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Article(id=1148989445648474841, tenantId=1146029695717560320, journalId=1146031712061968385, issueId=1148989441470952447, articleNumber=null, orderNo=null, doi=10.12211/2096-8280.2023-058, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1692374400000, receivedDateStr=2023-08-19, revisedDate=1699286400000, revisedDateStr=2023-11-07, acceptedDate=null, acceptedDateStr=null, onlineDate=1751870031034, onlineDateStr=2025-07-07, pubDate=1714406400000, pubDateStr=2024-04-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1751870031034, onlineIssueDateStr=2025-07-07, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1751870031034, creator=13701087609, updateTime=1751870031034, updator=13701087609, issue=Issue{id=1148989441470952447, tenantId=1146029695717560320, journalId=1146031712061968385, year='2024', volume='5', issue='2', pageStart='217', pageEnd='395', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1751870030037, creator=13701087609, updateTime=1752057315553, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1149774973969068078, tenantId=1146029695717560320, journalId=1146031712061968385, issueId=1148989441470952447, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1149774973969068079, tenantId=1146029695717560320, journalId=1146031712061968385, issueId=1148989441470952447, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=385, endPage=395, ext={EN=ArticleExt(id=1149999710019399870, articleId=1148989445648474841, tenantId=1146029695717560320, journalId=1146031712061968385, language=EN, title=Applications of the recombinant human collagen type Ⅲ-based trimerization motif in the design of vaccines to fight against SARS-CoV-2 and influenza virus, columnId=1149992670190514179, journalTitle=Synthetic Biology Journal, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Glycoproteins with enveloped viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, and human immunodeficiency virus (HIV), display a trimeric conformation. Different from the monomeric form, the trimeric proteins exhibit superior immunogenicity. Several trimerization motifs, such as Foldon derived from phage T4 fibritin, have been used to promote the formation of trimeric proteins with natural conformations. Although the Foldon-induced trimeric proteins are stable, their high immunogenicity limits applications in the development of vaccine antigens. In a previous study, we developed a recombinant human collagen type Ⅲ protein and determined its crystal structure, revealing a triple-helix conformation. However, the potential of this recombinant protein as a trimerization motif remained unknown. In this study, we demonstrated that the recombinant humanized type Ⅲ collagen (Rh3C) was able to act as a trimerization motif, facilitating the spontaneous trimer formation of the Rh3C-conjugated receptor-binding domain (RBD) within the spike (S) protein of SARS-CoV-2. This trimeric protein could induce a stronger SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a immune response, when compared with the monomeric RBD protein in the immunized mice. Notably, the Rh3C-RBD protein, when adjuvanted with the novel STING agonist CF501, also elicited significantly higher neutralizing antibody responses against both the pseudotyped SARS-CoV-2 (D614G) and its variant Omicron (BA.2.2) in the immunized mice. To showcase the broad applications of the Rh3C trimerization motif, we further demonstrated that the Rh3C-conjugated HA1 of the influenza virus could also elicit a stronger antibody response than free HA1. Considering the wide distribution of the Rh3C protein in human bodies, its use as a trimerization motif would not induce an immune response due to immune tolerance, thereby allowing the immune response to concentrate on targeted viral proteins. Therefore, this Rh3C-based trimerization motif holds great potential for the design and optimization of vaccines consisting of trimeric protein antigens.

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新冠病毒等多种包膜病毒的糖蛋白在天然状态下呈现三聚体形式。三聚体蛋白通常较单体蛋白具有更优的免疫原性。目前使用非人蛋白来源的Foldon等三聚体基序可促使目的蛋白形成稳定三聚体，但这些基序的强免疫原性会削弱目的蛋白的免疫应答，限制了其在疫苗抗原设计中的应用。在本研究中，发现并利用重组人源化Ⅲ型胶原蛋白（Rh3C）作为新型三聚体基序，利用合成生物学方法，与新冠病毒刺头（spike）蛋白受体结合域（RBD）融合表达后促使RBD形成三聚体。这种胶原-RBD（Rh3C-RBD）三聚体蛋白在免疫小鼠后，较RBD单体蛋白诱导产生了更高滴度的结合抗体和中和抗体。此外，我们还进一步验证了该Rh3C基序与流感病毒HA1蛋白融合后，较HA1单体蛋白可在小鼠体内诱导更强效的抗体应答。因此，这种新型的Rh3C基序，有望广泛用于三聚体疫苗抗原的设计和优化。

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姜世勃（1953—），男，教授，博士生导师。研究方向为微生物学。E-mail：
陆路（1982—），男，研究员，博士生导师。研究方向为微生物学。E-mail：
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刘泽众（1990—），男，青年研究员，博士生导师。研究方向为微生物学。E-mail：

