CN114835803A - 一种人工抗体的制备方法 - Google Patents
一种人工抗体的制备方法 Download PDFInfo
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- CN114835803A CN114835803A CN202210491741.9A CN202210491741A CN114835803A CN 114835803 A CN114835803 A CN 114835803A CN 202210491741 A CN202210491741 A CN 202210491741A CN 114835803 A CN114835803 A CN 114835803A
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Abstract
本发明涉及一种人工抗体的制备方法，其特征在于，从冠状病毒保守基因或微卫星中筛选靶标siRNA，由两条互补的正反义siRNA链合成带loop环的小发夹shRNA，并合成以受体结合域RBD为配体的ACE2，然后将ACE2分别连接到shRNA的正反义链上，合成由shRNA区和ACE2区构成的人工抗体，其中双价ACE2起中和RBD和靶向递送shRNA的作用，shRNA通过ACE2连接病毒，并随着病毒的感染进入靶细胞，不但可避免shRNA的非特异性递送给未感染细胞的副作用，而且还能产生抗变异毒株和以ACE2中和病毒的作用，另外以两分子ACE2和1分子shRNA合成的人工抗体及shRNA的免疫佐剂作用可使ACE2具有更强的抗原性，刺激机体产生的抗‑ACE2又具有抗病毒作用。
Description
技术领域
本发明涉及一种广谱抗病毒人工抗体的制备方法，属于传染病防治的生物制药领域。
背景技术
新型冠状病毒的主要结构包括单股正链核酸(ssRNA)、刺突蛋白(S)、膜蛋白(M)、包膜蛋白(E)和核壳蛋白(N)，其中S蛋白的N端由结构域(S1-NTD)和受体结合域(S1-RBD)组成，新型冠状病毒通过其受体结合域S1-RBD与宿主细胞受体ACE2结合引起感染。
ACE2为I型跨膜糖蛋白，由805个氨基酸组成，包括跨膜区、胞内羧基端和胞外氨基端，冠状病毒通过其S1-RBD与ACE2的细胞外催化结构域互动结合，导致细胞内吞、膜融合，使病毒进入表达ACE2或含有ACE2受体的细胞。
RNA干扰(RNAi)作为一种高效的序列特异性基因沉默技术，正在给疾病的治疗带来难以想象的应用前景，已有多种siRNA药物被FDA审批上市。小干扰RNA(siRNA)大约21～23bp，以参与RNA干扰(RNAi)的方式调节基因表达,特异性降解与之互补的靶信使RNA(mRNA)，但无论是细胞水平还是活体内，使用siRNA进行基因干扰均要克服很多困难：1)膜通透性：siRNA带有大量的负电荷，分子量大(～13KD)，自身很难穿过细胞膜，运输siRNA主要靠化学修饰和一些运输载体；2)抗核酸酶降解：siRNA由大量的核糖核酸分子构成，很容易被外界的RNA酶降解，如果在设计siRNA及选择运输载体时不对碱基进行特定的化学修饰或采取载体保护方法，则在siRNA进入作用位点前即被RNA酶降解；3)靶向递送及载体：siRNA的作用位点主要在靶细胞浆内，所以需将siRNA特异性递送给靶细胞浆，如果不能有效地选用合适的靶向递送载体并及时使siRNA从内涵体释放到胞浆，通常可激活细胞免疫反应，导致干扰素等细胞因子的释放，所以，如何有效地将siRNA运输并释放到靶细胞浆是影响RNAi效果的瓶颈问题，有些siRNA能导致序列或浓度依赖性非特异性基因沉默即脱靶，因此，在设计siRNA时，应同时考虑靶向递送、基因抑制效果以及尽可能选择低脱靶效应的序列。
在COVID-19的防治中，如果能以合适的靶向递送载体将nCoVsiRNA稳定地、特异地依次递送给靶器官、靶组织、靶细胞、定位于靶细胞、穿越靶细胞膜、释放到靶细胞浆，并对多种变异毒株广谱有效，就应能更好地开展COVID-19的靶向基因治疗。
所以本发明要设计和合成一种广谱抗病毒人工抗体。
发明内容
本发明的目的是要提供一种人工抗体及其合成方法和用途，使合成抗体的双价ACE2能与RBD结合而起中和病毒和靶向递送shRNA作用，并使被ACE2靶向递送的shRNA随着病毒的感染进入靶细胞，起抗变异毒株作用。
本发明的目的通过以下技术方案实施：
筛选共有基因：从数据库记载的各种致病性冠状病毒及其变异毒株中筛选不随病毒变异而改变的共有基因，包括保守基因、超保守基因和/或保守微卫星。
筛选siRNA：从共有基因中预选多对以该共有基因为干扰靶标的siRNA，使siRNA包含有保守基因、超保守基因和/或保守微卫星。
合成siRNA：合成2条互补的21-25nt的寡核苷酸siRNA及起间隔作用的碱基序列。
合成shRNA：将已合成的2条互补寡核苷酸多肽siRNA和起间隔作用的碱基序列进一步合成由中间碱基序列间隔成loop环的小发夹shRNA双链。
优选siRNA：将合成的shRNA构建干扰载体，检测其mRNA表达、蛋白表达和干扰效果，经siRNA设计、合成、筛选、迭代设计和验证，优选具有高沉默效率的siRNA。
合成优选的siRNA和shRNA：采用优选的siRNA序列按上述合成siRNA、shRNA，包括为增加稳定性和避免脱靶进行的化学修饰。
合成ACE2多肽或蛋白：合成的ACE2包括但不限于全长ACE2、第741-763位氨基酸的跨膜ACE2、第764-805位氨基酸的胞内ACE2、第1-740位氨基酸的胞外ACE2以及经氨基酸序列密码子优化的ACE2多肽或蛋白。
