc cgttgaagcc tgcttttttg gtagacacac atcttgtcat atgatataat 540
ggtttcgcca aaaatcaata atcagacaac aagatgaaca ttaaaaaatt cgcgaaacag 600
gcgaccgttc tgaccttcac caccgcgctg ctggcgggcg gtgcgaccca ggcgttcgct 660
aaagaaacca accagaaacc gtataaagaa acctacggta tttctcacat tacccgccac 720
gatatgctgc agatcccgga acagcagaaa aacgaaaaat accaggttcc ggaattcgac 780
agctccacca tcaaaaacat cagcagcgcg aaaggcctgg atgtttggga ttcttggccg 840
ctgcagaacg ctgatggcac cgtcgcgaac taccacggct atcatattgt gttcgcgctg 900
gcgggcgacc cgaaaaacgc ggatgacacc tctatctaca tgttctacca gaaagttggt 960
gaaaccagta tcgacagctg gaaaaacgca ggccgtgttt tcaaagatag cgataaattc 1020
gatgcgaacg atagcatcct gaaagatcag actcaggaat ggagcggtag cgctaccttc 1080
acctccgacg gcaaaattcg tctgttctac accgatttca gcggcaaaca ctatggtaaa 1140
cagaccctga ccaccgcgca ggtgaacgtg tccgcgtccg actcctctct gaacatcaac 1200
ggcgttgaag attacaaatc tatcttcgat ggtgacggca aaacttatca gaacgttcag 1260
cagttcatcg atgaaggcaa ctactcttct ggcgataacc acaccctgcg tgatccgcac 1320
tacgttgaag ataaaggcca caaatacctg gttttcgaag ctaacaccgg caccgaagat 1380
ggctaccagg gtgaagaaag cctgttcaac aaagcgtatt acggtaaatc taccagcttt 1440
ttccgtcagg aatcccagaa actgctgcag agcgataaaa aacgtaccgc ggaactggcg 1500
aacggcgcgc tgggcatgat cgaactgaac gatgattaca ccctgaaaaa agttatgaaa 1560
ccgctgatcg ctagcaacac cgttaccgat gaaatcgaac gtgcgaacgt tttcaaaatg 1620
aacggcaaat ggtacctgtt caccgactct cgtggtagca aaatgaccat cgatggtatc 1680
acctctaacg acatctacat gctgggttac gttagcaaca gcctgaccgg tccgtacaaa 1740
ccgctgaaca aaaccggtct ggttctgaaa atggacctgg atccgaacga cgttaccttc 1800
acctatagcc atttcgcggt gccgcaggcg aaaggtaaca acgtggtgat cacctcctac 1860
atgaccaacc gtggtttcta cgctgataaa cagagcacct tcgcgccgag cttcctgctg 1920
aacatcaaag gtaagaaaac cagcgtcgtt aaagacagca tcctggaaca gggccagctg 1980
accgttaaca aataaatact aacttgagcg aaacgggaag gtaaaaagac aaaaagttgt 2040
ttttaatacc tttaagtgat accagatggc attgcgccat ctggcagagt gattaactaa 2100
acatcgcagt aatcgaggcg cttgccagag agtggaaatg aacgttaaac ccgaccatcg 2160
cgccgctggc accttcatcg acatcaatac gttctatatc cagcgcgtga acggtaaaaa 2220
tgtagcgatg agtttcgcct ttcggcggtg ctgcgccatc gtacccggtt ttaccaaagt 2280