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刘泽众（1990—），男，青年研究员，博士生导师。研究方向为微生物学。E-mail：

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刘泽众（1990—），男，青年研究员，博士生导师。研究方向为微生物学。E-mail：

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[(a) Schematic representation of the design of Rh3C-conjugated SARS-CoV-2 RBD (Rh3C-RBD) as a trimeric vaccine antigen.(b) Predicted trimeric structure of Rh3C-RBD using AlphaFold2. The entire structure is assembled from multiple segment predictions. The magnified images show the triple-helix motifs of the collagen tandem repeat and the trimeric conformation of RBD. (c) Schematic illustration of the Rh3C-RBD structure. (d) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis for the visualization of the Rh3C-RBD protein. (e) Representative elution chromatograph of the Rh3C-RBD protein using a calibrated Superose 6 Increase 10/300 column. (f) Determination of the molecular weight of Rh3C-RBD through the particle size analysis.]

, figureFileSmall=paQCzVYaBz+2pO3A2Vm08Q==, figureFileBig=vfwZ9igWXPx3eOjSA8BAxA==, tableContent=null), ArticleFig(id=1172891978107859286, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=CN, label=图1, caption=基于Rh3C连接的SARS-CoV-2 RBD融合蛋白作为三聚体疫苗抗原的设计、构建和鉴定

［（a）Ⅲ型胶原蛋白Rh3C同SARS-CoV-2 RBD融合蛋白的设计。（b）利用AlphaFold2预测的Rh3C-RBD三聚体结构。整个结构由多次的分段预测结果拼接而成。放大的图分别展示了串联重复的胶原三螺旋基序结构和RBD三聚体结构。（c）Ⅲ型人源化胶原蛋白Rh3C同RBD融合表达后的三聚体结构模式图。（d）Ⅲ型人源化胶原蛋白Rh3C同RBD融合表达后的SDS-PAGE图片。（e）凝胶过滤色谱分析Rh3C-RBD分子量。（f）粒径分析Rh3C-RBD融合蛋白获得蛋白分子量］

, figureFileSmall=paQCzVYaBz+2pO3A2Vm08Q==, figureFileBig=vfwZ9igWXPx3eOjSA8BAxA==, tableContent=null), ArticleFig(id=1172891978313380184, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=EN, label=Fig. 2, caption=Evaluation of the humoral immunity induced by Rh3C-RBD and RBD in the mice

[(a) Overview of the immunization protocol. Mice were vaccinated at day 0 and day 14, with serum collected at day 21; (b) Quantification of the SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a in different serum dilutions from day 21 using the enzyme-linked immunosorbent assay (ELISA); (c) Determination of the RBD-specific IgG, IgG1, and IgG2a titers in sera collected at day 21. Statistical analysis was conducted using one-way ANOWA. *** indicating the statistical significance at P<0.0001.]

, figureFileSmall=xleyRT3c5KuLpFmgxkTSwg==, figureFileBig=opkE692mMNloS198CURbsA==, tableContent=null), ArticleFig(id=1172891978439209304, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=CN, label=图2, caption=Rh3C-RBD和RBD免疫小鼠后体液免疫应答的评价

［（a）小鼠的免疫程序，分别在第0和14天进行免疫，在第21天收集小鼠血清；（b）免疫两次后第7天检测小鼠血清中针对RBD特异性的IgG、IgG1和IgG2a抗体；（c）RBD特异性的IgG、IgG1和IgG2a抗体滴度。One-way ANOWA用于分析统计学差异。***P<0.0001。每组小鼠n=6］

, figureFileSmall=xleyRT3c5KuLpFmgxkTSwg==, figureFileBig=opkE692mMNloS198CURbsA==, tableContent=null), ArticleFig(id=1172891978573427033, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=EN, label=Fig. 3, caption=Evaluation of the neutralizing antibodies against the pseudovirus SARS-CoV-2 (D614G) in the sera of immunized mice

[Assessment of the neutralizing antibody titers against SARS-CoV-2 (D614G) in the sera of mice immunized with CF501/RBD or CF501/Rh3C-RBD (a), Alum/RBD or Alum/Rh3C-RBD (b), CF501/RBD or CF501/Rh3C-RBD (c), and Alum/RBD or Alum/Rh3C-RBD (d). Statistical analysis was performed using the Student t test. ** indicating the statistical significance at P<0.001.]

