WO2021249031A1 - 表达新型冠状病毒s蛋白的口服重组酵母及其制备与应用 - Google Patents

表达新型冠状病毒s蛋白的口服重组酵母及其制备与应用 Download PDF

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WO2021249031A1
WO2021249031A1 PCT/CN2021/088592 CN2021088592W WO2021249031A1 WO 2021249031 A1 WO2021249031 A1 WO 2021249031A1 CN 2021088592 W CN2021088592 W CN 2021088592W WO 2021249031 A1 WO2021249031 A1 WO 2021249031A1
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protein
rbd
yeast
gene
recombinant
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黄金海
张丽琳
郭艳余
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天津大学
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Priority to CA3182052A priority patent/CA3182052A1/en
Priority to EP21823134.8A priority patent/EP4166649A1/en
Priority to US18/001,576 priority patent/US20230302118A1/en
Priority to AU2021287664A priority patent/AU2021287664A1/en
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    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention belongs to the technical field of biological genetic engineering, and relates to an oral recombinant yeast expressing a novel coronavirus S protein and an application thereof.
  • Corona Virus Disease 2019 is an acute respiratory infectious disease caused by a novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) infection.
  • SARS-CoV-2 is a single-stranded positive-stranded RNA virus, classified as Nidovirales (Nidovirales), Coronaviridae (Coronaviridae), Orthocoronavirinae (Orthocoronavirinae). And SARS (Severe Acute Respiratory Syndrome), MERS (Middle East) Respiratory Syndrome) belongs to the beta coronavirus.
  • the genome is 29,903bp, encoding the main structural proteins including Spike glycoprotein (S), Envelop (E), nucleocapsid phosphoprotein N (nucleocapsid phosphoprotein N), membrane glycoprotein (M) and so on.
  • SARS-CoV and MERS-CoV spike protein S binds to host cells through different receptor-binding domains (RBDs).
  • the S protein of MERS-CoV binds to host cell dipeptidyl peptidase IV (dipeptidyl peptidase 4, DPP-4, also known as CD26) [1]
  • SARS-CoV is the same as SARS-CoV-2
  • the blood vessels on the cell surface Angiotensin converting enzyme 2 (ACE2) is the binding site of S protein RBD [2,3] .
  • ACE2 Angiotensin converting enzyme 2
  • Studies have shown that there are differences in the RBD area between SARS-CoV and SARS-CoV-2, which mainly exist in the C-terminal 318-507aa [4] . The two do not have complete protection, so it is necessary to develop specific protection against the new coronavirus. preparation.
  • yeast cells More mature heterologous protein expression systems include yeast cells, prokaryotic expression systems and baculovirus insect cell expression systems. Compared with the latter two, yeast cells have the advantages of mature post-translational modification ability and short culture cycle, safety, convenience, and low production cost [5] .
  • the yeast surface display technology (Yeast surface display, YSD) is used to display foreign proteins on the surface of yeast cells to prepare oral protective preparations.
  • yeast cell wall polysaccharide has a regulatory effect on the body's immune system, can resist viruses, enhance immune function, and promote the development of immune organs. Therefore, the development of yeast oral preparations has a good application prospect.
  • One of the objectives of the present invention is to provide a novel coronavirus spike protein S surface display type recombinant yeast and its application, and specifically use it for the development of oral preparations.
  • the second objective of the present invention is to provide a method for preparing the oral recombinant yeast.
  • the third objective of the present invention is to provide the use of the oral recombinant yeast.
  • a truncated body of a novel coronavirus S protein preferably amino acids 300 to 1000, sequence feature SEQ ID No. 1, including RBD domains from 330 to 521 and FP fusion peptides from 816 to 833 .
  • the gene expressing the protein truncated body contains the RBD domain of spike protein S, that is from base 991 to 1563, and the FP domain, that is from base 2449 to base 2499, and its nucleotides
  • the sequence is SEQID No.2.
  • the recombinant yeast plasmid GPD-S(RBD-FP)-TU was constructed, with the sequence characteristic of SEQ ID No. 3, consisting of the gene fragment described in claim 3 and the POT-GPD-TU vector.
  • the method for preparing new coronavirus S protein recombinant yeast the in vitro constructed S protein truncated complete transcription unit GPD-S(RBD-FP)-TU is integrated into the yeast genome through homologous recombination, and the Aga1-Aga2 surface display system is used
  • the S protein is displayed on the surface of yeast cells to obtain a recombinant yeast strain ST1814G-S (RBD-FP) with S protein surface display type, and the obtained strain is used to prepare oral recombinant yeast.