cggtacgcgt ctgcaaaacg ccgtctggca ttgctaccag accagagcca aacccttgcg 2340
gtaatacgcg ggtatcagcg ggtaagttaa caactaccca gtgccaccag ccggagccgg 2400
ttggcgcatc cgggtcgtag caggtgacaa caaaactttt cgttcccgca ggaacatcat 2460
cccacgccag atgc 2474
Claims (10)
1.一种用于治疗和预防HPV感染疾病的核酸序列，其特征在于，包括：
1:1的序列HPV16-AVLS1和序列HPV16-AVLC1；
以及，1:1的序列HPV18-AVLS1和序列HPV18-AVLC1；
其中，序列AVLS1和序列AVLC1均包括两个串联的蛋白E6分子、两个L1短肽，两个L2的短肽、两个串联的蛋白E7分子、一个PADRE序列及一个adjuvant序列；序列AVLS1的N端带有小鼠IgK分泌肽序列；序列AVLC1的N端带有泛素分子；
所述L1短肽选自SEQ ID No：1~SEQ ID No：4所示序列中的至少一种；
所述L2短肽选自SEQ ID No：5~SEQ ID No：8所示序列中的至少一种。
2.根据权利要求1所述的核酸序列，其特征在于，所述PADRE序列为SEQ ID No：9所示序列；
和/或，所述adjuvant序列为佐剂肽Beta-defensin-3，为SEQ ID No：10所示序列；
和/或，所述小鼠IgK分泌肽序列为SEQ ID No：11所示序列；
和/或，所述泛素分子的序列为SEQ ID No：12所示序列。
3.根据权利要求1或2任一项所述的核酸序列，其特征在于，HPV16融合蛋白E6序列引入C70G和I135T突变，HPV18融合蛋白E6序列引入C65G和I130T突变； HPV16的E7蛋白序列引入C24G和E26G突变，HPV18的E7蛋白序列引入C27G和E29G突变。
4.根据权利要求3所述的核酸序列，其特征在于，各个元件之间使用AGA或AAY连接子；佐剂肽使用刚性连接子EAAAK连接。
5.根据权利要求1所述的核酸序列，其特征在于，所述核酸序列为SEQ ID No：13~ SEQID No：16所示四条序列之一。
6.mRNA序列，其特征在于，所述mRNA序列为权利要求1~5任一项所述核酸序列转换的mRNA序列。
7.重组载体，其特征在于，包括表达载体和权利要求1~5任一项所述的核酸序列；所述表达载体为无抗生素标记的AVL0318载体。
8.扩增权利要求7所述重组载体的制备方法，其特征在于，包括如下步骤：
1）将E6/E7蛋白、L1/L2多肽与IgK，Ubquitin，PADRE和adjuvant的氨基酸序列进行拼接合成，合成疫苗序列HPV16-AVLS1、HPV16-AVLC1、HPV18-AVLS1和HPV18-AVLC1四条核酸序列；合成为SEQ ID No：13~ SEQ ID No：16所示的序列；
2）将所述四条核酸序列利用HindIII和XhoI酶切位点连入载体PUC57；再把疫苗序列亚克隆到表达载体AVL0318；获得四个重组载体AVL0318-HPV16/18-AVLS1/AVLC1；
3）使用大肠杆菌AVL-DH5α(SacB)对AVL0318-HPV16/18-AVLS1/AVLC1的质粒进行扩增；
其中，所述大肠杆菌AVL-DH5α(SacB)的基因组序列中含有能够组成型表达的SacB基因，且不含有抗生素筛选标记；能够表达SacB基因的序列为SEQ ID No：17。
9.根据权利要求8所述的制备方法，其特征在于，所述大肠杆菌菌株AVL-DH5α(SacB)的制备方法：在大肠杆菌的attB位点插入SacB基因；
基因编辑是通过包括下述步骤的方法实现：
1）分别PCR扩增插入位点上游、下游的同源臂基因序列、p5/6 6/6-SacB基因序列，以上述三个序列为模板重叠延伸PCR扩增长片段SacB-CRISPR核苷酸序列；所述SacB-CRISPR的核苷酸序列为SEQ ID No.18所示序列；
2）将表达Cas9质粒、sgRNA和长片段SacB-CRISPR核苷酸序列共转化到大肠杆菌DH5α感受态细胞中，进行基因编辑和同源重组修复，挑取单克隆培养后PCR测序验证，最后通过温度敏感消除工具质粒和编辑菌的抗性，即得可用于无抗生素筛选目标质粒的AVL-DH5α(SacB)菌株。
10.一种新型HPV治疗性核酸疫苗，其特征在于，包括权利要求1~5任一项所述的核酸序列，或权利要求6所述的mRNA序列，或权利要求7所述的重组载体和权利要求8~9所述制备方法获得的重组载体。
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