CN113234149B - 全人源的新冠IgA单链抗体及其应用 - Google Patents
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Abstract
本发明涉及生物医药技术领域，具体公开了全人源的新冠IgA单链抗体及其应用。本发明通过BCR全长测序，通过分析不同康复者共有而在正常人群中没有的BCR序列，筛选出一类新冠特异性的IgA BCR抗体全长。同时基于抗体全长序列设计特定抗体特异性引物，进行扩增富集后并进行单克隆的制备与后续的克隆筛选测序，获得了BCR序列全长，该序列进行表达后可获得具有中和活性的病毒特异性抗体。通过获得的全人源抗体可以寻找一种能与新冠特异性结合的单链抗体，这类特异识别新冠的单链抗体本身或者其可变区序列，经过直接表达或经基因工程改造成其它的抗体形式，可以作为抗新冠药物开发、疫苗生产、检测标志物开发等新冠相关应用中。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及全人源的新冠IgA单链抗体及其应用。
背景技术
新型冠状病毒是一种具有包膜的、不分节段的正链单股RNA病毒，颗粒呈圆形或椭圆形，直径约80～120nm，属于网巢病毒目冠状病毒科Betacoronavirus。病毒粒子被宿主细胞提供的脂质双层所包裹，其中含有核酸及核衣壳蛋白，有三种主要蛋白：包膜蛋白(E蛋白)、膜蛋白(M蛋白)和刺突蛋白(S蛋白)。此病毒每组基因组长度约三万个核苷酸左右，基因序列显示SARS-CoV-2属于乙型冠状病毒属谱系β(Betacoronavirus Lineageβ,Sarbecovirus)进化树中分支较长的一种病毒，与中华菊头蝠中发现的冠状病毒相似，例如MERS-CoV或SARS-CoV。对病毒的生物遗传学分析显示，同属人类冠状病毒的SARS病毒分离株AY274119相比MERS病毒分离株KC164505、JX869059等在亲缘关系上距离SARS-CoV-2病毒更近。
2019年末，该病爆发并向全球扩散。病毒潜伏期平均大约3-7天，最长不超过14天。大多数患者的表现以下呼吸道症状为主，常见临床表现包括发热、四肢乏力、干咳等症状，其他表现包含鼻塞、流鼻涕、头痛、咽痛、咳血，咳痰、或腹泻等。有部分患者仅表现为低热、轻微乏力等，无肺炎表现。还有部分患者无任何临床表现。病毒感染严重后，会引发多种并发症包含急性呼吸窘迫综合征(ARDS)、脓毒症休克、全身炎症反应综合征、难以纠正的代谢性酸中毒、急性心肌损伤，和出凝血功能障碍等。
针对这场危及全人类的群体大流行，揭示人体对新冠侵染后的免疫过程，获取新冠病毒的B细胞受体对于新冠特异性药物的筛选、病毒疫苗的生产研发及其重要。B细胞受体(B cell receptor，BCR)是B细胞抗原识别决定性表面分子，其本质是一种膜表面免疫球蛋白(membrane immunoglobulin,mIg)。BCR具有抗原结合特异性，每个个体的BCR多样性高达5×10^13，构成容量巨大的BCR库，赋予个体识别各种抗原、产生特异性抗体的巨大潜能。
BCR的结构包括重链和轻链。BCR的重链(heavy chain，H)由65～100种可变区(VH)、2种多变区(DH)、6种结合区(JH)和恒定区(CH)四部分基因片段组成；轻链(lightchain，L)由可变区、结合区和恒定区三部分基因片段组成。发育过程中的B细胞在重组酶(RAG1、RAG2)作用下，形成了多样性高达1～2×10^11的BCR。同时，由其形成互补决定区(complementarities determining region，CDR)：CDR1、CDR2和CDR3区氨基酸序列的多样性，特别是编码CDR3的基因，由于其位于轻链V、J或重链V、D、J片段的连接处，可以通过V(D)J的重排和(或)两个基因片段的连接间丢失或插入数个核苷酸，进一步增加BCR的多样性，而形成具有功能的BCR编码基因(B细胞克隆)。
BCR测序是通过高通量测序技术检测靶向扩增后的BCR重链和轻链，全面解析BCR基因重排碱基序列，以及各序列的丰度的测序技术。BCR测序常用于评价各种免疫相关疾病和遗传性突变引起的某个物种所有B细胞或特定B细胞激活介导的细胞免疫反应中BCR基因重排碱基序列，以及各序列的丰度，用于研究不同B细胞克隆的转录情况和相互间关系，从而揭示更深层次的B细胞功能特异性，继而解释体液免疫应答耐受以及高频突变在B细胞应答识别抗原异常等相关生命现象。传统的BCR测序都是采用使用Illumina的测序仪进行2×300bp或2×150bp的双端测序方法来对BCR进行测序，该方法的测序准确率高，但对于部分长度超过600bp的BCR序列，该方法仅能获得两端的序列，会存在中间部分关键可变区序列缺失的问题。