, figureFileSmall=gMqAnTYkuFiLIfHJPW905Q==, figureFileBig=YthiMPWwUUI9nXmfhZ58xQ==, tableContent=null), ArticleFig(id=1172891978720227675, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=CN, label=图3, caption=免疫后小鼠血清中针对SARS-CoV-2假病毒中和抗体的评价（每组小鼠n=6）

［（a）以CF501为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（D614G）假病毒中和抗体滴度。（b）以Alum为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（D614G）假病毒中和抗体的评价。（c）以CF501为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（BA.2.2）假病毒中和抗体滴度。（d）以Alum为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（BA.2.2）假病毒中和抗体的评价。t检验用于分析统计学差异。**P<0.001］

, figureFileSmall=gMqAnTYkuFiLIfHJPW905Q==, figureFileBig=YthiMPWwUUI9nXmfhZ58xQ==, tableContent=null), ArticleFig(id=1172891978791530845, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=EN, label=Fig.4, caption=Design and construction of the Rh3C-conjugated influenza virus HA1 (Rh3C-HA1) and evaluation of its immunogenicity

[(a) Design of the Rh3C-conjugated influenza virus HA1 (Rh3C-HA1); (b) SDS-PAGE analysis for the expressed Rh3C-HA1 protein; (c) Overview of the immunization protocol. Mice were vaccinated at day 0 and day 14, with serum collection at day 21; (d and e) ELISA analyses of the HA1-specific IgG in the serum collected at day 21 from mice immunized with CF501/RBD or CF501/Rh3C-RBD and Alum/RBD or Alum/Rh3C-RBD. Statistical analysis was performed using the two-way ANOWA. *** and * indicating the statistical significance at P<0.0001 and P<0.05, respectively.]

, figureFileSmall=d8sgF5It0HsL0MyXNHiDew==, figureFileBig=nAif6c33k4wF4psqxfkgSQ==, tableContent=null), ArticleFig(id=1172891978858639711, tenantId=1146029695717560320, journalId=1146031712061968385, articleId=1148989445648474841, language=CN, label=图4, caption=基于Rh3C连接的H1N1 HA1融合蛋白作为三聚体流感疫苗抗原的设计构建及其免疫原性评价

［（a）基于Rh3C连接的H1N1 HA1重组融合蛋白（Rh3C-HA1）的设计。（b）SDS-PAGE图片验证Rh3C-HA1蛋白的表达。（c）小鼠的免疫程序。（d）以CF501为佐剂免疫小鼠后血清中针对HA1特异性的IgG抗体水平。（e）以Alum为佐剂免疫小鼠后血清中针对HA1特异性的IgG抗体水平。利用Two-way ANOWA进行统计学分析。***P<0.0001，*P<0.05］

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基于重组人Ⅲ型胶原蛋白的三聚体抗原疫苗策略在新冠和流感疫苗中的应用
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刘泽众 1, 2 , 周洁 1 , 朱赟 3 , 陆路 1 , 姜世勃 1
合成生物学 | 研究论文 2024,5(2): 385-395
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合成生物学 | 研究论文 2024, 5(2): 385-395
基于重组人Ⅲ型胶原蛋白的三聚体抗原疫苗策略在新冠和流感疫苗中的应用
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刘泽众1, 2 , 周洁1, 朱赟3, 陆路1 , 姜世勃1
作者信息
  • 1 复旦大学基础医学院，上海 200032
  • 2 复旦大学药学院药理系，上海 201203
  • 3 中国科学院生物物理研究所，北京 100101

    • 刘泽众（1990—），男，青年研究员，博士生导师。研究方向为微生物学。E-mail：

通讯作者:

姜世勃（1953—），男，教授，博士生导师。研究方向为微生物学。E-mail：
陆路（1982—），男，研究员，博士生导师。研究方向为微生物学。E-mail：
Applications of the recombinant human collagen type Ⅲ-based trimerization motif in the design of vaccines to fight against SARS-CoV-2 and influenza virus
Zezhong LIU1, 2 , Jie ZHOU1, Yun ZHU3, Lu LU1 , Shibo JIANG1
Affiliations
  • 1 School of Basic Medical Sciences，Fudan University，Shanghai 200032，China
  • 2 Department of Pharmacology，School of Pharmacy，Fudan University，Shanghai 201203
  • 3 Institute of Biophysics，Chinese Academy of Sciences，Beijing 100101，China
出版时间: 2024-04-30 doi: 10.12211/2096-8280.2023-058
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新冠病毒等多种包膜病毒的糖蛋白在天然状态下呈现三聚体形式。三聚体蛋白通常较单体蛋白具有更优的免疫原性。目前使用非人蛋白来源的Foldon等三聚体基序可促使目的蛋白形成稳定三聚体，但这些基序的强免疫原性会削弱目的蛋白的免疫应答，限制了其在疫苗抗原设计中的应用。在本研究中，发现并利用重组人源化Ⅲ型胶原蛋白（Rh3C）作为新型三聚体基序，利用合成生物学方法，与新冠病毒刺头（spike）蛋白受体结合域（RBD）融合表达后促使RBD形成三聚体。这种胶原-RBD（Rh3C-RBD）三聚体蛋白在免疫小鼠后，较RBD单体蛋白诱导产生了更高滴度的结合抗体和中和抗体。此外，我们还进一步验证了该Rh3C基序与流感病毒HA1蛋白融合后，较HA1单体蛋白可在小鼠体内诱导更强效的抗体应答。因此，这种新型的Rh3C基序，有望广泛用于三聚体疫苗抗原的设计和优化。