  • PCR amplification of SARS-CoV-2 spike protein S-encoding gene refer to the SARS-CoV-2 virus gene sequence NC_045512.2, synthesize the S gene, the sequence feature is SEQ No. 2; based on pcDNA3.1-CoV -S plasmid is used as a template, and primers are designed to amplify the S protein encoding gene S (RBD-FP) for yeast vector connection;
  • the POT-GPD-TU vector is linearized by BamHI single enzyme digestion, and the S gene fragment is seamlessly cloned and connected to the surface to display expression
  • the recombinant plasmid GPD-S(RBD-FP)-TU was obtained, and its sequence feature was SEQNo.3.
  • the recombinant plasmid was transformed into E.coli DH5a, and the S gene detection primer was used for PCR and sequencing verification , To obtain positive clones;
  • the beneficial effects of the present invention is based on the spike protein S that triggers the initiation of infection when the new coronavirus binds to the ACE2 receptor of the host cell.
  • Short-body surface display oral recombinant yeast preparation. It stimulates the body's protective immune response through oral route, and uses yeast cell wall polysaccharides to regulate the body's innate immune system to exert more effective immune protection.
  • oral recombinant yeast preparations have low cost, can achieve large-scale scale-up production, are safe and reliable, have good application development prospects, and provide options for immune prevention and control of new coronaviruses.
  • novel coronavirus S protein oral recombinant yeast preparation described in the present invention is the first report in China.
  • the construction of surface display strains and preparation of preparations are innovative to a certain extent, and provide new ideas and preparations for the prevention and control of COVID-19.
  • Figure 1 A schematic diagram of the structure of the gene encoding the S protein of the novel coronavirus
  • Figure 3 Detection of transformants after GPD-S(RBD-FP)-TU plasmid transforms into E. coli; lanes 1-10 represent the results of PCR detection of colonies of different E. coli transformants, and CK+ is based on the S (RBD-FP) gene
  • the PCR product is the amplification of the template, and CK- is the ddH 2 O control;
  • FIG. 4 GPD-S(RBD-FP)-TU complete transcription unit splicing pattern diagram
  • FIG. 5 Growth of GPD-S(RBD-FP)-TU in SD-leu medium after yeast transformation
  • Figure 6 Genotype verification of ST1814G-S (RBD-FP) recombinant yeast; the genotypes were verified after yeast transformation corresponding to transformants 2 and 3 of E. coli. Lanes 1-6 are 6 different yeast transformants, CK+ is PCR amplification with plasmid GPD-S(RBD-FP)-TU as template, CK- is negative control with ddH 2 O as template;
  • Figure 7 Western blot verification of ST1814G-S (RBD-FP) yeast strain, two bands appeared after the S protein truncated body was detected by His antibody. The larger molecular weight is the glycosylated modified S protein truncated body, and the smaller molecular weight is the product of protease cleavage; lanes 3-1, 3-4 and 3-6 correspond to 3 different yeast transformants;
  • Figure 8 Immunofluorescence observation of S (RBD-FP) protein in ST1814G-S (RBD-FP) recombinant yeast; the ST1814G empty strain was used as a control;
  • Figure 9 The growth curve of ST1814G-S (RBD-FP) recombinant yeast and its relationship with the protein expression trend; a is the growth curve of the bacterial cell, with ST1814G as the control, the growth trend of the protein-expressing strain is consistent with the control group; b For the analysis of protein expression at different culture time points, lanes 1-5 respectively represent culture for 1 day to 5 days;
  • Figure 10 Detection of specific IgA and IgG in the serum of BALB/c mice after oral administration of yeast recombinant bacteria;
  • the S protein coding gene consists of two parts: S1 subunit and S2 subunit.
  • the S1 subunit contains the RBD domain, which can bind to the PD domain of the host cell ACE2 receptor (peptidase domain);
  • the S2 subunit contains the fusion peptide FP (hydrophobic fusion peptide) sequence, which helps the virus and the host cell membrane to close and fuse, similar
  • the structure also exists in SARS-CoV [8,9] .
  • the primers were designed and synthesized, CoV-SF (SEQ ID NO.4: GACGATAAGGTACCAGGATCCATGAAGTGTACGTTGAAATCCT) and CoV-SR (SEQ ID NO.5: gaattccaccacactggatccTCTGCCTGTGATCAACCTAT), according to the reported viral genome Sequence (Genbank No. MT407658.1), the gene fragment of S protein was artificially synthesized, and pcDNA3.1-CoV-S plasmid was constructed. Using this plasmid as a template, the S protein coding gene S( RBD-FP).
  • the PCR amplification system is:
  • the PCR product size is 2106bp.
  • the PCR results are shown in Figure 2.
  • the rightmost lane is the amplification result of the S (RBD-FP) gene.
  • the POT-GPD-TU vector was linearized by BamHI and then connected to the S (RBD-FP) gene.