人工抗体的合成：以二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键或马来酰亚胺-巯基键等偶联方法将已合成的shRNA和ACE2连接、合成为化合物；或根据shRNA的核苷酸序列和ACE2的氨基酸序列从氨基酸水平直接合成ACE2-shRNA-ACE2。
人工抗体的提纯：以高效液相色谱、反向高效液相色谱或离子交换色谱进行提纯。
人工抗体的验证：在体外细胞水平检测人工抗体对2种或以上不同变异毒株的抗病毒效果，观察是否具有以保守基因为靶标的广谱抗变异毒株作用；检测人工抗体在动物体内是否具有靶向递送shRNA的作用以及是否能刺激宿主产生ACE2-Ab。
本发明的有益效果在于：
首次设计抗冠状病毒变异靶标siRNA/shRNA，首次以ACE2作为靶向递送载体，首次以shRNA和ACE2合成具有靶向递送、中和病毒和产生ACE2-Ab作用的人工抗体。
本发明基于保守基因为不同毒株的共有基因从不随病毒变异的病毒保守基因、超保守基因和/或保守微卫星中筛选siRNA，并以这种siRNA合成广谱抗变异毒株靶标shRNA。
进而，基于配体RBD和受体ACE2的关系设计以ACE2靶向递送shRNA的人工抗体。
进而，以shRNA和ACE2合成人工抗体，使之由短发夹shRNA区和双链ACE2区构成，其中ACE2起递送shRNA和结合RBD的作用，使shRNA随病毒感染进入靶细胞，这种以ACE2和病毒共同递送shRNA的新方法，不但可避免将shRNA非特异性递送给未感染细胞的副作用，而且还产生广谱抗变异毒株的作用。
进而，原本siRNA/shRNA带负电荷、脂溶性，通透性和稳定性差，将siRNA/shRNA和ACE2合成人工抗体后，则优化了siRNA/shRNA的通透性和稳定性，易被递送。
进而，以2个ACE2分子和1个shRNA分子合成的ACE2siRNA，因ACE2以双价结合RBD，所以实质上是以双价ACE2中和病毒，从而阻断病毒感染靶细胞。
进而，因人工抗体中含有2个ACE2分子和1个shRNA分子，以及shRNA除了抗病毒外还能产生免疫佐剂的作用(shRNA也是主要佐剂之一)，所以人工抗体比单分子ACE2的抗原性更强，能刺激宿主产生更高效价的ACE2-Ab。因ACE2-Ab能与病毒竞争靶细胞的ACE2受体，所以人工抗体产生的ACE2-Ab具有阻断病毒感染的作用。
进而，体外细胞实验表明合成的人工抗体对2种不同变异毒株同时有效，说明具有以保守基因为靶标的抗变异毒株作用；体内动物实验表明人工抗体在动物体内具有靶向递送的RNAi作用，刺激产生的ACE2-Ab可通过封闭ACE2受体而抑制病毒感染。
附图说明
图1是本发明制备人工抗体的技术线路图。
图2是本发明人工抗体的结构示意图。
图3是本发明人工抗体及其应用示意图。
在图1中，经保守基因筛选、以保守基因为靶标的广谱抗变异毒株靶标siRNA筛选、shRNA合成、ACE2合成，最终合成本发明的人工抗体ACE2-shRNA-ACE2。
在图2中，1为loop环，2为由两条互补的正反义链形成的shRNA，3为两条ACE2多肽(蛋白)，两条ACE2多肽分别与shRNA的正反义链相连接。shRNA被ACE2保护并被ACE2靶向递送至病毒受体结合域RBD或ACE2受体通道，然后随病毒RBD通过ACE2通道而特异性进入靶细胞浆，降解病毒靶基因。
在图3中，1是loop环，由间隔小发夹shRNA正反义链的碱基序列间隔形成；2是shRNA，由两条正反义链siRNA互补结合形成，该siRNA以冠状病毒保守基因为干扰靶标，具有广谱抗变异毒株的靶向基因治疗作用；3是与shRNA的正反义链相连接的ACE2；4是冠状病毒；5是冠状病毒S蛋白受体结合域RBD；6是细胞7表达的ACE2受体；8是不表达ACE2的细胞。如图2所示，由loop环1、shRNA 2和ACE2 3构成ACE2siRNA；冠状病毒4通过其受体结合域RBD5与受体ACE2 6特异性结合而感染细胞7，但冠状病毒4不会感染不表达ACE2的细胞8；ACE2siRNA中的ACE2 3与细胞7表达的ACE2 6一样，也能与冠状病毒4的RBD 5结合，所以ACE2 3能与ACE2 6竞争结合RBD 5，使ACE2 3具有抑制病毒4感染细胞7的作用；ACE2siRNA在后期又起疫苗的作用，因为ACE2 3能刺激宿主产生抗-ACE2,抗-ACE2能封闭ACE2 6，从而产生抑制病毒4感染细胞7的作用；更重要的是，通过ACE2 3的桥接，形成了“shRNA 2-ACE23-RBD 5-病毒4”的复合物，使shRNA 2随病毒4进入细胞7，靶向干扰病毒4在细胞7中复制，避免因shRNA 2非特异性进入未感染病毒4的细胞8而产生的毒副作用。
具体实施方式
下面结合图1、2和3，对本发明的具体实施方法作详细的描述，但这些范例性的描述并不对本发明的权利要求所限定的保护范围构成任何限制。
一、设计以超保守基因、保守基因或保守微卫星为靶标的siRNA
1、超保守基因、保守基因及保守微卫星的设计
如技术线路图1所示，从Genbank数据库(http://www.NCBI.nlm.nih.gov/genome/)中下载β冠状病毒属(特别是新型冠状病毒及其变异毒株)全基因组(cDNA)序列，在全基因组序列中搜寻最长的公共子序列，获得超保守基因或保守基因；利用Clustal W软件对Genbank数据库下载的全基因组进行序列比对，检测不同序列之间的相似度，筛选保守微卫星序列；利用MEGA6.0分子进化遗传分析软件，用邻接法(Neighbour-Jioning，N-J)对下载的冠状病毒氨基酸序列构建氨基酸种系分子进化树，对氨基酸序列的分子变异特征进行分析，推断保守基因序列。
