WO2022077591A1 - 一种重组腺病毒在制备预防病毒的药物中的用途 - Google Patents

一种重组腺病毒在制备预防病毒的药物中的用途 Download PDF

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WO2022077591A1
WO2022077591A1 PCT/CN2020/125324 CN2020125324W WO2022077591A1 WO 2022077591 A1 WO2022077591 A1 WO 2022077591A1 CN 2020125324 W CN2020125324 W CN 2020125324W WO 2022077591 A1 WO2022077591 A1 WO 2022077591A1
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sars
coronavirus
adenovirus
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黄文林
周晓鸿
田烁
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广州达博生物制品有限公司
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • adenovirus has been used in the research and development of various vaccines and achieved good results.
  • the present invention provides the use of a recombinant adenovirus in the preparation of a virus-preventing medicine.
  • the replication-deficient adenovirus contains CMV promoter and BGH gene polyA sequence.
  • the medicine for preventing coronavirus is a medicine for preventing SARS-CoV-2 coronavirus, and the medicine includes the sequence from the S gene of SARS-CoV-2 coronavirus.
  • the sequence of the S gene is SEQ ID NO: 3.
  • the medicine includes the truncated S1C sequence of the S gene of the SARS-CoV-2 coronavirus, and the PCR primers of the truncated body are V1 and V2, wherein the sequence of V1 is SEQ ID NO: 1, and the sequence of V2 is SEQ ID NO:2.
  • the PCR product of the S1C sequence was digested with SmaI/HindIII and then ligated into the pShuttle plasmid.
  • the recombinant combined adenovirus backbone plasmid was transfected into HEK293 cells and expressed.
  • the described preparation method comprises the following steps:
  • step (2) (2) recombining the gene fragment of step (1) with replication-deficient adenovirus;
  • the gene of the target virus of the prevention in the described step (1) is a fragment of the S gene of the SARS-CoV-2 coronavirus.
  • the means for extracting the target fragment in the step (1) is PCR.
  • the step (2) is to clone the extracted target fragment into the pShuttle plasmid carrying the secreted peptide, and then link and combine it with the adenovirus backbone plasmid.
  • the adenovirus backbone plasmid is pBHGlox(delta)E1,3Cre.
  • the packaging cell in the step (3) is a cell line or cell line that integrates the E1 region gene of the C subtype 5 adenovirus.
  • Figure 1 shows the sequencing results of recombinant pShuttle.
  • Figure 2 shows the mRNA expression of SARS-CoV-2 vaccine.
  • M is Marker
  • 1 is blank control (without template)
  • 2 is negative control (template is RNA extracted from cells without vaccination)
  • 3 is experimental group 1 (template is inoculated with 5 ⁇ 10 8 vp/mL Ad / RNA extracted from cells after 48h of S1C);
  • 4 is experimental group 2 (template is RNA extracted from cells after inoculation with 1 ⁇ 10 9 vp/mL Ad/S1C for 48 h);
  • 5 is experimental group 3 (template is inoculated with 2 ⁇ 10 9 vp RNA extracted from cells after 48h of Ad/S1C).
  • Figure 4 shows the secretion of SARS-CoV-2 vaccine to the level of extracellular proteins.
  • control is the cell without vaccination
  • E10A is the cell inoculated with other adenovirus products
  • Ad/S1C is the cell inoculated with 2 ⁇ 10 9 vp/mL Ad/S1C for 48h
  • Medium is the cell culture supernatant
  • Whole cell is the Whole cell lysate.
  • Figure 5 shows the immune neutralizing antibody titers of SARS-CoV-2 vaccine animals.
  • Figure 6 shows the immune-specific antibody titers of SARS-CoV-2 vaccine animals.
  • 293 cells were purchased from ATCC under the catalog number CRL-1573.
  • the amplification primers are V1 and V2, their respective sequences are shown in SEQ ID NOs: 1-2, VI and V2 are a pair, and the C-terminal fragment S1C of the S1 gene is amplified.
  • Ad/S1c The S1C fragment (955-2055) of the SARS-CoV-2 S gene cloned into an adenovirus vector.
