【目的】：构建并鉴定表达SARS-CoV-2 B.1.618突变株S蛋白受体结合域（RBD）的重组腺病毒rAd-RBD与表达甲型流感病毒（Influenza A virus，IAV ）H1N1核衣壳蛋白（nucleoprotein，NP)蛋白的重组腺病毒rAd-NP，通过动物实验与假病毒中和实验对两种重组腺病毒进行免疫原性评估，通过两种重组腺病毒的单独免疫与联合免疫的不同免疫方案的比较，分析rAd-NP对rAd-RBD之间是否存在免疫协同效应。

【方法】：1.重组腺病毒rAd-RBD与rAd-NP的构建与鉴定：合成SARS-CoV-2 B.1.618突变株RBD序列、IAV H1N1 NP序列及其上下游引物，将RBD序列与NP序列的PCR产物双酶切后连接至pShuttle-CMV穿梭载体上，筛选阳性克隆重组穿梭载体质粒pShuttle-NP与pShuttle-RBD后，进行PCR及目的基因测序验证。将线性化后的携带目的基因的重组穿梭载体质粒电转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183感受态细胞中进行同源重组，筛选得到携带目的基因的prAd-RBD和prAd-NP重组腺病毒质粒。通过PacI酶切和测序对重组获得的prAd-RBD和prAd-NP进行鉴定。将鉴定后的prAd-RBD和prAd-NP质粒通过PacI线性化、纯化后，转染至HEK293细胞中，包装出重组腺病毒。提取重组腺病毒的基因组DNA，通过PCR和琼脂糖电泳对目的基因进行鉴定。在确认目的基因已重组至腺病毒基因组后，将重组腺病毒感染细胞，感染48h后提取细胞总RNA，通过RT-PCR法鉴定重组腺病毒中的目的基因能否在细胞中正常转录。在确认目的基因正常转录后，进一步通过Western Blot（WB）和免疫荧光对RBD和NP蛋白的表达进行鉴定。2. 重组腺病毒的扩增浓缩与纯化： HEK293细胞扩增重组腺病毒rAd-RBD与rAd-NP，将rAd-RBD与rAd-NP病毒收获液用超滤管离心浓缩并用 Core 700层析柱进行纯化。通过WB，透射电镜观察以及PCR法鉴定纯化后产物是否为重组腺病毒rAd-RBD与rAd-NP，用qPCR法检测纯化后的rAd-RBD与rAd-NP病毒滴度。3. 重组腺病毒免疫原性研究：将20只6-8周龄的BALB／c雌性小鼠随机分成四组，分别是阴性对照组，rAd-NP组，rAd-RBD组及rAd-NP、rAd-RBD联合免疫组。采用后肢内侧肌肉注射方式分别免疫相同剂量的PBS缓冲液，rAd-NP，rAd-RBD，rAd-NP和rAd-RBD。免疫剂次为1剂免疫，分别采集免疫前1天，免疫后14天与免疫后21天的血清，通过ELISA效价测定和SARS-CoV-2 Delta株和Omicron株假病毒中和实验评价疫苗免疫后的IgG和中和抗体水平。采集免疫后21天的小鼠脾细胞进行流式细胞术检测，评价疫苗在小鼠中的细胞免疫水平，最终完成重组腺病毒rAd-RBD与rAd-NP免疫原性的评估以及不同免疫方案间效果的比较。

【结果】：1.重组腺病毒rAd-RBD与rAd-NP的构建与鉴定： PCR及测序结果显示，成功构建重组穿梭载体质粒pShuttle-NP与pShuttle-RBD。pShuttle-NP与pShuttle-RBD成功与腺病毒骨架质粒pAdeasy-1重组，成功构建重组腺病毒质粒prAd-NP与prAd-RBD。重组腺病毒骨架质粒转染HEK293细胞48h后，HEK293细胞出现了明显的细胞病变效应（cytopathic effect，CPE），72h待细胞全部病变后收获病毒液与细胞沉淀，经病毒基因组DNAPCR鉴定，感染后HEK293细胞总RNART-PCR鉴定，感染后HEK293细胞总蛋白的WB鉴定及感染后MA104细胞免疫荧光鉴定，目的基因RBD序列与NP序列可以在宿主细胞中正常复制、转录、翻译、表达。重组腺病毒rAd-RBD与rAd-NP构建成功。2. 重组腺病毒的扩增浓缩与纯化：rAd-RBD与rAd-NP经扩增后各收获1L病毒收获液，用超滤管各浓缩至约50mL，用Core 700层析柱纯化收集收集LP Data View 软件实时监测到的rAd-RBD，rAd-NP病毒浓缩液上样后的第一个流穿峰，纯化后产物经WB法，透射电镜观察以及PCR法鉴定为携带目的基因的重组腺病毒。用qPCR检测rAd-RBD与rAd-NP浓缩纯化后滴度分别为1.5×10¹¹Copies/mL与4.7×10¹²Copies/mL。3. 重组腺病毒免疫原性研究：ELISA效价测定实验结果表明，rAd-NP和rAd-RBD均能诱导小鼠产生对目的蛋白RBD/NP IgG抗体水平的升高，假病毒中和实验证明，rAd-RBD存在Delta株以及Omicron株的假病毒的中和作用，在流式细胞术实验中，rAd-NP能够诱导小鼠产生较好的T细胞免疫应答而rAd-RBD则不能，通过两种重组腺病毒的单独免疫与联合免疫的不同免疫方案的比较发现，联合免疫的对目的蛋白RBD/NP IgG抗体水平，对假病毒的中和抗体水平，流式细胞术检测到的总T细胞数量，CD4⁺细胞数量，CD8⁺细胞数量均优于rAd-RBD免疫组。