三聚体抗原  /  人胶原蛋白  /  新冠病毒  /  流感病毒  /  疫苗设计

Glycoproteins with enveloped viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, and human immunodeficiency virus (HIV), display a trimeric conformation. Different from the monomeric form, the trimeric proteins exhibit superior immunogenicity. Several trimerization motifs, such as Foldon derived from phage T4 fibritin, have been used to promote the formation of trimeric proteins with natural conformations. Although the Foldon-induced trimeric proteins are stable, their high immunogenicity limits applications in the development of vaccine antigens. In a previous study, we developed a recombinant human collagen type Ⅲ protein and determined its crystal structure, revealing a triple-helix conformation. However, the potential of this recombinant protein as a trimerization motif remained unknown. In this study, we demonstrated that the recombinant humanized type Ⅲ collagen (Rh3C) was able to act as a trimerization motif, facilitating the spontaneous trimer formation of the Rh3C-conjugated receptor-binding domain (RBD) within the spike (S) protein of SARS-CoV-2. This trimeric protein could induce a stronger SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a immune response, when compared with the monomeric RBD protein in the immunized mice. Notably, the Rh3C-RBD protein, when adjuvanted with the novel STING agonist CF501, also elicited significantly higher neutralizing antibody responses against both the pseudotyped SARS-CoV-2 (D614G) and its variant Omicron (BA.2.2) in the immunized mice. To showcase the broad applications of the Rh3C trimerization motif, we further demonstrated that the Rh3C-conjugated HA1 of the influenza virus could also elicit a stronger antibody response than free HA1. Considering the wide distribution of the Rh3C protein in human bodies, its use as a trimerization motif would not induce an immune response due to immune tolerance, thereby allowing the immune response to concentrate on targeted viral proteins. Therefore, this Rh3C-based trimerization motif holds great potential for the design and optimization of vaccines consisting of trimeric protein antigens.

