WO2023155236A1 - 一种EBNA3A截短mRNA相关疫苗及其制备方法和应用 - Google Patents

一种EBNA3A截短mRNA相关疫苗及其制备方法和应用 Download PDF

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WO2023155236A1
WO2023155236A1 PCT/CN2022/078158 CN2022078158W WO2023155236A1 WO 2023155236 A1 WO2023155236 A1 WO 2023155236A1 CN 2022078158 W CN2022078158 W CN 2022078158W WO 2023155236 A1 WO2023155236 A1 WO 2023155236A1
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mrna
seq
vaccine composition
amino acid
acid sequence
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曾木圣
赵舸昕
孔祥炜
卜国龙
李紫倩
冯国开
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中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所）
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  • the invention belongs to the fields of biotechnology and medicine, and in particular relates to an EBNA3A truncated mRNA-related vaccine and its preparation method and application.
  • EBV is the first discovered human tumor virus, which mainly infects B cells and epithelial cells, and is currently found to have the strongest host cell transformation ability of infectious disease pathogens, resulting in about 2% of human malignant tumors. EBV infects more than 90% of the world's population. Since its discovery in Burkitt lymphoma 57 years ago, EBV has been found to be closely related to the etiology of various lymphoid and epithelial cancers, such as nasopharyngeal carcinoma (NPC), gastric cancer (GC), Burkitt lymphoma ( BL), Hodgkin's lymphoma (HL) and post-transplantation lymphoproliferative disease (PTLD).
  • NPC nasopharyngeal carcinoma
  • GC gastric cancer
  • BL Burkitt lymphoma
  • HL Hodgkin's lymphoma
  • PTLD post-transplantation lymphoproliferative disease
  • EBV is a major cause of infectious mononucleosis and may play a role in the pathogenesis of several autoimmune diseases, including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis and Sjogren's syndrome.
  • Nasopharyngeal carcinoma is a squamous cell epithelial neoplasm originating from the lateral wall of the nasopharynx, with more than 100,000 new cases worldwide each year, about 80% of which occur in East and Southeast Asia.
  • NPC is divided into two histological variants, squamous cell carcinoma (SCC) and undifferentiated carcinoma of the nasopharyngeal type (UCNT).
  • SCC squamous cell carcinoma
  • UCNT undifferentiated carcinoma of the nasopharyngeal type
  • LEL lymphoepithelioid
  • EBV lymphocytic infiltration
  • EBV-associated gastric cancer (approximately 10% of all gastric cancers) is also EBV positive and has similar pathological changes in LEL, with about 83,000 new cases per year.
  • the purpose of the present invention is to provide a therapeutic vaccine for EBV-related tumors based on EBN3A.
  • the first aspect of the present invention provides a kind of mRNA, the nucleotide sequence of described mRNA is as shown in SEQ ID NO.8 or SEQ ID NO.14.
  • the second aspect of the present invention provides a protein, the amino acid sequence of the protein is a) or b):
  • amino acid sequence shown in a) has been modified by one or more amino acid substitutions, deletions or additions and has the same or similar functions;
  • the third aspect of the present invention provides a DNA molecule, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO.7 or SEQ ID NO.13.
  • the fourth aspect of the present invention provides an expression vector containing the DNA molecule described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a recombinant cell containing the expression vector described in the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides an mRNA vaccine composition, which includes the mRNA described in the first aspect of the present invention, the protein described in the second aspect of the present invention, the DNA described in the third aspect of the present invention, the The expression vector of the fourth aspect or the recombinant cell of the fifth aspect of the present invention.
  • the vaccine composition further includes a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • the carrier is a liposome.
  • the carrier is a cationic liposome, including cationic lipids and helper lipids.
  • the cationic lipid comprises at least one of DOTAP, DOTMA, DOEPC, DC-Chol, DDAB, DODMA and DLinDMA.
  • the helper lipid is a neutral helper lipid.
  • the neutral helper lipid includes at least one of DSPC, DOPE, DOPC, DOPG, DOPS and cholesterol.
  • the liposome is: DOTMA:DOPA; the molar ratio is 1:10-10:1.
  • the preparation method of the vaccine composition is: mixing mRNA with liposomes, wherein the charge ratio of mRNA to liposomes is: 5:1-1:10; The cationic lipid mass ratio is 27:1 ⁇ 1:18.