  • the collection of viruses adopts the method of picking plaques: adding low-melting point agarose to the culture medium, and usually small plaques can be seen under the microscope on the 10th to 21st day after transfection. After plaque formation, the plaques were picked up with agarose and placed in 1 mL of fresh medium overnight. Usually 3-6 plaques are picked, and then the titers are compared, and the one with the highest titer is used for subsequent experiments.
  • the virus in the medium was added to the fresh 293 cell culture medium for a small amount of virus amplification.
  • plaques appeared in the cells again the cells and the supernatant were collected, and the virus was collected by repeated freezing and thawing three times, and this virus was used as the P1 generation virus.
  • the 293 cells were infected with the P1 generation virus, and the infection was carried out for three consecutive generations, and the virus was amplified in large quantities in the P4 generation. After the plaques were formed, the virus was collected, and the virus was purified and concentrated by column chromatography.
  • Example 2 After infecting A549 cells with the SARS-CoV-2 vaccine purified in Example 2 at 5 ⁇ 10 8 vp/mL, 1 ⁇ 10 9 vp/mL, and 2 ⁇ 10 9 vp/mL for 2 hours, the virus suspension was discarded. Cell culture medium was added, and the cells were incubated at 37°C and 5% CO 2 . Cell lysates were collected 48 h after infection, and their mRNA expression levels were detected by RT-PCR. Blank control (without template) and negative control (template was RNA extracted from cells without vaccination) were set.
  • the protein expression level was detected by Western blot, and the whole cell lysate without vaccination and the whole cell lysate inoculated with other adenovirus products (Guangzhou Dabo Biological Products Co., Ltd.) were set as controls.
  • the protein expression levels in the cell culture supernatant and whole cell lysate were detected by ELISA, and the whole cell lysate without vaccination and the whole cell lysate inoculated with other adenovirus products (Guangzhou Dabo Biological Products Co., Ltd.) were set as controls.
  • Intramuscular injection in the inner thigh Intramuscular injection in the inner thigh.
  • Immunization procedure immunization was performed once every 6 days, for a total of 3 times; the mouse serum was collected on the 12th day, the 18th day and the 24th day respectively.
  • mice serum was collected on the 12th day, the 18th day and the 24th day, respectively.
  • Neutralizing antibody titer detection method SARS-CoV-2 pseudovirus neutralization method.
  • Example 2 the SARS-CoV-2 vaccine obtained in Example 2 was selected.
  • mice The test subjects Balb/C mice were grouped as follows:
  • High-dose vaccine + thymopentin group vaccine stock solution 1 ⁇ 10 9 vp/only + thymopentin;
  • Low-dose vaccine group vaccine stock solution 2 ⁇ 10 8 vp/vaccine
  • High-dose vaccine group vaccine stock solution 1 ⁇ 10 9 vp/vaccine
  • Physiological saline control group inject the same volume of normal saline with other groups of reagents.
  • Vaccine stock solution concentration 1 ⁇ 10 11 vp/mL.
  • the concentration of thymopentin in the vaccine injection is 1.6 mg/mL.
  • Intramuscular injection in the inner thigh Intramuscular injection in the inner thigh.
  • Immunization procedure immunization was performed once every 6 days, for a total of 3 times; the mouse serum was collected on the 18th day and the 24th day respectively.
  • mice serum was collected on the 18th day and the 24th day, respectively.
  • Immunogenicity detection method The titer of the specific binding antibody to the RBD region of the S protein was determined by ELISA, and the results are shown in Figure 6.
  • Serum titer against the new crown Spike RBD 18 days after immunization, the serum titer is about 2500-3400. The serum titer was about 8000-32000 24 days after immunization.
  • the virus suspension was discarded, the cell culture medium was added, and the cells were cultured at 37° C. and 5% CO 2 .
  • Cell lysates were collected 48 h after infection, and indirect ELISA was used to detect the cross-reaction of antigens expressed by each SARS-CoV-2 vaccine with the convalescent sera of COVID-19 patients.