PacBio公司正式推出Sequel II新一代测序系统后，依赖于PacBio SMRT测序技术独有的CCS测序模式，能够通过滚环测序提升reads准确度，同时结合聚合酶试剂的优化，酶读长大幅提升，在获得高精准度HiFi reads的同时能够保证10kb以上的插入片段读长，将解决原有的Illumina等二代测序平台下无法完整覆盖整个片段区域的问题。通过进行HiFi测序，可以获得准确性达到99.5％以上的长读长序列。
目前，针对新冠病毒侵染人体后引起的BCR免疫反应，常规的方法是采用二代测序平台对抗体的可变区进行测序后筛选新冠相关抗体，但是受限于测序的读长，仅能够获得可变区的部分序列，无法进行准确的抗体类型识别与编码翻译识别，而基于三代测序平台PacBio的新冠病毒BCR全长扩增测序可轻松读取病毒侵染后的BCR全长序列，突破了二代测序读长较短的局限性，提高了针对新冠病毒特异性BCR抗体的分辨能力，极大的提高了新冠特异性BCR抗体鉴定的分辨率与准确率。特别是在PacBio公司正式推出Sequel II新一代测序系统后，依赖于PacBio SMRT测序技术独有的HiFi测序模式，能够通过滚环测序提升reads准确度，目前采用HiFi技术进行BCR抗体全长测序的准确率可以达到99.5％以上，通过进行HiFi测序，可以获得极高准确性的新冠BCR抗体全长序列，用于新冠相关BCR抗体序列筛选、基于全长抗体序列的表达翻译与后续新冠中和反应评估等。
BCR一般包括与膜结合的免疫球蛋白分子和Ig-α/Ig-β信号转导组件，通过二硫键连接着免疫球蛋白分子和信号转导组件。BCR包括以下两部分：1.某一亚型(IgD、IgM、IgA、IgG或者IgE)的膜结合免疫球蛋白(mIg)。除了C端疏水的膜结合区和胞内区，这些膜结合免疫球蛋白和分泌型的免疫球蛋白单体是相同的，有两条重链(IgHs)和两条轻链(IgLs)；2.信号转导组件：Ig-α/Ig-β的异二聚体(CD79)，由二硫键连接。两个亚基都是跨膜蛋白，在胞内区都有免疫受体酪氨酸的活化基序(ITAM)。
免疫球蛋白A(Immunoglobulin A，缩写：IgA)，是血清中的含量仅次于IgG，占血清免疫球蛋白的10～20％，存在于黏膜组织，例如消化道、呼吸道以及泌尿生殖系统。黏膜组织具有黏膜层淋巴组织，会制造出IgA以避免遭到病原的入侵，也存在于唾液、泪液以及乳汁当中，尤其是初乳，其IgA的含量相当高。IgA是机体黏膜防御系统的主要成分，广泛分布于乳汁、唾液以及胃肠道、呼吸道、泌尿生殖道黏膜分泌液中。它能抑制微生物在呼吸道上皮附着，减缓病毒繁殖，有重要的免疫屏障作用，对某些病毒、细菌和一般抗原具有抗体活性，是防止病原体入侵机体的第一道防线。
目前为止，未能通过筛选方式获得新冠特异性的IgABCR抗体全长。
发明内容
本发明目的在于提供一种全人源的新冠IgA单链抗体，这类特异识别新冠的单链抗体本身或者其可变区序列，经过直接表达或经基因工程改造成其它的抗体形式后，可以作为抗新冠药物开发、疫苗生产、检测标志物开发等进行新冠相关应用。
本发明提供一种全人源的新冠IgA单链抗体，包括如SEQ ID No：2所示的氨基酸序列。
本发明还提供，编码上述新冠IgA单链抗体的基因序列，优选地，包括如SEQ IDNo：1所示的核苷酸序列。
本发明还提供，包含上述新冠IgA单链抗体中所述基因序列的文库。
本发明还提供，上述文库的制备方法，对不同新冠康复者共有而在正常人群中没有的BCR序列进行分析并筛选出新冠特异性的IgABCR抗体序列。
进一步地，所述分析筛选过程为：对新冠康复者与正常人群进行BCR全长测序，将测序的原始数据进行HiFi的一致性校正后，获得了质量值在Q20以上的BCR全长一致性序列，经过与BCR数据库中的抗体恒定区序列进行比对后，获得了每个样本测序数据中的不同类别的BCR抗体序列。
本发明还提供，包含上述新冠IgA单链抗体中所述基因序列的表达载体。
本发明还提供，包含上述新冠IgA单链抗体中所述基因序列的宿主细胞。
本发明还提供上述新冠IgA单链抗体在制备新冠病毒治疗药物、药物载体及检测标记物中的应用。
相比现有技术，本发明的有益效果为：
1.本发明通过使用BCR全长扩增子测序的方法对新冠康复者与健康人群进行了BCR全长扩增建库测序，获得了质量值在Q20以上的BCR全长扩增子序列，获得的BCR序列包括完整的启动子至终止密码子的的区域，所获得的序列为全人源，无需进一步进行整合即可进行后续的表达验证。
2.本发明通过PacBio HiFi测序获得高质量的BCR序列，经与数据库进行比对后，根据人的BCR序列在恒定区的保守性，对获得的BCR全长序列进行转录方向校正与分类，构建新冠康复者与建库人群不同类别的BCR抗体全长数据库；传统的二代BCR测序技术仅进行部分可变区的测序，无法准确进行抗体的聚类筛选。