共找到以下3段最长及次长的超级保守子序列(没有插入或删除的完全相同的子序列)，其长度为22～30bp,与小RNA长度相当，但在高等生物特别是人类中不包含这3段子序列。
具体序列如下：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacaca gagt(22bp)；
SEQ ID NO.3(Subsequence 3)＝caggcgtttgttggttgattaa(22bp)。
共找到以下3段最长及次长的保守子序列，它们的长度为22～30bp,与小RNA长度相当，但在高等生物特别是人类中不包含这3段保守子序列：
SEQ ID NO.4(Subsequence 1)＝gttttacgacaacgatgttggtttaggaca(30bp)；
SEQ ID NO.5(Subsequence 2)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 3)＝ggttcagagagtctcctattta(22bp)。
共找到以下5个由核苷酸重复多次的保守微卫星位点，微卫星分别为CTCTCT、AGAGAG、AAAAAAA、TATATA、CACACA。
2、筛选以超保守基因、保守基因或保守微卫星为靶标的siRNA
利用Clustal W软件或其他软件，将上述设计的超保守基因、保守基因及保守微卫星与常规筛选的siRNA进行基因序列比对，检测不同序列之间的相似度，设计既为超保守基因、保守基因或保守微卫星，又为RNAi靶位点的多对siRNA(设计以超保守基因、保守基因或保守微卫星为靶标的siRNA)。
(1)以超保守基因和保守微卫星为靶标的siRNA(S1/S2)：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacaca gagt(22bp)；
(2)以保守基因和保守微卫星为靶标的siRNA(S3/S4)：
SEQ ID NO.5(Subsequence 3)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 4)＝ggttcagagagtctcctattta(22bp)。
通过上述设计，获得以超保守基因、保守基因或保守微卫星为干扰靶标的在理论上抗冠状病毒变异毒株的siRNA，命名为siRNA1/2/3/4。
3、筛选常见变异毒株的共同靶标siRNA
根据Genbank数据库(http://www.NCBI.nlm.nih.gov/genome/)的β冠状病毒属(特别是新型冠状病毒及其变异毒株)的完整基因组(cDNA)序列，利用多种shRNA在线设计软件(例如http://www.ambion.com/techlib/misc/siRNAtools.html)获得长度约为19nt的多个siRNA备选序列，根据RNA结合的Tm值及特异性比对结果，优选siRNA。例如，对新冠病毒变异毒株B.1.617.1、B.1.1.529、B.1.1.7、P.1、BA.2、B.1.351、B.1.525、B.1.617.2、C.1.2、B.1.621.1、B.1.621、P.2、N.9、C.37.1、C.37、B.1.427、B.1.351.2和B.1.351.3共18株变异毒株的E、M、N、ORF1ab和S基因进行s iRNA筛选，获得表1所示的共同靶标SEQ IDNO.7～39，其中SEQ ID NO.7～10、SEQ ID NO.16～18、SEQ ID NO.20～22、SEQ ID NO.30～32均为score较高的优选siRNA，可作为靶向新冠病毒变异毒株的候选靶标。
表1从18株新冠病毒变异毒株中筛选的共同靶标siRNA(SEQ ID NO.7～39)
二、验证siRNA功能
1、合成siRNA/shRNA
当siRNA有效干扰SARS-CoV-2的S基因的mRNA表达时，会形成失去感染性的S蛋白缺陷型病毒。当siRNA有效干扰SARS-CoV-2的N基因的mRNA表达时，会抑制病毒的包装、复制。当siRNA有效干扰SARS-CoV-2的ORF1a或1b基因的mRNA表达时,会影响病毒RNA聚合酶(RdRp)或蛋白质加工酶(3CLpro)的合成。而M和E基因是病毒的膜基因，膜缺陷对病毒的抑制作用可能不明显。所以本发明选用靶向N基因的siRNA(SEQ ID NO.16-18)、靶向ORF1ab基因的siRNA(SEQ ID NO.20-22)、靶向S基因的siRNA(SEQ ID NO.30-32)，以及SEQ ID NO.1～2、SEQ ID NO.5～6进行合成。另外根据pSilencer4.1.CMV.neo干扰载体的多克隆酶切位点设计能表达发夹结构的shRNA模板，每个模板由两条大部分互补的55bp的单链DNA构成，退火互补后能形成带有BamH I和Hind III酶切位点粘性末端的DNA双链，用于与线性化的pSilencer4.1.CMV.neo的连接。然后按设计的siRNA及其shRNA模板，委托公司合成。
2、shRNA表达载体的构建
将上述合成的shRNA分别与线性化的干扰载体pSilencer4.1.CMV.neo进行连接和鉴定，构建shRNA表达质粒，转化DH5a，获得shRNA表达载体。
3、shRNA表达(干扰)载体的效果鉴定
根据合成的siRNA/shRNA及其构建的表达质粒，选择相应的靶基因进行合成或PCR扩增，然后构建荧光标签载体，并分别与shRNA表达质粒共转染293T细胞，进行鉴定。
PCR扩增的常规方法如下：