  • the N end of the S (RBD-FP) gene was connected to Aga2 and expressed in tandem.
  • the C-terminal of the gene has a His tag on the vector .
  • the ligation product was obtained using the seamless cloning kit (C112-01, Vazyme) and transformed into E. coli DH5 ⁇ .
  • E. coli transformants were screened by Amp resistance plate, and S gene detection primers (SF 331: aatattacaaacttgtgccct (SEQ ID NO. 6) and SR 524: aacagttgctggtgcatgtag (SEQ ID NO. 7)) were used for PCR verification and sequencing.
  • the vector GPD-S (RBD-FP)-TU was digested with BsaI, and at the same time, the homology arm plasmid (URR1 and URR2) and the selectable marker plasmid (LEU) were digested with BsmB I.
  • the URRs homology arm, LEU selective tag, GPD-S (RBD-FP)-TU transcription unit were spliced according to the specific prefix and suffix sequence, and T4 ligase was overnight at 16°C.
  • the ligation and splicing products are used for yeast transformation.
  • 2Yeast transformation add 100 ⁇ L 0.1M lithium acetate to the centrifuged cell pellet, resuspend the cells, and centrifuge at 12000rpm for 20s to discard the supernatant. Add 50 ⁇ L of 0.1M lithium acetate, resuspend the cells, and collect the cells by centrifugation at 12000rpm for 20s. Then add 240 ⁇ L of 50% PEG4000, 36 ⁇ L of 1M lithium acetate, 100 ⁇ g of salmon sperm DNA, and 2 ⁇ g of fragmented DNA in the centrifuge tube, and shake vigorously to completely mix.
  • yeast genome was taken as a genome template for PCR amplification.
  • the PCR amplification system is as follows:
  • the genomic DNA was used as a template for PCR amplification, and the plasmid GPD-S(RBD-FP)-TU was used as a positive control, and ddH 2 O was used as a negative control.
  • the experimental results are shown in Figure 5.
  • the 2 and 3 E. coli transformants of plasmid GPD-S(RBD-FP)-TU were digested and ligated to transform yeast cells, although both can be selected in SD-leu It can be grown on a healthy medium, but only plasmid 3 can obtain a recombinant yeast strain ST1814G-S (RBD-FP) with the correct genotype.
  • the correct recombinant yeast numbers are No.1, No.4 and No.6 ( Figure 6).
  • Collect 2mL 48h YPD culture solution centrifuge at 6000rpm for 1min to collect the bacteria, add acid-washed glass beads, 60 ⁇ L PEB buffer to resuspend the bacteria, break the walls 5 times by the glass bead method, add 15 ⁇ L 5 ⁇ SDS loading buffer, boiling water bath Centrifuge at 12000 rpm for 5 min at 4°C for 10 min, carefully aspirate the supernatant, and use 8 ⁇ L of the supernatant for 12% SDS-PAGE electrophoresis.
  • the protein separation gel is transferred to the PVDF membrane of the same size by wet transfer method, the transfer condition is 300mA 100min; after the transfer is completed, the PVDF membrane is sealed with 5% skim milk at room temperature for 1h ; Completely immerse the membrane in the mouse anti-His monoclonal antibody (HT501, Transgene) diluted 1:2000 with 5% BSA, and incubate overnight at 4°C; recover the primary antibody and rinse the PVDF membrane 3 times with TBST buffer for 10 minutes each ; Use 5% BSA diluted 1:5000 goat anti-mouse HRP-labeled secondary antibody (LK2003, S.e Biotech) to incubate at room temperature for 1 h; rinse the PVDF membrane with TBST buffer 3 times; avoid light, drop a chemiluminescent chromogenic substrate onto the front of the PVDF membrane (34075, ThermoFisher), exposed to Bio-rad chemiluminescence imager to observe protein expression
  • HT501 mouse anti-His monoclo
  • the control group ST1814G did not show fluorescence ( Figure 8 top); while the experimental group ST1814G-S (RBD-FP) had obvious green fluorescence ( Figure 8 bottom), indicating that the S protein was successfully expressed and Localized on the surface of recombinant Saccharomyces cerevisiae cells.
  • mice SPF BALB/c mice were divided into three groups, each with 6 mice, and they were fed ST1814G-S (RBD-FP) No. 4 yeast strain on day 1, 6, 11, and 23, 10 7 cfu/mouse
  • ST1814G blank yeast strain was used as a control.
  • blood was collected from the eyeball to detect the levels of IgG and IgA antibodies.