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Abstract

提供了重组腺病毒在制备预防冠状病毒的药物中的用途，该重组腺病毒为E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒。具体而言，该药物包括来自SARS-CoV-2冠状病毒S基因的截短体S1C序列，可用于开发SARS-CoV-2冠状病毒疫苗。

Description

一种重组腺病毒在制备预防病毒的药物中的用途 技术领域
本发明属于生物医药领域，具体涉及一种重组腺病毒在制备预防病毒的药物中的用途。
背景技术
人腺病毒(humanAdenovirus,HAdV)是1953年首次从急性呼吸道疾病病人身上分离、鉴定出来的一类病原体，可以感染人类呼吸道、胃肠道、泌尿系统或眼部等多种组织，导致急性呼吸道感染(ARI)、急性胃肠炎、肾炎、眼角膜结膜炎等多种不同疾病。
腺病毒是一类上呼吸道亲和性高的非整合性病毒。它能够通过CAR受体(coxsackie/adenovirus receptor，柯萨奇/腺病毒受体)进入上皮细胞，较容易引起呼吸道粘膜免疫反应，产生IgM抗体。另外，E1与E3基因缺失的腺病毒，无法在普通细胞中扩增，能够作为携带大分子的载体。
目前腺病毒已经应用于多种疫苗的研发中并取得了较好的效果。
中国专利201610696322.3中公开了一种基于黑猩猩腺病毒载体的埃博拉病毒疫苗。其公开的复制缺陷型黑猩猩腺病毒载体包括改造的黑猩猩腺病毒AdC68基因组序列，其中E1缺失。该专利采用的黑猩猩腺病毒具有高免疫原性，不仅能诱导特异性体液免疫反应，而且能诱导特异性细胞免疫反应，此外，不会受人体内预存抗人血清型腺病毒中和抗体的影响，因此是一种理想的疫苗载体。
中国专利201811262788.8中公开了一种腺病毒载体系统及重组腺病毒构建方法，包括：PCR扩增获得两侧含有同源重叠区的目的基因片段，与PmeI线性化的腺病毒质粒进行DNA组装，得到含有外源目的基因的腺病毒质粒；或者先将多个基因片段克隆到穿梭质粒，再使用限制性内切酶切下含全部目的基因的片段，与PmeI线性化的pKAd5f11pES-PmeI进行DNA组装，获得含有外源目的基因的腺病毒质粒。
但目前将腺病毒应用与冠状病毒疫苗的生产中的研究较少。
中国专利202010193587.8中公开了一种以人5型复制缺陷腺病毒为载体的新型冠状病毒疫苗。所述疫苗以E1、E3联合缺失的复制缺陷型人5型腺病毒为载体，以整合腺病毒E1基因的HEK293细胞为包装细胞系，携带的保护性抗原基因是经过优化设计的2019新型冠状病 毒(SARS-CoV-2)S蛋白基因(Ad5-nCoV)。该疫苗对2019新型冠状病毒具有良好的免疫保护效果。
发明内容
本发明利用携带分泌肽的缺陷型腺病毒载体，使目的基因在体内表达后，能被分泌至细胞外，从而激活体液免疫。本发明可用于SARS-CoV-2冠状病毒疫苗的开发。
一方面，本发明提供了一种重组腺病毒在制备预防病毒的药物中的用途。
所述的重组腺病毒为E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒；所述的预防病毒的药物为预防冠状病毒的药物。
所述的药物中还包括所预防的冠状病毒的基因片段；所述的冠状病毒为SARS-CoV-2，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV或MERS-CoV。
所述的复制缺陷型腺病毒内装CMV启动子和BGH基因polyA序列。
所述的预防冠状病毒的药物为预防SARS-CoV-2冠状病毒的药物，所述的药物中包括来自SARS-CoV-2冠状病毒S基因的序列。
所述的S基因的序列为SEQ ID NO：3。
所述的药物中包括来自SARS-CoV-2冠状病毒S基因的截短体S1C序列，所述截短体的PCR引物为V1和V2，其中V1的序列为SEQ ID NO：1，V2的序列为SEQ ID NO：2。