  • the vaccine composition is provided in a physiologically administrable form and is suitable for oral, intramuscular, intravenous, subcutaneous or dermal injection applications.
  • the seventh aspect of the present invention provides the application of biological materials in the preparation of products, and the biological materials are any one of (a1) to (a6):
  • the function of the product is any one of (b1) to (b6):
  • the product is a medicament.
  • the drug is preferably a vaccine.
  • the invention provides an EBNA3A truncated mRNA and related mRNA vaccines prepared based on the truncated mRNA.
  • the applicant analyzed and compared the expression intensity of different truncated sequences of EBNA3A, and found that the expression of truncated body C is the strongest; and compared with the full length of EBNA3A, truncated body C has a better immune effect; and prepared mRNA based on truncated body C Vaccines have been found to significantly inhibit tumor development, improve survival time, antibody responses, and immune responses, and are also relatively safe. It shows that mRNA vaccine can provide good protection to mice, and can be used to develop therapeutic vaccines for EBV-related tumors.
  • Figure 1 shows the expression of different antigen truncations in 293T cells.
  • Figure 2 is the PCS particle size data of 3A-RNP.
  • Figure 3 shows the Zeta potential of 3A-RNP.
  • Figure 4 is a schematic diagram of the design of the backbone sequence of the mRNA vaccine.
  • Figure 5 is an enzyme-linked immunospot test (ELISPOT) comparison of the effect of the full-length mRNA vaccine encoding truncated C and EBNA3A in stimulating the mouse cellular immune response.
  • ELISPOT enzyme-linked immunospot test
  • Figure 6 is the antigen expression detection of B16-luc stable strain expressing the full length of EBNA3A.
  • Figure 7 is a schematic diagram of the experimental design of tumor formation in mice.
  • Figure 8 is the statistics of tumor development in mice injected with 3A-RNP.
  • Figure 9 is the detection of tumor development in mice injected with 3A-RNP.
  • Figure 10 is the survival time statistics of mice injected with 3A-RNP.
  • Figure 11 is the antibody response of mice injected with 3A-RNP.
  • Figure 12 shows the cellular immune response of mice injected with 3A-RNP.
  • Figure 13 is the safety assessment of mice injected with 3A-RNP.
  • EBV M81 GenBank: AFY97830.1
  • this example designed the coding region containing the EBNA3A antigen-enriched fragment, and mutated the nuclear sequence (K378A, K397A, R379A, R398A).
  • T cell epitopes are considered to be more enriched mainly in 250-600aa
  • the applicant analyzed and compared the expression intensities of different truncated sequences, and selected the fragment with the strongest expression for the next step Immunological control experiment.
  • RNA sequence is shown in SEQ ID NO.8; the amino acid sequence is shown in SEQ ID NO.9), and the c-terminus all have Flag as a label for detection (the DNA sequence is shown in SEQ ID NO.10; The RNA sequence is shown in SEQ ID NO.11; the amino acid sequence is shown in SEQ ID NO.12).
  • the target antigen production detection method (Western-Blot) is as follows:
  • the pcaggs plasmid encoding the antigen can be transfected when the cell confluency reaches more than 60%.
  • Primary antibody incubation add primary antibody (anti-Flag, sigma or anti-beta-actin, CST) diluted 1:1000 with 5% BSA, and incubate overnight at 4°C with slow shaking. After removing the primary antibody, add an appropriate amount of PBST (0.1% tween20) and wash for 10 minutes on a side-swing shaker; wash 3 times in total.
  • primary antibody anti-Flag, sigma or anti-beta-actin, CST
  • Secondary antibody incubation add secondary antibody (anti-mouse, CST, or anti-rabbit, CST) diluted 1:3000 with 5% BSA, and incubate overnight at 4°C with slow shaking. After removing the primary antibody, add an appropriate amount of PBST (0.1% tween20) and wash for 10 minutes on a side-swing shaker; wash 3 times in total.