trimeric antigen  /  humanized collagen  /  SARS-CoV-2  /  influenza virus  /  vaccine design
刘泽众, 周洁, 朱赟, 陆路, 姜世勃. 基于重组人Ⅲ型胶原蛋白的三聚体抗原疫苗策略在新冠和流感疫苗中的应用. 合成生物学, 2024 , 5 (2) : 385 -395 . DOI: 10.12211/2096-8280.2023-058
Zezhong LIU, Jie ZHOU, Yun ZHU, Lu LU, Shibo JIANG. Applications of the recombinant human collagen type Ⅲ-based trimerization motif in the design of vaccines to fight against SARS-CoV-2 and influenza virus[J]. Synthetic Biology Journal, 2024 , 5 (2) : 385 -395 . DOI: 10.12211/2096-8280.2023-058
正文
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新冠病毒（severe acute respiratory syndrome coronavirus2，SARS-CoV-2）和流感病毒（influenza virus）等呼吸道病毒持续威胁人类健康。提高疫苗抗原的免疫原性，诱导强效的免疫应答，对于开发新冠疫苗和流感疫苗至关重要。新冠病毒的刺突（spike，S）蛋白包含S1和S2两个亚基。S1亚基包含受体结合域（receptor-binding domain，RBD）负责识别宿主细胞的受体［1］。S2亚基负责介导病毒膜与宿主细胞膜融合［1-2］。新冠病毒的S蛋白，特别是其上的RBD结构域抗原区，是开发疫苗的主要抗原靶点之一［3-4］。在天然情况下，新冠病毒的S/RBD蛋白呈现同源三聚体的形式［5］。目前有较多研究证明S三聚体与RBD三聚体较S单体与RBD单体具有更好的免疫原性，可在机体中诱导更高滴度的中和抗体［6-7］。与此类似，流感病毒的糖蛋白HA也包含HA1和HA2两个亚基，分别负责介导病毒与受体的识别和病毒膜与靶细胞膜的融合，是疫苗抗原设计的主要靶点［8］。HA在天然情况下也同样为同源三聚体结构。因此，设计新冠病毒/流感病毒的三聚体疫苗抗原，对于提升疫苗效果具有重要意义。
目前人们已经开发了一些具有三聚体结构特征的蛋白基序，例如Foldon基序。Foldon是T4噬菌体次要纤维蛋白（fibrin）的C末端区域，其可自发形成典型的三聚体［9-10］。Foldon亚基内部通过非共价键作用共同维持稳定结构，三聚体稳定性高，目前已在1型艾滋病病毒（HIV-1）gp140［11-13］、SARS-CoV-2 RBD［14-15］等三聚体蛋白上用作三聚体标签基序。然而，虽然Foldon可有效促使目的蛋白呈现三聚体形式，但其分子量较大，具有强免疫原性［16］。将Foldon和病毒蛋白的融合三聚体抗原免疫人体后会产生对Foldon蛋白的强烈免疫应答，削弱了对病毒蛋白的抗原反应［16］。因此仍需开发其他的三聚体策略用于疫苗抗原的设计。
合成生物学技术在近年来迅速发展，并在许多领域展示出巨大的应用价值。人们通过集合基因工程、分子生物学、结构生物学等手段，可以精确设计和合成具有特定结构和功能的蛋白质，比如重组人源化胶原蛋白［17-18］。胶原蛋白在人体中含量丰富，参与形成皮肤、骨头、韧带、角膜等组织。在人体中，至少有28种类型的胶原蛋白［19］。其中Ⅲ型胶原蛋白广泛分布于皮肤、血管等组织［20］。在前期研究中，我们发现了人Ⅲ型胶原蛋白三螺旋区中一个重要的功能区（Gly483-Pro512）并解析了其主要区域的晶体结构，发现该功能区能折叠成典型的三股螺旋结构，具有强效的细胞黏附特性［21］。随后，我们进一步设计和表达了该功能区16重复串联的融合蛋白-重组人源化Ⅲ型胶原蛋白（Rh3C），发现Rh3C具有更强的细胞黏附特性，提示了其具有更加稳定的三螺旋构象［21］。由于Ⅲ型胶原蛋白广泛分布于人体中，较难在人体中产生免疫应答，且可有效形成三聚体，故而在本文中，我们将SARS-CoV-2的RBD蛋白与该Rh3C基序连接后进行融合表达，发现该融合蛋白（Rh3C-RBD）可形成三聚体构象。小鼠免疫后发现，Rh3C-RBD三聚体Rh3C较RBD单体蛋白可更加强效地诱导RBD特异性结合抗体和中和抗体。此外，我们也进一步在流感HA1抗原中验证了该策略，发现与该重组胶原融合后的HA1蛋白在小鼠体内较单体形式的HA1也可诱导较高的IgG抗体水平。因此，以上结果提示这种新型的Rh3C基序，有望广泛用于三聚体疫苗抗原的设计和优化。
1 材料与方法