引物设计：设计上、下游引物，在上游引物5'端添加起始密码，为将扩增产物克隆到pEGFP-N1中，引物的5'端添加用于与载体发生同源重组的同源臂。
靶基因扩增：按上海生工试剂盒所提供的基因扩增反应体系及反应条件进行基因扩增、产物回收和纯化，获得扩增产物。
pEGFP-N1的线性化：复苏含有pEGFP-N1质粒的DH5a菌种，按试剂盒提取质粒，测定浓度后进行酶切，0.8％琼脂糖凝胶电泳鉴定并回收线性化的载体。
将扩增的靶基因与荧光标签载体(pEGFP-N1)进行连接：使用金斯瑞公司的同源重组试剂盒进行连接，连接结束后可保存在-20℃备用或马上进行转化。
shRNA干扰载体的效果鉴定：分别将干扰载体(pSilencer-shRNA)和荧光标签载体(pEGFP-N/S/ORF1ab)共转染293T细胞，干扰载体与标签载体的质量比为1：2，同时设立对照，转染后48h观察细胞内GFP蛋白的融合表达，根据荧光强度评价干扰效果：
流式细胞检测：为定量分析不同干扰载体的干扰效果，用流式细胞术检测，分析荧光蛋白表达细胞在总细胞数中的比例。
Westernbolt分析：①细胞收集与裂解：以RIPA裂解细胞。②SDS-PAGE蛋白电泳：制备SDS-PAGE胶，将样品加入等体积的2xSDS缓冲液，沸水煮5min，冰浴2min，12000xg，10min。③Western blot检测：经转膜、封闭、一抗结合、洗涤、二抗结合和显色，观察结果。
RT-PCR检测mRNA：采用相对荧光定量RT-PCR法检测转染细胞中靶基因的相对表达量，根据标准曲线，由CT值换算目的基因以及B-actin内参基因拷贝数，以B-actin内参基因校正病毒基因mRNA相对表达量(目的基因拷贝数/B-actin拷贝数)，定量评价干扰效果。
4、获得高沉默效率的siRNA/shRNA
经上述设计、合成、筛选、迭代设计、再合成和细胞水平的验证，优选具有较高沉默效率的siRNA/shRNA，其序列分别为SEQ ID NO.1(命名为shRNA1，下同)、SEQ ID NO.2(shRNA2)、SEQ ID NO.5(shRNA5)，以及靶向N基因的SEQ ID NO.16(shRNA16)、靶向ORF1ab基因的SEQ ID NO.21(shRNA21)、靶向S基因的SEQ ID NO.30(shRNA30)，其沉默效率分别为78％、76％、88％、89％、84％、90％。
三、合成以保守基因为靶标的shRNA
根据上述筛选的共同靶标(SEQ ID NO.1-39)和优选的具有高沉默效率的序列SEQID NO.5(shRNA5)、SEQ ID NO.16(shRNA16)、SEQ ID NO.21(shRNA21)和SEQ ID NO.30(shRNA30)，委托生物公司将具有高沉默效率的序列合成shRNA，每个shRNA合成2条互补的19-25nt的寡核苷酸多肽siRNA，以及合成起间隔作用的9nt的碱基序列，然后将所合成的siRNA和碱基序列连接成由中间碱基序列间隔成loop环的小发夹shRNA双链，所合成的shRNA双链的每条单链均可以分别连接ACE2。
例如，可分别将SEQ ID NO.1(shRNA1)、SEQ ID NO.2(shRNA2)和SEQ ID NO.5(shRNA5)合成5'-ttaatacgacctctctgttggattttgacattcaagagatgtcaaatccaacagagaggtcgtattaa-3'(SEQ ID NO.40)、5'-ggttcgcaacttcacacagagtttcaagagaactctgtgtgaagttgcgaacc-3'(SEQ ID NO.41)和5'-ggtt cggt tgtta tatac gata ttcaagaga tatc gtatataaca accg aacc-3'(SEQ ID NO.42)，其中“TTCAAGAGA”为loop环，其左、右侧分别为互补的正反义链，SEQ ID NO.40～42分别示意shRNA1、shRNA2和shRNA5的合成。同理优选高沉默效率的siRNA，合成shRNA，在其3'和/或5'分别连接ACE2或其多肽。
四、靶向递送载体ACE2的设计和合成
1、ACE2的氨基酸序列
从GeneBank数据库(http://www.ncbi.nlm.gov/genbank)中查找人ACE2基因序列信息，ACE2由805个氨基酸组成，其中第1-740位氨基酸序列位于细胞膜外，第741-763位氨基酸位于跨膜区，第764-805位氨基酸位于细胞内。在构成ACE2蛋白的20种氨基酸中，亮氨酸占9.4％，半胱氨酸和组氨酸分别占1.0％和2.0％，以及带负电荷氨基酸残基(天冬氨酸+谷氨酸)和带正电荷氨基酸残基(精氨酸+赖氨酸)等，SARS-CoV及SARS-CoV 2通过病毒受体结合区与ACE2的细胞外催化结构域互动结合，这种互动能够导致细胞内吞、膜融合，从而使得SARS-CoV进入高表达ACE2或含有ACE2受体的细胞内。
2、ACE2的受体作用
ACE2为脂溶性的I型跨膜糖蛋白，有氨基末端催化结构域和羧基末端结构域，其中N端在细胞膜外，C端在细胞膜内，分为N末端信号肽区、羧基多肽酶活化区和跨膜区。当冠状病毒Spike蛋白接触ACE2催化结构域的亚结构域I的顶端(不影响亚结构域II和肽酶活性)时，ACE2的外结构域被裂解，跨膜结构域被内在化，使病毒-宿主细胞进一步融合，所以认为跨膜区与病毒-受体复合物从细胞膜转运到细胞质有关。
3、本申请ACE2的设计