3.本发明针对获取的不同类型的BCR序列，基于不同类别抗体恒定区BCR序列的一致性，通过定位位于恒定区上的终止密码子位置，将获得的抗体直接翻译为蛋白质氨基酸序列，在蛋白质水平上对抗体进行比较；传统的二代BCR测序方法不涉及BCR序列的恒定区，无法进行准确的翻译，仅能在DNA序列的水平上进行比较。
4.本发明通过对新冠康复者与正常人群的不同类别的抗体蛋白质氨基酸序列进行比较，找到新冠康复者共有而在正常人群中没有的BCR抗体，这些抗体为新冠特异性抗体；其抗体DNA序列为全人源的新冠特异性抗体全长序列，在经过直接表达或经基因工程改造成其它的抗体形式后，可以作为抗新冠药物开发、疫苗生产、检测标志物开发等进行新冠相关应用中；传统的新冠相关应用均是基于可变区的部分序列进行基因工程改造后再进行表达，由于其序列并非全人源，且必须经过改造，可能引入某些未知的安全问题。
5.本发明通过对获取的新冠特异性抗体序列设计特异性引物，该引物扩增区域包括启动子至终止密码子的全部区域，使用该引物在新冠康复者的cDNA作为模板进行PCR扩增后，将扩增产物转移至大肠杆菌表达载体中，通过构建单克隆后进行一代测序，基于扩增片段大小筛选获取的目标序列进行测序，在测序数据中找到了单一的新冠特异性的抗体DNA序列，获取的DNA序列为单一的纯的全人源特定抗体DNA序列，无需进行人工合成即可直接作为抗新冠药物开发、疫苗生产、检测标志物开发的原始DNA反应物。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，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，还可以根据这些附图获得其他相关的附图。
图1是本发明新冠相关特异性抗体筛选与获取原理示意图。
图2是本发明特异性引物扩增富集新冠相关BCR序列电泳图示意图。
图3是本发明单克隆制备培养示意图。
图4是本发明单克隆扩增筛选电泳图。
具体实施方式
以下实例用于说明本发明，但不限制本发明的范围。在不背离本发明精神和实质的前提下，对本发明的方法、步骤或条件所作的修改或替换，均属于本发明的范围。
如图1所示，本发明提供的全人源的新冠单链抗体获取流程如下：
1.通过进行新冠康复者与健康人群的BCR全长扩增子建库测序，获取质量值Q20以上的BCR全长序列，分别构建新冠康复者与健康人群的BCR抗体库；
2..在获得高质量的BCR抗体序列后，与抗体数据库进行比对，构建BCR抗体恒定区序列确定抗体类别，由于同类型的BCR抗体相似性非常高，且进行翻译时翻译的终止密码子位置也是一致，基于终止密码子位置将BCR抗体序列翻译为蛋白质氨基酸序列；
3.通过比较新冠康复者共有，而健康人群没有的BCR蛋白质氨基酸序列，找到新冠特异性相关的不同类别的BCR序列；
4.基于找到的新冠特异性序列设计特异引物，进行扩增特异性富集新冠相关的抗体序列；
5.将抗体序列转移至感受态细胞中，制备单克隆，挑选单克隆进行扩增后，选择特定片段大小的扩增产物进行一代测序，基于一代测序的结果筛选获得的全人源的新冠相关的BCR序列。
在本发明中，获得了新冠特异性IgA序列1条，获得的新冠特异性IgA序列如SEQ IDNo：1所示，经翻译后的氨基酸序列如SEQ ID No：2所示。所有的序列均包含VDJ区域，在5’端包括起始密码子ATG，在3’端包括终止密码子TGA，该抗体序列为全人源序列，可安全的应用于后续疫苗生产与抗体药物研发等应用中。
根据本发明的新冠特异性IgA中特异性核苷酸或氨基酸序列，可在体外人工合成与此相同的抗体轻重链基因的核苷酸序列或编码相同氨基酸的核苷酸序列，从而获得相同的抗体基因或用于相关基因的改造，而获得IgA抗体或相关蛋白。
在本发明中，提出的包含上述新冠IgA单链抗体中所述基因序列的文库，所述文库为：通过对新冠康复者与健康人群的BCR全长扩增子建库测序，获取质量值Q20以上的BCR全长序列，分别构建新冠康复者与健康人群的BCR抗体库；经与数据库进行比对后，根据人的BCR序列在恒定区的保守性，对获得的BCR全长序列进行转录方向校正与分类，构建新冠康复者与建库人群不同类别的BCR抗体全长数据库。
在本发明中，提出包含所述基因序列的表达载体，根据本领域的常识，载体可以为原核细胞表达载体、真核细胞表达载体或昆虫细胞表达载体。
在本发明中，提出包含所述表达载体的宿主细胞，根据本领域的常识，所述的宿主细胞可以为原核表达细胞、真核表达细胞或昆虫细胞，所述原核表达细胞优选大肠杆菌。
实施例一：新冠康复者与正常人群BCR全长扩增测序