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1.1 实验材料
EXPi293悬浮细胞、Huh-7细胞、HEK293T细胞，EasyTrans转染试剂，HRP标记的兔抗小鼠IgG、IgG1、IgG2a二抗，Luciferase检测试剂盒。
1.2 蛋白的表达与纯化
将Rh3C（GERGAPGFRGPAGPNGIPGEKGPA GERGAP16）通过Linker（GAPGPCCGG-GGGGS4）分别与SARS-CoV-2（Delta）RBD（Arg319-Asn532）或者H1N1（WSN）的HA1（Asp18-Arg343）的基因序列进行连接，并在C末端加入6个组氨酸，在南京金斯瑞生物进行基因合成。然后将合成后的序列分别利用ECOR Ⅰ和NCO Ⅰ酶切位点插入到pFUSE-hIgG1-Fc2载体。分别命名为Rh3C-RBD和Rh3C-HA1质粒。然后利用EXPi293真核表达系统分别对这两种蛋白进行表达。具体方法参照Liu等［22］的方法。首先当EXPi293悬浮细胞密度为2×106时，利用EasyTrans（李记生物，上海）转染试剂对质粒进行转染。转染6天后，收集细胞上清，过0.45 μm的滤膜后，进行蛋白纯化。通过NI-NTA亲和色谱对蛋白进行纯化。首先将收集的细胞上清与NI-NTA在4 ℃孵育2 h后，利用10 mmol/L的咪唑进行杂蛋白的洗涤。最后利用500 mmol/L的咪唑洗脱目的蛋白。将洗脱的目的蛋白进行超滤浓缩后，利用SDS-PAGE鉴定蛋白的分子量和纯度。
1.3 蛋白分子量大小的鉴定
应用凝胶过滤色谱，使用Superose 6 increase 10/300凝胶柱在GE蛋白色谱纯化系统AKATA中分别上样纯化后的Rh3C-RBD蛋白和标准品蛋白。根据标准品蛋白分子量测算Rh3C-RBD 蛋白分子量大小。此外，利用粒径分析仪Zetasizer Nano System，根据操作说明，测量Rh3C-RBD蛋白分子量大小。
1.4 小鼠免疫
Balb/c小鼠购自北京维通利华有限公司。小鼠的性别为雌性，8周龄。小鼠免疫采用肌内注射方式。每组小鼠共6只，分别免疫5 μg的Rh3C-RBD/Rh3C-HA1和20 μg的CF501；5 μg的RBD/HA1和20 μg的CF501；5 μg的Rh3C-RBD/Rh3C-HA1和等体积的铝佐剂（Thermo Scientific）；5 μg的RBD/HA1和等体积的铝佐剂（Thermo Scientific）。小鼠共免疫两次，间隔14天。在第21天采集小鼠血清，存放于-20 ℃。
1.5 ELISA
参考Liu等［4，23］的方法进行ELISA实验。分别将1 μg/mL的RBD或HA1包被于ELISA板中，在4 ℃过夜包被。然后加入100 μL的含5% BSA的PBST，于37 ℃封闭2 h。将小鼠血清利用PBST进行系列倍比稀释后，加入到ELISA板中，于37 ℃孵育1 h。利用PBST进行洗涤5次后，分别加入HRP标记的兔抗小鼠的IgG、IgG1和IgG2a二抗。于37 ℃孵育1 h后，利用PBST洗涤5次。加入TMB底物显色，然后加入H2SO4进行终止。利用酶标仪检测ELISA板中每孔的A 450数值。
1.6 SARS-CoV-2假病毒的制备
SARS-CoV-2假病毒的制备参考Liu等［2，22］的方法。将生长良好的HEK293T细胞进行传代。24 h后，待细胞密度在60%～80%时，利用Vigofect转染试剂对PNL-4-3骨架质粒和PcDNA-3.1-SARS-CoV-2-S质粒进行共转染。8 h后更换新鲜DMEM培养基。48 h后收取细胞上清即为SARS-CoV-2假病毒。SARS-CoV-2假病毒存放于-80 ℃。
1.7 假病毒中和实验
将生长良好的Huh-7细胞铺96孔板，每孔5000个细胞。8 h后，对小鼠血清进行系列倍比稀释，然后加入等体积的SARS-CoV-2假病毒，于37 ℃孵育0.5 h。将血清和假病毒混合液加入到Huh-7细胞中。在12 h后，更换新鲜的DMEM培养基。继续培养48 h后。加入细胞裂解液作用0.5 h。然后加入Luciferase底物。检测Luciferase活性。
1.8 统计学分析
结果以平均值±标准误差表示。使用GraphPad Prism分别利用One way ANOWA、Student-t test、Two way ANOWA对实验数据进行统计学分析。
2 结果
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2.1 基于重组人源化Ⅲ型胶原蛋白（Rh3C）基序的SARS-CoV-2 RBD三聚体蛋白的设计、表达与鉴定