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Abstract

本发明提供了一种表达新型冠状病毒S蛋白的口服重组酵母及其制备与应用，其包含S蛋白的16位至1035位氨基酸，包含RBD结构域和FP融合肽。将体外构建的S蛋白截断体完整转录单位GPD-S(RBD-FP)-TU，通过同源重组整合于酵母基因组，利用Aga1-Aga2表面展示系统将S蛋白展示于酵母细胞表面，获得S蛋白表面展示型的重组酵母菌株ST1814G-S(RBD-FP)，并利用所得菌株制备口服重组酵母。

Description

表达新型冠状病毒S蛋白的口服重组酵母及其制备与应用 技术领域
本发明属于生物基因工程技术领域，涉及一种表达新型冠状病毒S蛋白的口服重组酵母及其应用。
背景技术
[根据细则26改正13.05.2021]　
新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)是由新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)感染导致的急性呼吸系统传染病。
SARS-CoV-2为单股正链RNA病毒，分类地位为巢病毒目(Nidovirales)冠状病毒科(Coronaviridae)正冠状病毒亚科(Orthocoronavirinae).与SARS(Severe Acute Respiratory Syndrome)、MERS(Middle East Respiratory Syndrome)同属于beta类冠状病毒。基因组29,903bp，编码主要的结构蛋白包括刺突蛋白(Spike glycoprotein,S)、囊膜蛋白(Envelop,E)、核衣壳蛋白(nucleocapsid phosphoprotein N)、膜蛋白(membrane glycoprotein,M)等。在SARS-CoV和MERS-CoV中刺突蛋白S通过不同的受体结合结构域(receptor-binding domains,RBDs)与宿主细胞结合。MERS-CoV的S蛋白与宿主细胞二肽基肽酶Ⅳ(dipeptidyl peptidase 4,DPP-4,also known as CD26)结合 [1]，而SARS-CoV与SARS-CoV-2一致，细胞表面的血管紧张素转化酶2(Angiotensin converting enzyme 2,ACE2)是S蛋白RBD的结合位点 [2,3]。有研究表明SARS-CoV与SARS-CoV-2的RBD区域存在差别，主要存在于C端318-507aa [4]，二者不具有完全的保护性，因而需要研发特异性的抗新型冠状病毒保护制剂。
比较成熟的异源蛋白表达系统包括酵母细胞、原核表达系统和杆状病毒昆虫细胞表达系统。相较于后两者，酵母细胞兼具成熟的翻译后修饰能力和培养周期短、安全、便捷、生产成本低等优势 [5]。利用酵母表面展示技术(Yeast surface display,YSD)将外源蛋白展示于酵母细胞表面，可制备口服型保护制剂。同时酵母细胞壁多糖对机体的免疫系统具有调节作用，可抗病毒、增强免疫功能，促进免疫器官发育，因而酵母口服制剂的开发具有良好的应用前景。
发明内容
本发明的目的之一在于提供一种新型冠状病毒刺突蛋白S表面展示型重组酵母及其应用，具体将其用于口服制剂的研制。
本发明的目的之二在于提供所述口服重组酵母的制备方法。
本发明的目的之三在于提供所述口服重组酵母的用途。
本发明目的是通过如下技术方案实现的：
一种表达新型冠状病毒S蛋白的重组酵母，ST1814G-S(RBD-FP)，其包含S蛋白的16位至1035位氨基酸。
一种新型冠状病毒S蛋白的截短体，优选为第300至1000位氨基酸，序列特征为SEQ ID No.1，为包含第330位至521的RBD结构域和第816至833位FP融合肽。
表达所述蛋白截短体的基因，包含刺突蛋白S的RBD结构域,即从第991位至1563位碱基和FP结构域，即从第2449位至2499位碱基，其核苷酸序列为SEQID No.2。
构建所述重组酵母的质粒GPD-S(RBD-FP)-TU，序列特征为SEQ ID No.3，由权利要求3所述基因片段和POT-GPD-TU载体组成。
制备新型冠状病毒S蛋白重组酵母的方法，将体外构建的S蛋白截短体完整转录单位GPD-S(RBD-FP)-TU，通过同源重组整合于酵母基因组，利用Aga1-Aga2表面展示系统将S蛋白展示于酵母细胞表面，获得S蛋白表面展示型的重组酵母菌株ST1814G-S(RBD-FP)，并利用所得菌株制备口服重组酵母。
具体包括以下步骤：
(1)SARS-CoV-2刺突蛋白S编码基因的PCR扩增：参考SARS-CoV-2病毒基因序列NC_045512.2，合成S基因，序列特征为SEQ No.2；以pcDNA3.1-CoV-S质粒为模板，设计引物扩增S蛋白编码基因S(RBD-FP)用于酵母载体连接；