从ACE2的氨基酸序列及ACE2的受体作用可知，ACE2表达细胞的细胞壁和细胞膜均存在ACE2受体通道，全长ACE2、跨膜区ACE2、胞内ACE2及胞外ACE2均属于穿膜多肽，具有靶向递送siRNA的功能，胞外ACE2的N端具有结合冠状病毒RBD的中和病毒功能。可设计并合成全长ACE2、第741-763位氨基酸的跨膜ACE2、第764-805位氨基酸的胞内ACE2、第1-740位氨基酸的胞外ACE2，以及优化氨基酸序列的ACE2多肽作为siRNA的靶向递送载体。
4本申请ACE2的合成
两个氨基酸脱水缩合形成肽键，多个氨基酸残基以肽键相连接形成多肽。可委托公司采用多肽合成仪自动化合成多肽，基本方法是，按合成多肽的序列逐个加入氨基酸，使肽链从C端到N端残基逐步延长，要求每一个氨基酸残基以一端保护和另一端活化的形式缩合，并且在每一轮肽链延长循环后除去氨基上的临时性保护基团，直至目标多肽的全部氨基酸序列缩合完毕。目前常用的固相合成多肽的反应原理是在密闭的防爆玻璃反应器中使氨基酸按照已知的序列从C端-羧基端向N端-氨基端的顺序不断添加所需氨基酸，进行合成反应，最终得到多肽。其步骤包括：①去保护：用碱性溶剂去除氨基的保护基团；②激活和交联：活化下一个氨基酸的羧基，使活化的单体羧基与游离的氨基交联，形成肽键，反复循环这两步反应，直到多肽合成完成。
五、以ACE2和shRNA合成人工抗体
1、人工抗体(2ACE2-shRNA)的设计
以胞外ACE2(SEQ ID NO.43)和shRNA[SEQ ID NO.5(shRNA5)、SEQ ID NO.16(shRNA16)、SEQ ID NO.21(shRNA21)和SEQ ID NO.30(shRNA30)]合成为例，将合成的siRNA/shRNA的正反义链的一端与loop环(5'-TTCAAGAGA-3’)相连，另一端分别与胞外ACE2(C端)相连，连接成“胞外ACE2-siRNA正义链-loop环-siRNA反义链-胞外ACE2”的结构，其中两条互补的正反义链会形成双链，但两条ACE2多肽不会形成双链，如图2所示，是带有两条ACE2多肽、两条siRNA正反义链和loop环的发夹状连接物，即ACE2-shRNA-ACE2，缩写为2ACE2-shRNA。
由上述序列合成的产物分别表达为2ACE2-shRNA5、2ACE2-shRNA16、2ACE2-shRNA21和2ACE2-shRNA30。因为病毒RBD通过C端结合ACE2的N端感染细胞，以及ACE2多肽与siRNA的结合可增加siRNA的通透性、稳定性及干扰效果，所以如图3所示，本设计中的ACE2不但能中和病毒、阻止病毒感染，而且能将siRNA/shRNA靶向递送给病毒受体结合域RBD，形成“shRNA-ACE2-RBD-病毒”的复合物，进而使shRNA被病毒递送，并使shRNA随着病毒的感染进入靶细胞，发挥靶向干扰作用。
2、胞外ACE2-shRNA-胞外ACE2(ACE2-shRNA)的合成
按照设计(图2)合成人工抗体，采用多肽与寡核苷酸的常规合成方法，将上述合成的多肽(ACE2)和寡核苷酸(shRNA/siRNA)以羧腙键、肟键、酰胺键、硫醚键、二硫键、磷酰键、腙键、酰脲键、磷酸二酯键、二硫代磷酸脂键、马来酰亚胺-巯基键等形式偶联成缀合物，包括多肽和寡核苷酸的正义链(5'末端、3'末端)或反义链(3'末端)以较牢固的共价键、较松散的离子键、疏水键或以带间隔臂的羧腙键进行非共价或共价交联，合成多肽-寡核苷酸偶联物(POCs)。目前最常用的合成POCs法是共价交联-液相片段合成法，已广泛应用于合成各种POCs，其主要步骤是：分别在固相基质上分别合成多肽和寡核苷酸，然后同时将两个合成物从固相基质上剥离，所剥离的多肽和寡核苷酸在溶液中通过反应活性基团进行偶联。合成POCs主要包括：①马来酰亚胺-巯基偶联：在多肽或寡核苷酸上修饰马来酰亚胺，在另一单体上修饰巯基，然后将两个单体加入同一溶液中即可反应得到POCs；②二硫键或硫醚键偶联：用巯基对寡核苷酸的5'或3'位进行修饰，然后与C端用溴乙酰基进行修饰的多肽在pH7.0的缓冲溶液中进行反应；二硫键偶联可以通过两个巯基直接氧化，或先通过联吡啶二硫醚等活化剂将巯基活化再与另一含有巯基的寡聚物偶联，常用二硫键合成siRNA与多肽的偶联物；③肟键偶联：醛基与氨基相互反应产生肟，该反应条件温和、反应效率高，并且可以直接生成双链DNA与特定多肽的偶联产物，同时，还可以通过双功能化的寡核苷酸与多肽或糖类通过肟键在核酸的5’与3’端同时连接上两条多肽，该方法不需要各种保护过程并能一步完成，用于合成了“肽-寡核苷酸-肽”产物，具体方法为在寡核苷酸的5’和3’均引入醛基，再与羟基胺修饰的多肽反应，得到“肽-寡核苷酸-肽”，且所得产率较高，这种双官能团化的寡核苷酸与多肽的一步反应不需要任何保护策略和交联剂，在微酸的条件下就能得到较高的产率；④酰胺键偶联：直接利用含有活化羧酸或硫酯的寡聚物与另一修饰有氨基的聚合物反应得到产物；⑤腙键偶联：需要先将肼基引入到多肽上，然后加入pH值为3～5之间的柠檬酸缓冲溶液，再与修饰有乙酰醛基的寡核苷酸进行反应，才可得到以腙键连接的POCs。
3、人工抗体(ACE2-shRNA)的提纯
色谱方法一直是纯化和分析多肽与寡核苷酸偶联物的最常用的方法之一。根据偶联物的复杂程度需选择不同的色谱方法进行分离，主要方法有高效液相色谱(HPLC)、反向高效液相色谱(RP-HPLC)，离子交换色谱(IEC，通常为阴离子交换色谱)，或者将其中的两个或几个进行串联使用，具体按操作说明。
4、以ACE2靶向递送shRNA的其他化合物的合成方法