  • secondary antibody anti-mouse, CST, or anti-rabbit, CST
  • the mRNA of '-UTR element, 3-UTR element and polyA tail element has a DNA sequence as shown in SEQ ID NO.13, an RNA sequence as shown in SEQ ID NO.14, and an amino acid sequence as shown in SEQ ID NO.15; And prepare the mRNA liposome mixture, and encode the mRNA (RNA sequence such as SEQ ID NO.16 institute of SEQ ID NO.
  • the liposome preparation method is: DOTMA and DOPE are dissolved in absolute ethanol (100mg DOTMA and 40.92mg DOPE are dissolved in 680 ⁇ l absolute ethanol) with 2: 1 molar ratio, then the ethanol solution is slowly added dropwise to 34ml distilled water , stirred continuously at 200rpm for 1 hour, then filtered through a 0.45 ⁇ m filter membrane, and stored at 4°C.
  • RNA-liposome complex The preparation method of the RNA-liposome complex is as follows: first dilute the mRNA to 200 ⁇ l with PBS, add the liposome suspension, mix well and let stand for 10 minutes. RNA and liposome charge ratio is 2:1.3 (per 40 ⁇ g mRNA solution adds 17.6 ⁇ l above-mentioned liposome suspension) the particle zeta potential of the RNA-liposome complex of formation is about-30mV (negatively charged), size is About 300-400nm.
  • mice were immunized with truncated C and EBNA3A full-length mRNA vaccines by tail vein injection (40 ⁇ g each time, administered 4 times on days 3, 6, 10, and 15 respectively), and 7 days after the last injection The spleen was taken to compare the immune response.
  • the enzyme-linked immunospot test (ELISPOT) detection method is as follows:
  • Lysis red the splenocyte sample of each mouse was lysed with 2ml erythrocyte lysate (biosharp) for 2 minutes, then the reaction was terminated with 10ml PBS, centrifuged, resuspended with an appropriate amount of 1640 containing 10% FBS, and the cells were counted.
  • the applicant established a stable B16-luc monoclonal cell line expressing the full length of EBNA3A, Then, a tumor-bearing mouse model was established by injecting 2*10 5 tumor cells into the tail vein, and the design diagram of the mouse tumor formation experiment is shown in FIG. 7 . The expression of EBN3A protein in the cell line was detected, and the results are shown in FIG. 6 . It can be seen that it has established a stable strain successfully.
  • 3A-RNP or blank liposome control were injected respectively, and the tumor growth was observed. It can be seen that the tumor development of mice injected with 3A-RNP was significantly inhibited compared with the control group ( Figure 8 & Figure 9); and the survival time of mice injected with 3A-RNP (Figure 10), antibody response (Figure 1 11) and immune response (Figure 12) were significantly better than the control group.