(2)Aga2基因与SARS-CoV-2刺突蛋白S编码序列S(RBD-FP)串联：通过BamHI单酶切线性化POT-GPD-TU载体，无缝克隆连接S基因片段至表面展示表达载体GPD-POT-TU上，获得重组质粒GPD-S(RBD-FP)-TU，其序列特征为SEQNo.3.重组质粒转化至E.coli DH5a中，用S基因检测引物进行PCR及测序验证，获得阳性克隆；
(3)S蛋白酵母重组菌株的构建：将重组质粒GPD-S(RBD-FP)-TU与同源臂URRs、筛选标签Leu编码序列进行酶切拼接，获得完整的包含S基因序列的重组基因，转化到酿酒酵母基因组，经营养缺陷型平板筛选后获得重组菌株，利用检测引物进行基因水平检测， Western blot、免疫荧光进行蛋白表达水平验证。
新型冠状病毒S蛋白的重组酵母在制备用于抗新型冠状病毒药物中的应用。
本发明有益效果：本发明基于新型冠状病毒与宿主细胞ACE2受体结合时触发侵染启动的刺突蛋白S，制成了包含受体结合域及与病毒致病相关FP结构域的S蛋白截短体表面展示型口服重组酵母制剂。通过口服途径激发机体保护性免疫反应，并借助酵母细胞壁多糖对机体先天性免疫系统的调节作用，发挥更加有效的免疫保护。与新型的腺病毒疫苗、mRNA疫苗相比，口服重组酵母制剂成本低、可实现大规模放大生产，安全可靠，具有良好的应用开发前景，为新型冠状病毒的免疫防控提供选择。
本发明中所述的新型冠状病毒S蛋白口服重组酵母制剂在国内属于首次报道，表面展示菌株构建及制剂制备具有一定的创新性，为COVID-19的防控提供新思路与制剂。
附图说明
图1：新型冠状病毒S蛋白编码基因结构模式图；
图2：S(RBD-FP)基因PCR扩增结果；
图3：GPD-S(RBD-FP)-TU质粒转化大肠杆菌后的转化子检测；1-10泳道分别代表不同的大肠杆菌转化子菌落PCR检测结果，CK+是以S(RBD-FP)基因PCR产物为模板的扩增，CK-为ddH 2O对照；
图4：GPD-S(RBD-FP)-TU完整转录单位拼接模式图；
图5：GPD-S(RBD-FP)-TU完成酵母转化后在SD-leu培养基中的生长情况；
图6：ST1814G-S(RBD-FP)重组酵母的基因型验证；分别对应大肠杆菌的2号和3号转化子进行酵母转化后验证基因型。泳道1-6分别为6个不同的酵母转化子，CK+为质粒GPD-S(RBD-FP)-TU为模板的PCR扩增，CK-为ddH 2O为模板的阴性对照；
图7：ST1814G-S(RBD-FP)酵母菌株的Western blot验证，S蛋白截短体经His抗体检测后出现两条带。分子量大的为糖基化修饰的S蛋白截短体，分子量小的为蛋白酶切后产物；泳道3-1，3-4和3-6对应3株不同的酵母转化子；
图8：ST1814G-S(RBD-FP)重组酵母中S(RBD-FP)蛋白的免疫荧光观察；以ST1814G空菌株为对照；
图9：ST1814G-S(RBD-FP)重组酵母的生长曲线及其与蛋白表达趋势的关系；a为菌体的生长曲线，以ST1814G为对照，表达蛋白的菌株生长趋势与对照组一致；b为不同培养时间点的蛋白表达情况分析，泳道1-5分别代表培养1d至培养5d；
图10：BALB/c小鼠口服酵母重组菌后血清中特异性IgA和IgG的检测；A：ELISA检测未口服组(Blank)、口服原始空白酵母(Host)与口服重组酵母(FP)后血清中IgG的情况；B：ELISA检测未口服组(Blank)、口服原始空白酵母(Host)与口服重组酵母(FP)后血清中IgA的情况。
具体实施方式
以下结合实例进一步阐述本发明，其中的内容不应理解为限定该发明的范围。实施例中所述试剂除特别说明，均为商业化试剂。
实施例1.GPD-S(RBD-FP)-TU载体的构建
(1)S(RBD-FP)基因的扩增
S蛋白编码基因由S1亚基和S2亚基两部分组成。其中S1亚基包含RBD结构域，可结合宿主细胞ACE2受体的PD域(peptidase domain)；S2亚基含有融合肽FP(hydrophobic fusion peptide)序列，帮助病毒与宿主细胞膜靠近并发生融合，类似的结构在SARS-CoV中同样存在 [8,9]。根据S基因序列结构(图1所示)，设计并合成引物，CoV-S-F(SEQ ID NO.4:GACGATAAGGTACCAGGATCCATGAAGTGTACGTTGAAATCCT)和CoV-S-R(SEQ ID NO.5:gaattccaccacactggatccTCTGCCTGTGATCAACCTAT)，根据已有报道的病毒基因组序列(Genbank号MT407658.1)，人工合成了S蛋白的基因片段，并构建了pcDNA3.1-CoV-S质粒，以该质粒为模板，扩增包含RBD和FP区域的S蛋白编码基因S(RBD-FP)。PCR扩增体系为：