同理可将shRNA与胞外ACE2、跨膜ACE2、胞内ACE2或密码子优化的ACE2多肽连接成化合物，包括但不限于“跨膜ACE2-shRNA-跨膜ACE2”、“胞内ACE2-shRNA-胞内ACE2”；或将shRNA/siRNA插入到ACE2多肽中间，合成“跨膜ACE2-shRNA-胞外ACE2”、“胞内ACE2-shRNA-胞外ACE2”；也可同理设计以ACE2或其优化的多肽为靶向递送载体的化合物，包括但不限于“ACE2-siRNA、胞外ACE2-siRNA、跨膜ACE2-siRNA、胞内ACE2-siRNA”。
六、人工抗体(ACE2-shRNA)的验证
1、体外验证广谱抗病毒效果
(1)病毒液的准备
在Vero E6细胞生长至30％汇合度的DMEM培养液(10％FBS)中加入病毒株，36℃、5％CO2培养箱培养5～7d，至出现细胞病变效应(CPE)时，分离病毒，用培养液配成103～105TCID50/ml病毒液备用。据此分别准备新冠病毒的两种变异毒株B.1.617.1和B.1.617.2的病毒液，用于验证ACE2-shRNA是否对2种或以上含有相同保守基因的变异病毒同时有效，以证明本发明的shRNA/siRNA是否具有广谱抗病毒效果。
(2)人工抗体(ACE2-shRNA)和病毒共培养
分别设立实验组和对照组，试验化合物抗B.1.617.1和B.1.617.2的效果。每组接种8孔板，每孔2×105个Vero-E6细胞、2mL DMEM培养液(10％FBS)，置36℃、5％CO2培养箱中培养至30％汇合度时更换培养液，同时加入试验化合物、B.1.617.1和B.1.617.2株病毒液。
其中实验组包括:①2ACE2-shRNA5组(0.1nmol 2ACE2-shRNA+0.6ml病毒液)；对照组包括：naked shRNA5组(0.1nmol naked shRNA5+0.6ml病毒液)、阳性对照组(0.6ml病毒液)、阴性对照组(0.6ml DMEM培养液)(表1-6)。②2ACE2-shRNA16组、2ACE2-shRNA21组(0.1nmol 2ACE2-shRNA16/21+0.6ml病毒液)、ACE2组(0.1nmol ACE2+0.6ml病毒液)、对照组同①,结果见表1a-6a。然后于培养1小时、24小时和72小时后每组各取上清液，以1:4、1:12、1:36、1:108、1:324、1:972、1:2916、1:8748倍稀释，进行RT-PCR检测。
(3)实时荧光RT-PCR检测各组病毒RNA
病毒核酸提取和核酸(ORF1ab/N)检测按照试剂盒说明书操作。
(4)病毒RNA检测结果
①B.1.617.1株检测结果
表1所示，各组细胞培养1h后，2ACE2-shRNA5组、naked shRNA5组、阳性对照组和阴性对照组的病毒RNA检测结果分别为1:12、1:12、1:108和阴性。
表2所示，各组细胞培养24h后，2ACE2-shRNA5组、naked shRNA5组、阳性对照组和阴性对照组的病毒RNA检测结果分别为1:36、1:108、1:324和阴性。其中2ACE2-shRN5A组和阳性对照组相比有显著性差异(p＜0.05)。
表3所示，各组细胞培养72h后，2ACE2-shRNA5组、naked shRNA5组、阳性对照组和阴性对照组的病毒RNA检测结果分别为1:324、1:8748、1:8748和阴性。其中2ACE2-shRNA5组和阳性对照组相比有显著性差异(p＜0.01)。
表1-3说明，2ACE2-shRNA5组具有明显的抗B.1.617.1株作用，说明与ACE2连接的shRNA能被递送至靶细胞内进行RNA干扰，而未与ACE2连接的shRNA不能进入靶细胞内，在胞外不能发挥RNA干扰的作用。
表1a-3a为2ACE2-shRNA16组和2ACE2-shRNA21组的培养液病毒RNA检测结果，明显底于阳性对照组，而ACE2则有利于病毒生长。
表1 2ACE2-shRNA5与B.1.617.1株共培养1小时培养液中病毒RNA RT-PCR检测结果(+/-)
表1a 2ACE2-shRNA16/21与B.1.617.1株共培养1小时培养液病毒RNA RT-PCR检测结果
表2 2ACE2-shRNA5与B.1.617.1株共培养24小时培养液病毒RNA RT-PCR检测结果(+/-)
表2a 2ACE2-shRNA16/21与B.1.617.1株共培养24小时培养液病毒RNA RT-PCR检测结果
表3 2ACE2-shRNA5与B.1.617.1株共培养72小时培养液病毒RNA RT-PCR检测结果(+/-)
表3a 2ACE2-shRNA16/21与B.1.617.1株共培养72小时培养液病毒RNA RT-PCR检测结果
②B.1.617.2株检测结果
表4所示，各组细胞培养1h后，2ACE2-shRNA5组、naked shRNA5组、阳性对照组和阴性对照组的病毒RNA检测结果分别为1:12、1:36、1:36和阴性。其中2ACE2-shRNA5组和阳性对照组相比有显著性差异(p＜0.05)。
表5所示，各组细胞培养24h后，2ACE2-shRNA5组、naked shRNA5组、阳性对照组和阴性对照组的病毒RNA检测结果分别为1:36、1:108、1:324和阴性。其中ACE2-shRNA组和阳性对照组相比有显著性差异(p＜0.01)。
表6所示，各组细胞培养72h后，2ACE2-shRNA5组、naked shRNA5组、阳性对照组和阴性对照组的病毒RNA检测结果分别为1:108、1:8748、1:8748和阴性。其中2ACE2-shRNA5组和阳性对照组相比有显著性差异(p＜0.01)。
表4-6说明，2ACE2-shRNA5组具有明显的抗B.1.617.2株作用，说明与ACE2连接的shRNA或siRNA能被递送至靶细胞内进行RNA干扰，而未与RBD连接的shRNA不能进入靶细胞内，从而不能发挥RNA干扰的作用。