新冠康复者与正常人群的BCR全长扩增测序参照前期申请的专利方法(公开号CN111662970A)进行，主要流程如下：
1.全血样本Total RNA的提取
取新鲜全血样本1mL，使用TriZol LS进行全血样本Total RNA提取，提取后使用Nanidrop 2000C测定RNA样本的浓度与纯度，使用Agilent 2100测定样本的完整性，达到合格标准的样本(总量>1μg，完整性RIN值>7)的样本进行后续实验。
2.总RNA中cDNA第一链的合成
实验操作流程如下：
1)Oligo dT逆转录引物与poly(A)结合，如表1所示。
表1：
轻弹混匀，瞬时离心，70℃孵育5min后立即置于冰上。
2)逆转录合成cDNA第一链，配制表2中的反应。
表2：
轻弹混匀，瞬时离心，42℃孵育75min。反应完成后立即置于冰上，加入1uL的BCRTemplate Switching Oligo，轻弹混匀，瞬时离心，42℃孵育15min。
3.BCR cDNA的全长扩增
BCR cDNA的全长扩增包括两轮半巢式扩增反应，第一轮扩增进行BCR序列的初步富集，第二轮扩增时采用内部的巢式引物，进一步提高扩增的特异性，使扩增条带单一。
1)BCR cDNA全长的第一轮PCR扩增
取一新的0.2mL PCR管，加入表3中的试剂。
表3：
充分混匀，瞬时离心，置于PCR仪上进行PCR反应：98℃2min；98℃20s、65℃15s、72℃45s，18cycles；72℃5min。
2)BCR cDNA全长的第二轮PCR扩增
取一新的0.2mL PCR管，加入表4中的试剂。
表4：
10组混合引物均需要单独进行扩增，可以提高扩增反应的稳定性。
充分混匀，瞬时离心，置于PCR仪上进行PCR反应：98℃2min；98℃20s、65℃15s、72℃30s，20cycles；72℃5min。
反应结束后按照AMPure磁珠的使用说明对扩增产物进行磁珠纯化，最后使用10μL洗脱缓冲液洗脱，取1μL纯化产物，使用无核酸酶水稀释5倍后进行Qubit定量。
4.BCR全长扩增子片段混样
根据Qubit定量结果，将同一样本的不同扩增产物进行等量混样，要求混样后的总量在1μg以上，用于后续建库。
5.文库构建
1)末端修复
取1μg的全基因组扩增子样本，进行末端修复反应体系的配制，配制表5中的反应。
表5：
充分混匀，瞬时离心，20℃孵育30min。
反应完成后，按照AMPure磁珠的使用说明进行1X磁珠纯化，去除反应时加入的酶与Buffer，最后使用14μL洗脱缓冲液洗脱，获得片段末端加A的粘性末端。
2)连接带barcode的测序接头
通过末端修复与加A后，在加入具有与A末端匹配的带barcode的测序接头，在连接酶的作用下即可实现接头的连接。反应体系如表6。
表6：
混匀，瞬时离心，20℃孵育60min，反应结束后65℃孵育10min，置于冰上。进行外切酶消化，反应体系如表7。
表7：
混匀，瞬时离心，37℃孵育60min，置于冰上。按照AMPure磁珠的使用说明进行磁珠纯化，最后使用20μL洗脱缓冲液洗脱，获得的适用于PacBio测序平台的哑铃型的环状文库。
3)文库质检与上机测序
取1μL文库进行Qubit定量，获得文库浓度；取1μL文库进行安捷伦2100的片段大小分析，将全长扩增文库在PacBio Sequel II测序平台上进行混合测序，每个样本获得了约60G的测序数据。
实施例二：新冠康复者与正常人群BCR免疫组库分析，获得新冠特异性IgA单链抗体
将测序的原始数据进行HiFi的一致性校正后，获得了质量值在Q20以上的BCR全长一致性序列，经过与BCR数据库中的抗体恒定区序列进行比对后，获得了每个样本测序数据中的不同类别的BCR抗体序列，同时基于恒定区的终止密码子位置，将DNA序列翻译为蛋白质多肽序列。通过比较新冠康复者共有的，而正常人群中没有的BCR多肽序列，获得了新冠特异性相关的抗体全长。本次获得的新冠特异性IgA序列1条，所有的序列均包含VDJ区域，在5’端包括起始密码子ATG，在3’端包括终止密码子TGA，该抗体序列为全人源序列，可安全的应用于后续疫苗生产与抗体药物研发等应用中。获得的新冠特异性IgA序列如SEQ IDNo：1所示，经翻译后的氨基酸序列如SEQ ID No：2所示。
实施例三：基于以新冠康复者样本设计特异性引物，进行单克隆BCR抗体库的构建
基于抗体库筛选的IgA抗体序列，在起始密码子的与终止密码子的外测设计特异性引物，通过PCR扩增特异性富集目标抗体序列。由于抗体存在多重重组的特性，获取的扩增产物是一类与引物匹配的抗体序列集合，无法直接进行一代测序与后续应用，需要将抗体序列进行克隆实验，获取单个抗体序列的单克隆，并挑选单克隆进行测序，验证所获得的单克隆序列为目标抗体。具体流程如下：