【结论】：1.成功构建携带SARS-CoV-2 B.1.618突变株RBD和IAV H1N1 NP基因的重组腺病毒质粒；2. 构建的重组腺病毒质粒能够在293细胞中成功包装出重组腺病毒rAd-RBD和rAd-NP；3. 包装出的重组腺病毒rAd-RBD和rAd-NP中携带的目的基因能够在细胞中成功转录和表达；4.重组腺病毒rAd-RBD和rAd-NP免疫小鼠后，能够引起小鼠体液免疫和细胞免疫反应：rAd-NP和rAd-RBD均能诱导小鼠产生血清抗RBD/NP IgG抗体水平的升高；rAd-RBD免疫小鼠后产生的中和抗体能够中和Delta株和Omicron株假病毒，对Omicron株的假病毒的中和效果要优于Delta株；rAd-NP能够诱导小鼠产生较好的T细胞免疫应答而rAd-RBD则不能。5. rAd-RBD单独免疫组，rAd-NP单独免疫组和rAd-RBD和rAd-NP联合免疫组免疫结果初步表明rAd-NP对rAd-RBD的免疫效果存在免疫协同及旁观者效应。

【Objective】: To construct and characterize recombinant adenovirus rAd-RBD expressing S protein receptor binding domain (RBD) of SARS-CoV-2 B.1.618 mutant strain and recombinant adenovirus rAd-NP expressing Influenza A virus (IAV) H1N1 nucleoprotein (NP) protein. The immunogenicity of the two recombinant adenoviruses was evaluated by animal experiments and pseudovirus neutralization experiments, and whether there is immune synergism between rAd-NP and rAd-RBD by comparing different immunization protocols of the two recombinant adenoviruses alone and in combination with immunization.

【Methods】: 1. Construction and identification of recombinant adenovirus rAd-RBD and rAd-NP: The RBD sequence of SARS-CoV-2 B.1.618 mutant strain, IAV H1N1 NP sequence and its upstream and downstream primers were synthesized, and the PCR products of RBD sequence and NP sequence were double digested and ligated to pShuttle-CMV shuttle vector, and the positive clones were screened for recombinant shuttle After screening for positive clones of the recombinant shuttle plasmid pShuttle-NP and pShuttle-RBD, PCR and target gene sequencing were performed. The linearized recombinant shuttle vector plasmids carrying the target genes were electrotransformed into BJ5183 receptor cells containing the adenovirus backbone plasmid pAdEasy-1 for homologous recombination, and the prAd-RBD and prAd-NP recombinant adenovirus plasmids carrying the target genes were screened. The recombinant prAd-RBD and prAd-NP were identified by PacI digestion and sequencing. The identified prAd-RBD and prAd-NP plasmids were linearized and purified by PacI and transfected into HEK293 cells to package the recombinant adenovirus. The genomic DNA of the recombinant adenovirus was extracted and the target genes were identified by PCR and agarose electrophoresis. After confirming that the target gene had been recombined into the adenovirus genome, the recombinant adenovirus was infected with cells, and total cellular RNA was extracted 48h after infection, and whether the target gene in the recombinant adenovirus could be transcribed normally in the cells by RT-PCR. After confirming the normal transcription of the target genes, the expression of RBD and NP proteins was further characterized by Western Blot (WB) and immunofluorescence. 2. Amplification and purification of recombinant adenovirus: HEK293 cells were amplified with recombinant adenovirus rAd-RBD and rAd-NP, and the harvested solution of rAd-RBD and rAd-NP viruses were concentrated by centrifugation in ultrafiltration tubes and purified with Core 700 chromatography columns were used for purification. The purified products were identified as recombinant adenovirus rAd-RBD and rAd-NP by WB, transmission electron microscopy and PCR.3. Recombinant adenovirus immunogenicity study: 20 BALB/c female mice aged 6-8 weeks were randomly divided into four groups: negative control group, rAd-NP group, rAd-RBD group and rAd-NP, rAd-RBD combined immunization group. The mice were immunized with the same dose of PBS buffer, rAd-NP, rAd-RBD, rAd-NP and rAd-RBD by intramuscular injection in the inner hind limbs. immunization doses were 1 dose of immunization, and sera were collected 1 day before, 14 days after and 21 days after immunization, respectively, and evaluated by ELISA potency assay and SARS-CoV-2 Delta strain and Omicron strain Pseudovirus neutralization assay was performed to evaluate the IgG and neutralizing antibody levels after vaccination. Spleen cells from mice at 21 days post-immunization were collected for flow cytometry to evaluate the level of cellular immunity of the vaccine in mice, and finally to complete the assessment of the immunogenicity of recombinant adenovirus rAd-RBD and rAd-NP and the comparison of the effects between different immunization regimens.