前期研究中，筛选获得了人源化Ⅲ型胶原蛋白Gly483-Pro512功能区，并通过将该区域进行16重复的串联表达获得性质更好的Rh3C［21］。本研究将Rh3C通过Linker与SARS-CoV-2的RBD蛋白以融合蛋白的形式表达（Rh3C-RBD），期望在胶原蛋白三聚化的作用下促使RBD以三聚体的形式表达，提高RBD的免疫原性［图1（a）］。利用AlphaFold2程序［24］，预测了Rh3C-RBD融合蛋白的三聚体结构［图1（b）］。结果显示，整个Rh3C-RBD分子可以形成规则的三聚体结构，长度大约140 nm。分子的前端为相互错位1个氨基酸的胶原三聚体结构，后端为3重中心轴对称的RBD三聚体结构，两个结构由柔性Linker连接。该分子整体结构与设计预期基本吻合［图1（c）］。接下来的实验结果显示，Rh3C-RBD蛋白可被高效重组表达［图1（d）］。SDS-PAGE呈现的Rh3C-RBD蛋白单体分子量与预期蛋白分子量一致，约为85 kDa左右。通过对该蛋白进行纯化，获得了高纯度的Rh3C-RBD蛋白。通过凝胶过滤色谱检测了所表达的Rh3C-RBD的分子量，约为274 kDa，为所预测分子量的3倍左右，提示了Rh3C-RBD以三聚体形式成功被表达［图1（e）］。进一步通过测量该蛋白的粒径，估算出了该蛋白在天然情况下的分子量大小，约为255 kDa［图1（f）］，与凝胶过滤色谱结果一致，符合预期的三聚体分子量。结果提示，Rh3C-RBD以三聚体的形式被表达及制备。
2.2 Rh3C-RBD三聚体和RBD单体免疫原性的比较
为了鉴定Rh3C-RBD三聚体是否比RBD单体具有更好的免疫原性，利用之前研发的新型STING激动剂CF501［23，25］和铝佐剂（Thermo）分别作为Rh3C-RBD三聚体和RBD单体的佐剂免疫Balb/c小鼠。在免疫两次后，对小鼠血清中的RBD特异性抗体进行检测。结果如图2所示。CF501佐剂组，Rh3C-RBD在小鼠体内所诱导产生的RBD特异性IgG、IgG1和IgG2a的抗体水平显著高于RBD单体蛋白诱导的抗体水平。CF501/Rh3C-RBD组小鼠血清针对RBD的特异性IgG平均抗体滴度为656 100，较CF501/RBD组小鼠血清的IgG抗体水平高约27倍（P<0.0001）Alum/Rh3C-RBD组小鼠血清针对RBD的特异性IgG平均抗体滴度为5467，较Alum/RBD组小鼠血清的IgG抗体水平高约4倍。同样CF501/Rh3C-RBD组小鼠所诱导的RBD特异性IgG1和IgG2a的抗体滴度分别为1 433 600和307 200，高出CF501/RBD组小鼠血清中抗体滴度14倍（P=0.0069）和53倍（P<0.0001）。这些数据提示了，融合Rh3C后的RBD三聚体较RBD单体可在小鼠体内诱导更加强效的特异性抗体免疫应答。
2.3 Rh3C-RBD三聚体和RBD单体抗原在小鼠体内诱导的中和抗体评价
为了进一步鉴定Rh3C-RBD三聚体是否会诱导更高的中和抗体水平，将SARS-CoV-2（D614G）的刺突蛋白表达在HIV骨架蛋白表面，构建了SARS-CoV-2的假病毒。并利用SARS-CoV-2（D614G）和Omicron（BA.2.2）假病毒系统检测小鼠血清中的中和抗体效价。结果如图3所示，CF501/Rh3C-RBD组小鼠血清中对D614G和BA.2.2假病毒的平均中和抗体效价为3620和2807，而CF501/RBD组小鼠的平均效价仅为191和64。同样地，Alum/Rh3C-RBD组小鼠血清对D614G假病毒的平均中和抗体效价为198，而Alum/RBD组小鼠血清的平均中和抗体效价仅为63。Alum/Rh3C-RBD和Alum/RBD组小鼠血清对BA.2.2假病毒都未产生强效的中和作用。以上数据提示了，Rh3C-RBD不仅可显著提高抗原特异性结合抗体，还可以有效增强中和抗体的免疫应答。
2.4 Rh3C三聚体策略在流感病毒抗原设计中的应用
为了评价该Rh3C是否可作为一种增加抗原免疫原性的通用元件，进一步将该Rh3C与流感病毒H1N1 HA1蛋白进行融合表达［图4（a）］。经过SDS-PAGE鉴定后，发现Rh3C-HA1成功被表达，并具有较高的纯度［图4（b）］。进一步将Rh3C-HA1和HA1分别与佐剂CF501或Alum联用免疫Balb/c小鼠［图4（c）］。免疫两次后利用ELISA检测HA1特异性的IgG抗体水平。结果如图4（d）、（e）所示。无论是利用CF501佐剂还是铝佐剂，Rh3C-HA1所诱导的HA1特异性IgG抗体滴度要显著性地高于HA1抗原诱导的IgG抗体水平。该结果提示，Rh3C三聚化策略不仅可提高SARS-CoV-2 RBD抗原诱发特异性结合抗体和中和抗体，还可提高流感HA1诱发的特异性结合抗体。有望成为一种通用的增强抗原免疫原性的三聚化策略。
3 讨论