1.目标抗体IgA抗体序列引物设计
基于测序筛选的目标序列抗体设计引物，该引物扩增的目标片段包括起始密码子与终止密码子区域，覆盖整个抗体表达区域的全长，包含实施例二公开抗体的所有序列，具体设计的引物序列如表8所示。
表8：IgA抗体序列引物
其中，Primer_ID_01～02为R端通用引物，该引物在恒定区内，所有IgA抗体序列均可与该引物进行匹配，使用时混合在一起作为R端通用引物使用；Primer_ID_03～04为F端引物，该引物序列基于筛选的新冠相关特异性抗体设计，与R端引物组合在一起为一对引物，用于目标抗体序列的扩增。
2.扩增富集获取新冠相关特异性抗体
本步骤使用的扩增模板为新冠康复者样本的单链cDNA样本。取一新的0.2mL PCR管，加入如表9所示试剂。
表9：
充分混匀，瞬时离心，置于PCR仪上进行PCR反应：98℃2min；98℃20s、65℃15s、72℃120s，35cycles；72℃5min。
反应结束后进行电泳检测，电泳结果示意图如图2所示。切胶获取片段大小在1.5～2k的目标特异性片段。
3.目标片段的单克隆制备
本步骤使用表10中的试剂。
表10：
LB固体培养基配制：取本品8克，溶解于250ml蒸馏水中，121℃高压灭菌15分钟，至不烫手时加入250μL氨苄青霉素，混匀倒平板备用(每个平板15mL左右)。
LB液体培养基配制：取本品1克，溶解于40ml蒸馏水中，121℃高压灭菌15分钟，分装至2mL无菌离心管中备用。
配制好培养基后进行载体连接反应，配制如表11中的反应体系。
表11：
轻弹管底混匀，低速瞬时离心收集所有液体于离心管底，室温25℃反应5min。反应结束后，将离心管置于冰上。
将Fast-T1感受态细胞从-70℃拿出，迅速置于冰上融化，取20μL感受态细胞加入目的载体连接反应产物，轻弹管壁混匀(避免用枪吸打)，冰上静置30min。
42℃水浴热激30s后，迅速置于冰上静置2min，切勿摇动离心管。
向离心管中加入200μL LB液体培养基(不含抗生素)，混匀后置于37℃，200rpm摇床中复苏5min。
复苏后颠倒混匀直接取200μL，涂布在含氨苄青霉素的LB固体培养基平板上，将平板正置于37℃培养箱10min，待菌液被完全吸收后，倒置平板，过夜培养，单克隆制备培养结果示意图如图3所示。
4.单克隆筛选与测序鉴定
在平板培养基上挑选单克隆菌落至10μL ddH2O中混匀作为模板；使用2×RapidTaq Master Mix(Vazyme#P222)进行扩增，反应体系如表12所示。
表12：
放置在PCR仪上进行如表13所示的扩增反应程序。
表13：
将扩增获得的扩增产物进行凝胶电泳检测，获得图4所示的电泳图，挑选扩增条带亮度高、扩增条带单一且片段大小在1.5～5k的扩增产物进行双端单克隆一代测序鉴定，根据一代测序的序列与目标抗体序列进行一致性比对，选择序列完全一致的单克隆进一步进行扩增放大，即获得了高纯度的单一的新冠相关抗体特异性序列，该序列可以直接转移至假病毒体系中，进行后续的抗体效价验证，也可以直接转移至抗体表达体系中进行表达。
序列表
<110> 武汉菲沙基因组医学有限公司
<120> 全人源的新冠IgA单链抗体及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1473
<212> DNA
<213> IgA
<400> 1
atggagttgg ggctgtgctg ggttttcctt gttgctattt tagaaggtgt ccagtgtgag 60
gtgcagctgg tggagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc 120
tgtgcagcct ctggattccc cttcaattcc cttaccatga actgggtccg ccaggctcca 180
ggggagggac tggagtggct ttcatacatt agtactagta gtaataacat attctacgca 240
gactctgtga agggccgatt caccgtctcc agagacaatg ccaagaattc actgtatctg 300
caaatgaaca gcctgagaga cgaagacacg gctgtgtatt actgtgcggg acacactggc 360
agcaactggt ttgactactg gggccaggga accctggtca ccgtctcctc agcatccccg 420
accagcccca aggtcttccc gctgagcctc tgcagcaccc agccagatgg gaacgtggtc 480