表4a-6a的结果与表4-6的结果明显一致，2ACE2-shRNA16组和2ACE2-shRNA21组的培养液病毒RNA检测结果明显底于阳性对照组，而ACE2有利于病毒生长。
表1-6和表1a-6a表明，2ACE2-shRNA5、2ACE2-shRNA16和2ACE2-shRNA21同时具有抗B.1.617.1和B.1.617.2的作用，说明其广谱抗变异毒株的效果。
表4 2ACE2-shRNA5与B.1.617.2株共培养1小时培养液中病毒RNA RT-PCR检测结果(+/-)
表4a 2ACE2-shRNA16/21与B.1.617.2株共培养1小时培养液病毒RNA RT-PCR检测结果
表5 2ACE2-shRNA5与B.1.617.2株共培养24小时培养液病毒RNA RT-PCR检测结果(+/-)
表5a 2ACE2-shRNA16/21与B.1.617.2株共培养24小时培养液病毒RNA RT-PCR检测结果
表6 2ACE2-shRNA5与B.1.617.2株共培养72小时培养液病毒RNA RT-PCR检测结果(+/-)
表6a 2ACE2-shRNA16/21与B.1.617.2株共培养72小时培养液病毒RNA RT-PCR检测结果
2、体内验证ACE2的靶向递送功能
(1)动物分组和接种
动物分组：选择6-8周龄、40克左右的SPF级雌性BALB/c小鼠，随机分为2ACE2-shRNA5组(接种2ACE2-shRNA5+B.1.617.2)、shRNA5组(接种shRNA5+B.1.617.2)、阳性对照组(接种B.1.617.2+生理盐水)和阴性对照组(仅接种生理盐水)，每组20只。
动物接种：经鼻腔喷雾接种40μl滴度为105/mlTCID50的B.1.617.2株病毒液，阴性对照组经鼻腔喷雾接种40μl生理盐水。以腹腔注射5％水合氯醛溶液麻醉，分别将0.1nmol的ACE2-shRNA、和shRNA缓慢注入小鼠气管，在感染后第7天每组处死10只小鼠，进行病毒检测；另外每组10只用于观察抗体。
(2)以细胞半数感染量(TCID50)的百分率检测病毒
将感染后第7天处死小鼠肺组织制备10％匀浆，各取100pl离心后上清，以10倍递次稀释，接种于VeroE6单层生长的96孔板，每孔30μl，每个稀释度接种4个孔，轻摇匀浆，放37℃吸附lh，以Hank氏液清洗，加培养液，放37℃C02培养箱培养，观察细胞病变效应(CPE)，计算各组VeroE6半数感染量(TCID50)的百分率，百分率越大病毒含量越多(表7-10)。
表7 2ACE2-shRNA5组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表8 shRNA5组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表9阳性对照组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表10阴性对照组小鼠肺组织匀浆致VeroE6半数感染量的百分率
(3)ACE2靶向递送的效果
从表7-10可知，各组小鼠肺匀浆致VeroE6半数感染量的百分率(101孔)分别为：2ACE2-shRNA5组为37.5％、shRNA5组为95.0％、阳性对照组为97.5％和阴性对照组为5.0％。因为RNAi主要发生在细胞浆内，shRNA5组中的shRNA不易通过细胞膜，所以几乎不起RNAi作用，其结果与阳性对照一致；而2ACE2-shRNA5组中的shRNA因被ACE2递送至细胞浆，所以其RNAi效果较好，其VeroE6半数感染量的百分率与阳性对照组相比具有显著性差异(p＜0.05)。按照上述TCID50试验方法，对2ACE2-shRNA16和2ACE2-shRNA21进行TCID50试验，结果发现2ACE2-shRNA16组、2ACE2-shRNA21组、shRNA16/21组、阳性对照组和阴性对照组的TCID50分别为35％、40％、92.5％、95％和7.5％，试验组的TCID50明显低于阳性对照组。
3、ACE2-Ab的检测及功能验证
(1)样本与检测方法
采集上述另外每组10只小鼠的第2、4、6周静脉血，分离血清，并混合每组相同周次的小鼠血清,保存于-20℃备用。以双抗原夹心法测定ACE2-Ab，具体按照试剂盒操作。
(2)检测结果
由表11可知，2ACE2-shRNA5组小鼠的肺组织匀浆ACE2-Ab显著高于对照组小鼠的肺组织匀浆ACE2-Ab(p＜0.05)，说明ACE2-shRNA组的ACE2能刺激小鼠产生ACE2-Ab。
表11 ACE2-shRNA组小鼠的肺组织匀浆ACE2-Ab检测结果(ng/L)
(3)ACE2-Ab的功能验证
以2ACE2-shRNA5组为试验组(均含ACE2-Ab)，shRNA组为对照组(含病毒但不含ACE2-Ab)。混合经ACE2-Ab检测后的每组第2、4、6周血清，倍比稀释各组混合血清，然后各取30μl接种于VeroE6单层生长的96孔板，试验组同时接种shRNA组的未稀释混合血清30μl，轻摇混匀，置37℃吸附lh，以Hank氏液清洗，加培养液，置37℃C02培养箱培养，1周内观察细胞病变效应(CPE)，以“+”表示细胞正常生长，见表12。
表12因ACE2-Ab中和VeroE6细胞表面受体ACE2所致的抗病毒感染结果(+/CPE)