Figure PCTCN2021088592-appb-000001
使用以下PCR程序进行扩增：
Figure PCTCN2021088592-appb-000002
PCR产物大小为2106bp。
PCR结果如图2所示，最右侧泳道为S(RBD-FP)基因的扩增结果。
(2)GPD-S(RBD-FP)-TU载体的构建
将S(RBD-FP)基因连接到实验室已构建的POT-GPD-TU载体上 [7]。POT-GPD-TU载体经BamHI酶切线性化后与S(RBD-FP)基因连接，S(RBD-FP)基因N端与Aga2连接并串联融合表达，基因C端带有载体上的His标签。利用无缝克隆试剂盒(C112-01,Vazyme)获得连接产物，并转化E.coli DH5α。大肠杆菌转化子经Amp抗性平板筛选，并用S基因检测引物(S-F 331:aatattacaaacttgtgccct(SEQ ID NO.6)和S-R 524:aacagttgctggtgcatgtag(SEQ ID NO.7))进行PCR验证及测序。
结果如图3所示，PCR扩增产物大小为579bp，阳性转化子为No.1-8和9号，选择其中2号和3号测序，与预期结果一致，表明GPD-S(RBD-FP)-TU质粒构建成功。
实施例2.新型冠状病毒酵母重组菌株ST1814G-S(RBD-FP)的构建及检测
(1)酵母转化片段的构建
用BsaI酶切载体GPD-S(RBD-FP)-TU，同时，BsmB I酶切同源臂质粒(URR1和URR2)和选择性标记质粒(LEU)。参照Dai课题组的实验步骤 [6]，将URRs同源臂、LEU选择性标签、GPD-S(RBD-FP)-TU转录单位，按照特异性前后缀序列进行拼接，T4连接酶16℃过夜连接，拼接产物(如图4)用于酵母转化。
(2)ST1814G-S(RBD-FP)重组酿酒酵母菌株的构建
①菌株活化：挑取ST1814G单菌落接种到3mL YPD液体培养基中，30℃、220rpm过夜培养。将过夜培养的菌液按1：50的比例转接到5mL新鲜的YPD培养基中，使其初始OD为0.1～0.2，30℃、220rpm培养至OD600为0.5～0.8。2500rpm离心5min收集5mL菌液的菌体，并用1mL无菌水洗涤细胞，弃去上清。
②酵母转化：向离心后的菌体沉淀中加入100μL 0.1M的醋酸锂，重悬细胞，12000rpm离心20s弃去上清。加入50μL 0.1M醋酸锂，重悬细胞，12000rpm离心20s收集菌体。然后在离心管中依次加入240μL 50％的PEG4000、36μL 1M醋酸锂、100μg鲑鱼精DNA、2μg片段DNA，剧烈震荡至完全混匀。30℃ 200rpm 30min，42℃ 25min，6000rpm离心15s收集菌体，去除转化液，加入1mL的YPD液体培养基于30℃孵育2h，2500rpm离心5min收集菌体，用50μL去离子水重悬细胞，尽可能温和的吹吸混匀，将菌体涂布 在SD-leu固体培养基表面，30℃培养箱生长2～3天，直至形成典型菌落。挑取单菌落在SD-leu平板划线纯化，同步接种至3mL YPD液体培养基中，30℃、220rpm过夜培养，用于基因型验证。
③ST1814G-S(RBD-FP)菌株的基因型验证
选择在SD-leu培养基中生长的酵母，提取其基因组PCR验证S(RBD-FP)基因是否成功整合到基因组。酵母基因组的提取方法参照文献进行 [10]。取100μL培养后的菌液6000rpm离心1min弃去上清，用100μL裂解液(200mM LiOAc,1％SDS)重悬细胞，70℃孵育5min.加入300μL无水乙醇混匀，于15000g离心3min收集细胞碎片。70％乙醇清洗后加入50-100μL ddH 2O重悬沉淀。15000g离心15s后吸取上清作为基因组模板进行PCR扩增。PCR扩增体系如下：
Figure PCTCN2021088592-appb-000003
使用以下PCR程序进行扩增：
Figure PCTCN2021088592-appb-000004
以基因组DNA为模板进行PCR扩增，同时以质粒GPD-S(RBD-FP)-TU为阳性对照，ddH 2O为阴性对照。
实验结果如图5所示，质粒GPD-S(RBD-FP)-TU的2号和3号大肠杆菌转化子经酶切、连接后转化酵母细胞，虽二者均可在SD-leu的选择性培养基生长，但仅3号质粒可获得基因型正确的重组酵母菌株ST1814G-S(RBD-FP)。正确的重组酵母编号为No.1,No.4和No.6(图6)。
④ST1814G-S(RBD-FP)菌株的表型验证
由于GPD-S(RBD-FP)-TU载体插入片段S(RBD-FP)的C端带有His标签，利用Western  blot抗His抗体检测目的基因S(RBD-FP)的表达情况，利用激光共聚焦显微镜观察蛋白展示情况。
Western Blot:
收集2mL 48h的YPD培养液，6000rpm离心1min收集菌体，加入酸洗玻璃珠，60μL PEB缓冲液重悬菌体，玻璃珠法破壁5次，加入15μL 5×SDS上样缓冲液，沸水浴5min，4℃下12000rpm离心10min，小心吸取上清，8μL上清用于12％SDS-PAGE电泳。
SDS-PAGE电泳结束后，通过湿转法将蛋白分离胶转移至与其同等大小的PVDF膜上，转膜条件为300mA 100min；转膜完成后，在室温下，用5％脱脂牛奶封闭PVDF膜1h；将膜完全浸没在用5％BSA 1:2000稀释的鼠抗His单克隆抗体(HT501,Transgene)中，4℃孵育过夜；回收一抗，用TBST缓冲液漂洗PVDF膜3次，每次10min；用5％BSA 1:5000稀释的羊抗鼠HRP标记二抗(LK2003,Sungene Biotech)室温孵育1h；TBST缓冲液漂洗PVDF膜3次；避光向PVDF膜正面滴加化学发光显色底物(34075,ThermoFisher)，通过Bio-rad化学发光成像仪曝光，观察蛋白表达情况。
免疫荧光：
收取500μL诱导48h的ST1814G-S(RBD-FP)菌液，4000rpm，离心5min收集菌体；取诱导48h后的ST1814G作为阴性对照；用500μL PBST洗涤菌体3次，4000rpm，5min，弃上清；分别用200μL PBST 1:2000稀释的6*His-Tag抗体重悬菌体，4℃旋转结合1h；回收一抗；500μL PBST洗涤菌体3次，同上离心弃上清；加入200μL PBST 1:5000稀释的FITC-标记的羊抗鼠二抗，37℃避光孵育30min，回收二抗；500μL PBST洗涤菌体3次，同上离心弃上清；用50μL PBS重悬菌体，取10μL菌液滴于干净的载玻片上，盖上盖玻片，四周涂上指甲油固定盖玻片，在激光共聚焦显微镜下观察。
结果如图7所示，重组酵母菌株No.1,No.4和No.6均可表达S蛋白。S蛋白分子量理论值为78KDa，但实际观测结果为135KDa和23KDa两条带。S蛋白有多个糖基化位点，分子量增加考虑糖基化所致。分子量变小推测被蛋白酶切割所致。免疫荧光结果如图8所示，对照组ST1814G未出现荧光(图8上)；而实验组ST1814G-S(RBD-FP)则有明显绿色荧光出现(图8下)，说明S蛋白成功表达且定位在重组酿酒酵母细胞表面。
⑤ST1814G-S(RBD-FP)菌株的生长及蛋白表达规律
取ST1814G-S(RBD-FP)菌株4号转化子单菌落，接种于20mL YPD液体培养基中，间隔24h取样2mL进行OD 600测定，并以最初时间点菌量最低值为标准，调整菌体量一 致，制备不同时间点的蛋白样品，进行Western blot检测。
结果如图9所示，从9-a图生长曲线分析，S(RBD-FP)蛋白截短体的表达对菌体的生长没有影响，菌体生长趋势与对照组一致，随培养时间的延长菌体量逐渐增加。同时，蛋白的表达量也随培养时间的延长而增加，培养5d时表达量最高(图9-b)。
实施例3.新型冠状病毒S蛋白重组酵母的制备
以ST1814G-S(RBD-FP)No.4号菌株为种子，取单菌落接种20mL YPD液体培养基，30℃培养过夜，再取过夜培养的种子液以1:100稀释接种至新鲜的1L YPD液体培养基中，培养3-4d后离心收集菌体，PBS清洗一次，经抽真空冷冻干燥后制得重组菌株干粉。
实施例4.新型冠状病毒S蛋白酵母重组菌株的应用
SPF级BALB/c小鼠分为三组，每组6只，分别于第1、6、11和23天喂饲ST1814G-S(RBD-FP)No.4酵母菌株，10 7cfu/只小鼠，以ST1814G空白酵母菌株为对照。第四次免疫后，眼球采血检测IgG和IgA抗体水平。
应用试验设计原核表达SARS-COV2S蛋白RBD结构域，获得纯化蛋白，抗体制备，应用纯化的RBD蛋白，羊抗小鼠IgG、IgA-HRP酶结合物，间接ELISA检测IgG和IgA水平。
结果如图10所示，喂食ST1814G-S(RBD-FP)No.4重组酵母组的血清中S蛋白特异性的IgG(图10A)和IgA(图10B)的水平明显升高，表明重组酿酒酵母菌株可以很好的激发机体的体液免疫和粘膜免疫，可作为新型免疫防护制剂，为新型冠状病毒防控提供选择。
尽管本发明内容是结合上述实施例进行说明，但本发明的实施方式并不受上述实施例的限制,本发明的范围由所附权利要求书限定，其他的任何在限定范围内所作的改变或修饰，均应为等效的置换方式，均包含在本发明的保护范围之内。
References:
[1].Meyerholz,D.K.,A.M.Lambertz and P.B.McCray,Dipeptidyl Peptidase 4 Distribution in the Human Respiratory Tract.The American Journal of Pathology[J].2016.186(1):p.78-86.