atcgcctgcc tggtccaggg cttcttcccc caggagccac tcagtgtgac ctggagcgaa 540
agcggacagg gcgtgaccgc cagaaacttc ccacccagcc aggatgcctc cggggacctg 600
tacaccacga gcagccagct gaccctgccg gccacacagt gcctagccgg caagtccgtg 660
acatgccacg tgaagcacta cacgaatccc agccaggatg tgactgtgcc ctgcccagtt 720
ccctcaactc cacctacccc atctccctca actccaccta ccccatctcc ctcatgctgc 780
cacccccgac tgtcactgca ccgaccggcc ctcgaggacc tgctcttagg ttcagaagcg 840
aacctcacgt gcacactgac cggcctgaga gatgcctcag gtgtcacctt cacctggacg 900
ccctcaagtg ggaagagcgc tgttcaagga ccacctgagc gtgacctctg tggctgctac 960
agcgtgtcca gtgtcctgcc gggctgtgcc gagccatgga accatgggaa gaccttcact 1020
tgcactgctg cctaccccga gtccaagacc ccgctaaccg ccaccctctc aaaatccgga 1080
aacacattcc ggcccgaggt ccacctgctg ccgccgccgt cggaggagct ggccctgaac 1140
gagctggtga cgctgacgtg cctggcacgc ggcttcagcc ccaaggatgt gctggttcgc 1200
tggctgcagg ggtcacagga gctgccccgc gagaagtacc tgacttgggc atcccggcag 1260
gagcccagcc agggcaccac caccttcgct gtgaccagca tactgcgcgt ggcagccgag 1320
gactggaaga agggggacac cttctcctgc atggtgggcc acgaggccct gccgctggcc 1380
ttcacacaga agaccatcga ccgcttggcg ggtaaaccca cccatgtcaa tgtgtctgtt 1440
gtcatggcgg aggtggacgg cacctgctac tga 1473
<210> 2
<211> 490
<212> PRT
<213> IgA_translation
<400> 2
Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe
35 40 45
Asn Ser Leu Thr Met Asn Trp Val Arg Gln Ala Pro Gly Glu Gly Leu
50 55 60
Glu Trp Leu Ser Tyr Ile Ser Thr Ser Ser Asn Asn Ile Phe Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
85 90 95
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Gly His Thr Gly Ser Asn Trp Phe Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Pro Thr Ser Pro Lys
130 135 140
Val Phe Pro Leu Ser Leu Cys Ser Thr Gln Pro Asp Gly Asn Val Val
145 150 155 160
Ile Ala Cys Leu Val Gln Gly Phe Phe Pro Gln Glu Pro Leu Ser Val
165 170 175
Thr Trp Ser Glu Ser Gly Gln Gly Val Thr Ala Arg Asn Phe Pro Pro
180 185 190
Ser Gln Asp Ala Ser Gly Asp Leu Tyr Thr Thr Ser Ser Gln Leu Thr
195 200 205
Leu Pro Ala Thr Gln Cys Leu Ala Gly Lys Ser Val Thr Cys His Val