由表12可知，阴性对照组因未接种病毒，所以各孔VeroE6均未出现CPE；阳性对照组在无ACE2-Ab中和作用的前提下接种了病毒(shRNA组未稀释混合血清)，所以各孔VeroE6均产生CPE；shRNA组因无ACE2-Ab的中和作用，所以仅在含病毒的自身血清稀释1:64倍后才不使VeroE6出现CPE；而试验组虽然和阳性对照组一样也接种了病毒，但因具有ACE2-Ab的中和作用，所以在血清即ACE2-Ab含量稀释1:128时才使VeroE6出现CPE。说明ACE2-Ab能中和VeroE6细胞表面的病毒受体ACE2，从而产生有抗病毒作用。
综上，本发明人工抗体的shRNA起抗变异毒株的作用；ACE2起靶向递送shRNA、双价结合RBD、抑制病毒通过RBD感染、保护shRNA及刺激宿主产生ACE2-Ab的作用，而ACE2-Ab具有封闭ACE2受体的作用。
序列表
<110> 杭州痴创生物科技有限公司
<120> 一种人工抗体制备方法
<141> 2022-04-03
<150> 2021113298574
<151> 2021-11-11
<160> 43
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Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
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Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser
740
Claims (10)
1.一种人工抗体的制备方法，其特征在于，合成以ACE2中和冠状病毒和靶向递送抗变异毒株共同靶标shRNA的人工抗体，包括分别合成以冠状病毒变异毒株保守基因或变异毒株共有基因为靶标的shRNA和以冠状病毒受体结合域RBD为配体的ACE2，然后将合成的ACE2分别连接到合成的shRNA的正反义链上，构成由ACE2和shRNA组成的人工抗体，使人工抗体通过其ACE2结合病毒S1-RBD从而阻断病毒通过其RBD感染表达ACE2的靶细胞，所构成的“shRNA-ACE2-RBD-病毒”结合物通过ACE2和RBD的结合使shRNA被病毒靶向递送至靶细胞并随着病毒的感染进入靶细胞浆，使shRNA在靶细胞内起广谱抗病毒的RNA干扰作用。
2.根据权利要求1所述的一种人工抗体的制备方法，其特征在于，所述以保守基因为靶标是指用于合成shRNA的siRNA基于数据库记载的各种致病性冠状病毒及其变异毒株的共有基因筛选，使合成的siRNA和/或shRNA靶向干扰所述共有基因，从而起广谱抗变异毒株的作用；所述共有基因包括但不限于超保守基因、保守基因和/或由保守微卫星拼接的基因。
3.根据权利要求1、2所述的一种人工抗体的制备方法，其特征在于，所述合成shRNA包括但不限于首先合成2条互补的约21-25nt的寡核苷酸多肽siRNA以及合成起间隔作用的碱基序列，然后将所合成的siRNA和碱基序列连接成由中间碱基序列间隔成loop环的小发夹shRNA双链，继之在shRNA双链的每条单链上各连接ACE2多肽或RBD多肽。
4.根据权利要求1所述的一种人工抗体的制备方法，其特征在于，所述以变异毒株保守基因或共有基因为靶标的siRNA包括但不限于SEQ ID NO.1～39。
5.根据权利要求1、4所述的一种人工抗体的制备方法，其特征在于，所述以变异毒株保守基因或共有基因为靶标的siRNA包括但不限于SEQ ID NO.5、SEQ ID NO.7～10、SEQ IDNO.16～18、SEQ ID NO.20～22、SEQ ID NO.30～32；所述以变异毒株保守基因或共有基因为靶标的siRNA优选为SEQ ID NO.5、SEQ ID NO.16、SEQ ID NO.21和SEQ ID NO.30。
6.根据权利要求1所述的一种人工抗体的制备方法，其特征在于，所述ACE2包括但不限于第1-740位氨基酸的胞外ACE2、第741-763位氨基酸的跨膜ACE2、第764-805位氨基酸的胞内ACE2、全长ACE2或经氨基酸密码子优化的ACE2及其多肽。
7.根据权利要求1、6所述的一种人工抗体的制备方法，其特征在于，所述人工抗体包括但不限于将所述ACE2分别连接到由SEQ ID NO.1～39合成的siRNA/shRNA所制备的化合物；包括但不限于将由SEQ ID NO.1～39合成的siRNA/shRNA分别插入到所述ACE2多肽中间所制备的化合物；包括但不限于以脂质纳米粒包裹所述化合物制备的siRNA药物。
8.根据权利要求1、7所述的一种人工抗体的制备方法，其特征在于，所述连接或合成包括但不限于将ACE2与shRNA的反义链3'末端、正义链5'末端或3'末端进行连接，包括但不限于以带间隔臂的羧腙键、二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键、马来酰亚胺-巯基键的化学偶联或共价偶联进行连接。
9.根据权利要求1、8所述的一种人工抗体的制备方法，其特征在于，所述人工抗体包括但不限于以shRNA的正反义链连接ACE2制备胞外ACE2-shRNA-胞外ACE2、跨膜ACE2-shRNA-跨膜ACE2、胞内ACE2-shRNA-胞内ACE2、全长ACE2-shRNA-全长ACE2，以及将shRNA/siRNA插入到ACE2多肽中间制备跨膜ACE2-shRNA-胞外ACE2、胞内ACE2-shRNA-胞外ACE2。
10.根据权利要求1、9所述的一种人工抗体的制备方法，其特征在于，所述人工抗体包括但不限于2ACE2-shRNA5、2ACE2-shRNA16、2ACE2-shRNA21、2ACE2-shRNA30。
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