所述的S1C序列的PCR产物经SmaI/HindIII酶切后连接至pShuttle质粒中。
所述的pShuttle质粒转化并经氨苄抗性筛选后，再将其与腺病毒骨架质粒pBHGlox(delta)E1,3Cre重组结合。
所述的重组结合后的腺病毒骨架质粒转染HEK293细胞后表达。
优选地，所述的预防病毒的药物为疫苗。
另一方面，本发明提供了一种预防病毒的药物的制备方法。
所述的制备方法包括以下步骤：
(1)设计并合成预防的目的病毒的基因，并提取目的片段；
(2)将步骤(1)的基因片段与复制性缺陷型腺病毒重组结合；
(3)转染包装细胞；
(4)空斑挑选重组腺病毒；
(5)经扩增、分离、纯化后，制成制剂。
所述的步骤(1)中的预防的目的病毒的基因为SARS-CoV-2冠状病毒S基因的片段。
可选择地，所述的步骤(1)中的提取目的片段的手段为PCR。
优选地，所述的SARS-CoV-2冠状病毒S基因的片段。所述的片段来自SARS-CoV-2冠状病毒S基因的截短体S1C序列，所述截短体的PCR引物为V1和V2，其中V1的序列为SEQ ID NO：1，V2的序列为SEQ ID NO：2。
所述的步骤(2)中复制性缺陷型病毒为毒为E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒。
所述的步骤(2)为将提取的目的片段克隆到携带分泌肽的pShuttle质粒，再将其与腺病毒骨架质粒连接结合。
优选地，所述的腺病毒骨架质粒为pBHGlox(delta)E1,3Cre。
所述的步骤(3)中的包装细胞为整合了C亚类5型腺病毒E1区基因的细胞系或细胞株。
附图说明
图1为重组pShuttle的测序结果。
图2为SARS-CoV-2疫苗mRNA表达情况。其中，M为Marker；1为空白对照(不加模板)；2为阴性对照(模板为不接种疫苗的细胞提取的RNA)；3为实验组1(模板为接种5×10 8vp/mL Ad/S1C 48h后细胞提取的RNA)；4为实验组2(模板为接种1×10 9vp/mL Ad/S1C48h后细胞提取的RNA)；5为实验组3(模板为接种2×10 9vp/mL Ad/S1C 48h后细胞提取的RNA)。
图3为SARS-CoV-2疫苗蛋白表达情况。其中control为不接种疫苗的全细胞裂解液；E10A为接种其它腺病毒产品的全细胞裂解液，Ad/S1C为接种2×10 9vp/mL Ad/S1C 48h后的全细胞裂解液。
图4为SARS-CoV-2疫苗分泌至细胞外蛋白水平。其中，control为不接种疫苗的细胞；E10A为接种其它腺病毒产品的细胞；Ad/S1C为接种2×10 9vp/mL Ad/S1C 48h后的细胞；Medium为细胞培养上清；Whole cell为全细胞裂解液。
图5为SARS-CoV-2疫苗动物免疫中和抗体效价。
图6为SARS-CoV-2疫苗动物免疫特异抗体效价。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中：
pShuttle载体与病毒骨架质粒pBHGlox(delta)E1,3Cre购自Microbix Biosystems，货号为PD-01-64。
293细胞购自ATCC，货号为CRL-1573。
Balb/C小鼠购自广东省实验动物中心。
实施例1 SARS-CoV-2冠状病毒疫苗的前期构建
(1)对SARS-CoV-2冠状病毒的S基因进行优化，优化后的S基因的序列为SEQIDNO：3。
(2)取得SARS-CoV-2S基因的优化序列后，用PCR方法对进行扩增。扩增引物为V1与V2，其各自的序列如SEQ ID NO：1-2所示，VI和V2为一对，扩增S1基因C端片段S1C。
(3)PCR产物经跑胶鉴定与切胶回收后，用SmaI与HindIII在37℃条件下进行酶切。同时用这两种酶对pShuttle载体进行酶切。