210 215 220
Lys His Tyr Thr Asn Pro Ser Gln Asp Val Thr Val Pro Cys Pro Val
225 230 235 240
Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser
245 250 255
Pro Ser Cys Cys His Pro Arg Leu Ser Leu His Arg Pro Ala Leu Glu
260 265 270
Asp Leu Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly
275 280 285
Leu Arg Asp Ala Ser Gly Val Thr Phe Thr Trp Thr Pro Ser Ser Gly
290 295 300
Lys Ser Ala Val Gln Gly Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr
305 310 315 320
Ser Val Ser Ser Val Leu Pro Gly Cys Ala Glu Pro Trp Asn His Gly
325 330 335
Lys Thr Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro Leu
340 345 350
Thr Ala Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro Glu Val His
355 360 365
Leu Leu Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu Val Thr
370 375 380
Leu Thr Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp Val Leu Val Arg
385 390 395 400
Trp Leu Gln Gly Ser Gln Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp
405 410 415
Ala Ser Arg Gln Glu Pro Ser Gln Gly Thr Thr Thr Phe Ala Val Thr
420 425 430
Ser Ile Leu Arg Val Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe
435 440 445
Ser Cys Met Val Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys
450 455 460
Thr Ile Asp Arg Leu Ala Gly Lys Pro Thr His Val Asn Val Ser Val
465 470 475 480
Val Met Ala Glu Val Asp Gly Thr Cys Tyr
485 490
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gagtttattc aggggtggg 19
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
acaggcgggc ggctcagtag 20
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gattccaagg catttcca 18
<210> 6
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agctctcaga gaggtgc 17
Claims (6)
1.一种全人源的新冠IgA单链抗体，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
2.编码权利要求1所述的新冠IgA单链抗体的基因。
3.根据权利要求2所述的新冠IgA单链抗体基因，其特征在于，其核苷酸序列如SEQ IDNO：1所示。
4.包含权利要求3所述新冠IgA单链抗体基因的表达载体。
5.包含权利要求3所述新冠IgA单链抗体基因的宿主细胞。
6.权利要求1所述的新冠IgA单链抗体在制备新冠病毒治疗药物、药物载体及检测标记物中的应用。
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