【Results】: 1. Construction and characterization of recombinant adenovirus rAd-RBD and rAd-NP: PCR and sequencing results showed that the recombinant shuttle vector plasmids pShuttle-NP and pShuttle-RBD were successfully constructed. pShuttle-NP and pShuttle-RBD were successfully recombined with the adenovirus backbone plasmid pAdeasy-1 and successfully constructed. After transfection of HEK293 cells with the recombinant adenovirus backbone plasmid for 48h, HEK293 cells showed a significant cytopathic effect (CPE), and after 72h when all the cells were diseased, the viral fluid and cell precipitation were harvested and identified by the viral genome DNAPCR. The target gene RBD sequence and NP sequence could be replicated, transcribed, translated and expressed normally in the host cells after the identification of viral genome DNAPCR, total RNART-PCR of HEK293 cells after infection, WB identification of total protein of HEK293 cells after infection and immunofluorescence identification of MA104 cells after infection. The recombinant adenovirus rAd-RBD and rAd-NP were successfully constructed. 2. Amplification and purification of recombinant adenovirus: rAd-RBD and rAd-NP were amplified and harvested by 1L each, concentrated to about 50mL each with ultrafiltration tubes, and purified by Core 700 chromatography column to collect the rAd-RBD and rAd-NP viruses monitored by LP Data View software in real time. The first flow-through peak of the concentrated solution after sampling, the purified product was identified as recombinant adenovirus carrying the target gene by WB method, transmission electron microscopy observation and PCR method. The titers of rAd-RBD and rAd-NP concentrated and purified by qPCR were 1.5×10¹¹Copies/mL and 4.7×10¹²Copies/mL, respectively. 3. Immunogenicity study of recombinant adenovirus: ELISA potency assay results showed that both rAd-NP and rAd-RBD induced the production of antibodies to the target protein RBD/NP IgG in mice. In flow cytometry experiments, rAd-NP was able to induce a better T-cell immune response in mice while rAd-RBD was not. immunization was found to be superior to the rAd-RBD immunization group in terms of antibody levels against the target protein RBD/NP IgG, neutralizing antibody levels against the pseudovirus, total T cell count, CD4⁺ cell count, and CD8⁺ cell count detected by flow cytometry.

【Conclusion】: 1. The recombinant adenovirus plasmids carrying SARS-CoV-2 B.1.618 mutant strain RBD and IAV H1N1 NP genes were successfully constructed; 2. The constructed recombinant adenovirus plasmids were able to successfully package recombinant adenovirus rAd-RBD and rAd-NP in 293 cells; 3. The recombinant adenovirus rAd-RBD and rAd-NP carried in the packaged target The recombinant adenoviruses rAd-RBD and rAd-NP were able to induce humoral and cellular immune responses in mice after immunization: both rAd-NP and rAd-RBD induced elevated levels of serum anti-RBD/NP IgG antibodies in mice; the neutralizing antibodies produced after immunization with rAd-RBD were able to neutralize Delta and Omicron strains of pseudoviruses. rAd-RBD immunized mice produced neutralizing antibodies that could neutralize Delta and Omicron strains of pseudovirus, and the neutralizing effect on Omicron strain of pseudovirus was better than that of Delta strain; rAd-NP could induce a better T-cell immune response in mice, while rAd-RBD could not. 5. The immunization results of rAd-RBD alone, rAd-NP alone and rAd-RBD and rAd-NP combined showed that the immunization effect of rAd-NP on The immunization effect of rAd-RBD was immunosynergistic and bystander effect.