(4)用T4连接酶将酶切后的PCR产物与pShuttle在16℃条件进行过夜连接。
(5)将连接产物转化大肠杆菌DH5a，利用氨苄抗性筛选阳性克隆，并挑选克隆进行菌落PCR鉴定。阳性菌落经培养后提取质粒，并送测序以确认序列。重组pShuttle的测序结果如图1所示。
Ad/S1c：克隆到腺病毒载体中的为SARS-CoV-2S基因的S1C片段(955-2055)。
实施例2 SARS-CoV-2冠状病毒疫苗的制备
(1)根据实施例1的方法获得正确的重组SARS-CoV-2S1C基因的pShuttle质粒后，将其与腺病毒骨架质粒pBHGlox(delta)E1,3Cre共同转染至293细胞中以包装重组腺病毒。
(2)病毒的收集采用挑空斑的方式：在培养液中加入低溶点琼脂糖，转染后一般在第10-21天可以在显微镜下看到小的空斑。空斑形成后将空斑与琼脂糖一起挑起，放入1mL新鲜培养基中过夜。通常挑取3-6个空斑不等，然后比较滴度，使用滴度最高的一个空斑进行后续实验。
(3)将培养基中病毒加入新鲜293细胞培养液中进行病毒少量扩增。至细胞再次出现空斑，收集细胞及上清，反复冻融三次收集病毒，以此病毒为P1代病毒。
(4)以P1代病毒感染293细胞，连续进行三代感染，至P4代进行病毒的大量扩增，待空斑形成后收集病毒并对病毒进行柱层析纯化和浓缩。
(5)纯化后获得的产物即为SARS-CoV-2疫苗。SARS-CoV-2疫苗包含SARS-CoV-2病毒S1C基因和缺陷型腺病毒。缺陷型腺病毒为E1与E3区完全缺失的C亚类的5型腺病毒，不能在普通的人体细胞内复制。
实施例3 SARS-CoV-2疫苗体外表达试验
将实施例2中纯化获得的SARS-CoV-2疫苗以5×10 8vp/mL、1×10 9vp/mL、2×10 9vp/mL感染A549细胞2h后，弃去病毒悬液，加入细胞培养液，37℃、5％CO 2条件下培养。感染后48h收集细胞裂解液，分别用RT-PCR法检测其mRNA表达水平，设置空白对照(不加模板)和阴性对照(模板为不接种疫苗的细胞提取的RNA)。用Western blot法检测其蛋白表达水平，设置不接种疫苗的全细胞裂解液和接种其它腺病毒产品的全细胞裂解液(广州达博生物制品有限公司)为对照。用ELISA分别检测细胞培养上清和全细胞裂解液中的蛋白表达水平，设置不接种疫苗的全细胞裂解液和接种其它腺病毒产品的全细胞裂解液(广州达博生物制品有限公司)为对照。
结果见图2-4。
结果表明：SARS-CoV-2疫苗以1×10 9vp/mL与2×10 9vp/mL感染A549细胞后，能够检测到相应S1C抗原的mRNA(图2)。此外，SARS-CoV-2疫苗以2×10 9vp/mL感染A549细胞后，能够检测到S1C抗原蛋白表达(图3)，且该蛋白大小为40-55KD之间。而且，ELISA结果显示，本疫苗体内表达的抗原主要被分泌至细胞外(图4)，有利于诱导体液免疫。
实施例4 SARS-CoV-2疫苗诱导的中和抗体效价
本实施例选用实施例2得到的SARS-CoV-2疫苗。
将试验对象Balb/C小鼠按照下述方式分组：
疫苗组与生理盐水对照组。
给药方式：大腿内侧肌肉注射。
疫苗原液浓度：1×10 11vp/mL。
小鼠剂量：1×10 9vp/只。
免疫程序：每6天免疫一次，共3次；分别取第12天，第18天、第24天小鼠血清。
安乐死取血程序：分别取第12天，第18天、第24天小鼠血清。
中和抗体效价检测方法：SARS-CoV-2假病毒中和法。
结果见图5。
该结果表明：将小鼠免疫血清稀释25倍后，小鼠免疫12天的血清能够中和59％的SARS-CoV-2假病毒，免疫18天的血清能够中和65％的SARS-CoV-2假病毒，免疫24天的血清能够中和91％的SARS-CoV-2假病毒。总体而言，Ad/S1C疫苗能够在小鼠体内诱导出高效价的SARS-CoV-2中和抗体，且第12天即能够检测到中和抗体的产生。