[2].Zhou,P.,et al.,A pneumonia outbreak associated with a new coronavirus of probable bat origin[J].Nature,2020.
[3].Ge,X.,et al.,Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor[J].Nature,2013.503(7477):p.535-538.
[4].Tian,X.,et al.,Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody[J].Emerging Microbes&Infections,2020.9(1):p.382-385.
[5].Chen,W.and G.Georgiou,Cell‐Surface display of heterologous proteins:From high‐throughput screening to environmental applications[J].Biotechnology and Bioengineering,2002.79(5):p.496-503.
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[8].Yan,R.,et al.,Structural basis for the recognition of the SARS-CoV-2by full-length human ACE2.Science(New York,N.Y.),2020:p.eabb2762.
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[10].
Figure PCTCN2021088592-appb-000005
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Claims (7)

  1. 一种表达新型冠状病毒S蛋白的重组酵母，ST1814G-S(RBD-FP)，其包含S蛋白的16位至1035位氨基酸。
  2. 一种新型冠状病毒S蛋白的截断体，优选为第300至1000位氨基酸，序列特征为SEQ ID No.1，为包含第330位至521的RBD结构域和第816至833位FP融合肽。
  3. 表达权利要求2所述蛋白截断体的基因，其特征在于，包含刺突蛋白S的RBD结构域，即从第991位至1563位碱基和FP结构域，即从第2449位至2499位碱基，其核苷酸序列为SEQ ID No.2。
  4. 构建权利要求1所述重组酵母的质粒GPD-S(RBD-FP)-TU，序列特征为SEQ ID No.3，由权利要求3所述基因片段和POT-GPD-TU载体组成。
  5. 制备新型冠状病毒S蛋白重组酵母的方法，其特征在于，将体外构建的S蛋白截断体完整转录单位GPD-S(RBD-FP)-TU，通过同源重组整合于酵母基因组，利用Aga1-Aga2表面展示系统将S蛋白展示于酵母细胞表面，获得S蛋白表面展示型的重组酵母菌株ST1814G-S(RBD-FP)，并利用所得菌株制备口服重组酵母。
  6. 根据权利要求5所述制备新型冠状病毒S蛋白重组酵母的方法，其特征在于，具体包括以下步骤：
    (1)SARS-CoV-2刺突蛋白S编码基因的PCR扩增：参考SARS-CoV-2病毒基因序列NC_045512.2，合成S基因，序列特征为SEQ No.2；以pcDNA3.1-CoV-S质粒为模板，设计引物扩增S蛋白编码基因S(RBD-FP)用于酵母载体连接；
    (2)Aga2基因与SARS-CoV-2刺突蛋白S编码序列S(RBD-FP)串联：通过BamHI单酶切线性化POT-GPD-TU载体，无缝克隆连接S基因片段至表面展示表达载体GPD-POT-TU上，获得重组质粒GPD-S(RBD-FP)-TU，其序列特征为SEQ No.3.重组质粒转化至E.coli DH5a中，用S基因检测引物进行PCR及测序验证，获得阳性克隆；
    (3)S蛋白酵母重组菌株的构建：将重组质粒GPD-S(RBD-FP)-TU与同源臂URRs、筛选标签Leu编码序列进行酶切拼接，获得完整的包含S基因序列的重组基因，转化到酿酒酵母基因组，经营养缺陷型平板筛选后获得重组菌株，利用检测引物进行基因水平检测，Western blot、免疫荧光进行蛋白表达水平验证。
  7. 新型冠状病毒S蛋白的重组酵母在制备用于抗新型冠状病毒药物中的应用。
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