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Ⅰ型包膜病毒的糖蛋白在天然状态大多以三聚体形式呈现，例如HIV-1 Env蛋白、流感病毒HA蛋白、新冠病毒S蛋白和呼吸道合胞病毒F蛋白等。制备和天然状态相近的三聚体抗原对于研发这些疫苗至关重要。目前应用最为普遍的是利用Foldon作为三聚化基序与目的蛋白融合表达，获得三聚体构象蛋白［26］。融合Foldon的三聚体蛋白虽然也可显著提高目的蛋白的免疫原性，但机体中会产生对Foldon本身的大量抗体，削弱了对目的蛋白的免疫应答［16］。Rh3C基序蛋白在免疫小鼠后，在小鼠体内可能会诱发对该Rh3C本身的抗体。Rh3C来自于全人源Ⅲ型胶原蛋白的部分片段，Ⅲ型胶原蛋白广泛存在于人体各组织器官中，具有安全无毒、免疫原性低等特征。由于T细胞和B细胞在发育过程中需经历阳性选择和阴性选择，存在中枢和外周免疫耐受机制，因此来自于人Ⅲ型胶原蛋白功能区天然序列的三聚化基序，较难在人体中产生特异性抗体。即使与佐剂配伍使用，引起针对自身胶原蛋白的抗体可能性也较低。因此融合Rh3C的抗原可让免疫应答更加聚焦目的蛋白。此外，该Rh3C可作为三聚化基序促使与之融合表达的目的蛋白呈现三聚体形式，增加目的蛋白的免疫原性。同时，该Rh3C三聚体策略具有通用性，可促使多种抗原形成三聚体。
新冠病毒的RBD单体蛋白免疫原性较差，通过利用多种策略将RBD构建成二聚体或三聚体可显著提高其免疫原性。例如通过串联表达的方式构建的RBD二聚体较RBD单体可显著诱导免疫应答［27］；将RBD同人IgG1 Fc融合表达后可获得RBD-Fc二聚体蛋白，除增加RBD免疫原性外，Fc还可发挥分子内佐剂的作用［4］；将RBD同Foldon融合表达形成稳定的三聚体也可显著增强抗体免疫应答［15］。本文所构建的Rh3C蛋白优势在于提供了一种来自于人Ⅲ型胶原蛋白片段的三聚体基序，在生物相容性和安全性等方面可能更具有优势。
如今，合成生物技术在当前科学研究和应用中扮演着越来越重要的角色。该技术的重要性在于提供了一种精确、可控的方法来设计和构建新的生物分子，从而实现对生物分子及生物体的精确操控和优化。例如，本研究中利用的基于合成生物技术设计的Rh3C，已被发现具有良好的生物学性能和广泛的应用场景，包括治疗阴道萎缩［28］、心血管支架涂层［29］、快速修复慢性糖尿病伤口感染［30］、抗炎与血管生成［31］、改善盆底功能障碍［18］、缓解皮肤光老化［32］、改善慢性子宫内膜炎［33］等。本研究利用基于合成生物技术设计的Rh3C基序，为三聚体疫苗抗原的设计和优化提供了新的思路和方法。目前所设计、表达和制备的Rh3C-RBD和Rh3C-HA1抗原蛋白，初步证明了较未融合该Rh3C基序的RBD或HA1抗原，在小鼠模型上诱导产生了更加强效的体液免疫应答。这显示了合成生物学技术的应用潜力，为各类病毒的三聚化抗原设计提供了一种有效的、普适的新型抗原设计策略。后续我们还将深入分析这类抗原蛋白的精细结构特征，并在灵长类动物模型中证明该类融合蛋白的免疫原性，包括评估针对胶原基序产生抗体的情况。同时，也还将设计开发更多种类和特性的该类基序蛋白，进一步提高疫苗的免疫原性和保护效果，为解决人类面临的越来越严峻的病原微生物挑战提供新的解决方案。
基金
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  • 国家重点研发计划(2022YFC2305800)
  • 国家自然科学基金(92169112)
  • 国家自然科学基金(82202490)
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参考文献 引证文献
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doi: 10.12211/2096-8280.2023-058
  • 接收时间：2023-08-19
  • 首发时间：2025-07-07
  • 出版时间：2024-04-30
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  • 收稿日期：2023-08-19
  • 修回日期：2023-11-07
基金
国家重点研发计划(2022YFC2305800)
国家自然科学基金(92169112)
国家自然科学基金(82202490)
作者信息
    1 复旦大学基础医学院，上海 200032
    2 复旦大学药学院药理系，上海 201203
    3 中国科学院生物物理研究所，北京 100101

通讯作者:

姜世勃（1953—），男，教授，博士生导师。研究方向为微生物学。E-mail：
陆路（1982—），男，研究员，博士生导师。研究方向为微生物学。E-mail：
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Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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