实施例5 SARS-CoV-2疫苗体内表达试验
本实施例选用实施例2得到的SARS-CoV-2疫苗。
将试验对象Balb/C小鼠按照下述方式分组：
低剂量疫苗+胸腺五肽组：疫苗原液2×10 8vp/只+胸腺五肽；
高剂量疫苗+胸腺五肽组：疫苗原液1×10 9vp/只+胸腺五肽；
低剂量疫苗组：疫苗原液2×10 8vp/只；
高剂量疫苗组：疫苗原液1×10 9vp/只；
生理盐水对照组：注射与其他组别的试剂同等体积的生理盐水。
疫苗原液浓度：1×10 11vp/mL。
胸腺五肽在疫苗注射液中浓度为1.6mg/mL。
给药方式：大腿内侧肌肉注射。
免疫程序：每6天免疫一次，共3次；分别取第18天、第24天小鼠血清。
安乐死取血程序：分别取第18天、第24天小鼠血清。
免疫原性检测方法：用ELISA法测定小鼠针对S蛋白RBD区域的特异结合抗体效价，结果见图6。
该结果表明：
(1)针对新冠Spike RBD的血清效价：免疫后18天血清效价约为2500-3400。免疫后24天血清效价约为8000-32000。
(2)胸腺五肽的添加大大增强了小鼠体内抗体的滴度。免疫后18天，小鼠血清效价从2500增至3400；免疫后24天，血清效价从8000增至32000。
实施例6 SARS-CoV疫苗携带抗原序列的免疫原性检测
将实施例2的SARS-CoV-2疫苗以1×10 7pfu/mL感染A549细胞2h后，弃去病毒悬液，加入细胞培养液，37℃、5％CO 2条件下培养。感染后48h收集细胞裂解液，用间接ELISA检测各个SARS-CoV-2疫苗表达的抗原与COVID-19患者恢复期血清的交叉反应。
结果表明：SARS-CoV-2疫苗以1×10 7pfu/mL感染A549细胞后，表达的抗原能够与COVID-19患者恢复期的血清发生交叉反应。这表明，实施例2的SARS-CoV-2疫苗具有良好的免疫原性。

Claims (10)

  1. 一种重组腺病毒在制备预防病毒的药物中的用途，其特征在于，所述的重组腺病毒为E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒；所述的预防病毒的药物为预防冠状病毒的药物。
  2. 根据权利要求1所述的用途，其特征在于，所述的药物中还包括所预防的冠状病毒的基因片段；所述的冠状病毒为SARS-CoV-2，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV或MERS-CoV。
  3. 根据权利要求1所述的用途，其特征在于，所述的复制缺陷型腺病毒内装CMV启动子和BGH基因polyA序列。
  4. 根据权利要求2所述的用途，其特征在于，所述的预防冠状病毒的药物为预防SARS-CoV-2冠状病毒的药物，所述的药物中包括来自SARS-CoV-2冠状病毒S基因的序列。
  5. 根据权利要求4所述的用途，其特征在于，所述的药物中包括来自SARS-CoV-2冠状病毒S基因的截短体S1C序列，所述截短体的PCR引物为V1和V2，其中V1的序列为SEQ ID NO：1，V2的序列为SEQ ID NO：2。
  6. 根据权利要求4所述的用途，其特征在于，所述的S1C序列的PCR产物经SmaI/HindIII酶切后连接至pShuttle质粒中。
  7. 根据权利要求4所述的用途，其特征在于，所述的S基因的序列为SEQ ID NO：3。
  8. 根据权利要求6所述的用途，其特征在于，所述的pShuttle质粒转化并经氨苄抗性筛选后，再将其与腺病毒骨架质粒pBHGlox(delta)E1,3Cre重组结合。
  9. 根据权利要求7所述的用途，其特征在于，所述的重组结合后的腺病毒骨架质粒转染HEK293细胞后表达。
  10. 根据权利要求1-9任一项所述的用途，其特征在于，所述的预防病毒的药物为疫苗。
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