  • mice injected with 3A-RNP was not significantly lower than that of the blank liposome control group, but due to tumor growth, the state of the mice would deteriorate and the body weight would decrease.
  • the 3A-RNP experimental group ( FIG. 13 ) shows that the safety of 3A-RNP is relatively high.
  • the mRNA vaccine prepared by truncated body C has good tumor suppression effect and immune effect.

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Abstract

一种EBNA3A截短mRNA相关疫苗及其制备方法和应用，所述截短mRNA的核苷酸序列如SEQ ID NO.8或SEQ ID NO.14所示；相对于EBNA3A全长，截短体C具有更好的免疫效果；并基于截短体C制备mRNA疫苗，发现其可以显著抑制肿瘤发展，提高生存时间、抗体反应和免疫反应，也具有较高的安全性。表明mRNA疫苗可以给小鼠提供良好的保护性，可以用于开发EBV相关肿瘤的治疗疫苗。

Description

一种EBNA3A截短mRNA相关疫苗及其制备方法和应用 技术领域
本发明属于生物技术和医药领域，具体涉及一种EBNA3A截短mRNA相关疫苗及其制备方法和应用。
背景技术
EBV是第一个发现的人类肿瘤病毒，主要感染B细胞和上皮细胞，是在目前发现具有最强的宿主细胞转化能力的传染病病原体，导致了大约2％人类恶性肿瘤的发生。EBV感染了超过90％的世界人口。自从57年前在伯基特淋巴瘤中发现以来，EBV被发现与多种淋巴癌和上皮癌的病因密切相关，例如鼻咽癌癌(NPC)、胃癌(GC)、伯基特淋巴瘤(BL)、霍奇金淋巴瘤(HL)和移植后淋巴组织增生性疾病(PTLD)等。除此之外，EBV是传染性单核细胞增多症的主要原因，并且在几种自身免疫性疾病的发病中可能发挥作用，其中包括多发性硬化症、系统性红斑狼疮、类风湿性关节炎和干燥综合征等。
鼻咽癌(NPC)是一种鳞状细胞上皮肿瘤，起源于鼻咽侧壁，全球每年新增病例超过100,000例，其中约80％发生在东亚和东南亚。NPC分为两种组织学变体，即鳞状细胞癌(SCC)和鼻咽型未分化癌(UCNT)。在NPC流行地区，超过95％为未分化癌，表现出淋巴上皮样(LEL)外观和显著的淋巴细胞浸润，其中(EBV)感染被认为是NPC发育所必需和最重要的病原学因素。无论地理位置和发病率如何，所有未分化NPC病例都与EBV相关，并且EBV基因组存在于每个恶性细胞中。此外。EBV相关型胃癌(大约占胃癌总数的10％)也是EBV阳性，并具有相似的LEL病理变化，每年约有83,000例新病例。
近几十年来，由于流行地区生活水平和医疗水平的提高，特别是临床使用个体化化疗和调强放疗(IMRT)，鼻咽癌的发病率和死亡率均显着下降极大地提高了鼻咽癌患者的治愈率。然而，NPC治疗中的局部复发和远处转移依旧是较大挑战，接受放化疗的鼻咽癌患者生活质量较差，并伴有骨抑制等严重副作用。而免疫治疗的毒副作用低，前景广阔，成为了改善鼻咽癌患者及其他EBV相关肿瘤患者预后的重要治疗策略。
随着对疫苗技术的深入研究和对EBV免疫学的日益了解，已经开发出越来越多的EBV候选疫苗。在过去20年间，有多种针对EBV及EBV相关肿瘤的预防或治疗性疫苗被设计和研究。但是，到目前为止，均没有成功上市的有效治疗性疫苗能够被应用于患者。
发明内容
本发明的目的在于提供一种基于EBN3A的EBV相关肿瘤的治疗性疫苗。
本发明所采取的技术方案是：
本发明的第一方面，提供一种mRNA，所述mRNA的核苷酸序列如SEQ ID NO.8或SEQ ID NO.14所示。
本发明的第二方面，提供一种蛋白质，所述蛋白质的氨基酸序列为a)或b)：
a)如SEQ ID NO.9或SEQ ID NO.15所示；
b)a)所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列；
c)与a)或b)所限定的氨基酸序列具有99％以上、95％以上或90％以上同源性且具有相同功能的氨基酸序列。
本发明的第三方面，提供一种DNA分子，所述DNA分子的核苷酸序列如SEQ ID NO.7或SEQ ID NO.13所示。
本发明的第四方面，提供一种表达载体，含有本发明第三方面所述的DNA分子。
本发明的第五方面，提供一种重组细胞，含有本发明第四方面所述的表达载体。
本发明的第六方面，提供一种mRNA疫苗组合物，所述疫苗组合物包括本发明第一方面所述mRNA、本发明第二方面所述蛋白质、本发明第三方面所述DNA、本发明第四方面所述表达载体或本发明第五方面所述重组细胞。
在本发明的一些实施方式中，所述疫苗组合物还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
在本发明的一些实施方式中，所述载体为脂质体。
在本发明的一些实施方式中，所述载体为阳离子脂质体，包括阳离子脂质和辅助脂质。
在本发明的一些实施方式中，所述阳离子脂质包括DOTAP、DOTMA、DOEPC、DC-Chol、DDAB、DODMA和DLinDMA中的至少一种。
在本发明的一些实施方式中，所述辅助脂质为中性辅助脂质。
所述中性辅助脂质包括DSPC、DOPE、DOPC、DOPG、DOPS和胆固醇中的至少一种。
在本发明的一些优选实施方式中，所述脂质体为：DOTMA:DOPA；摩尔比为1:10～10:1。
在本发明的一些优选实施方式中，所述脂质体为：DOTMA:DOPA＝2:1。
在本发明的一些实施方式中，所述疫苗组合物的制备方法为：将mRNA与脂质体混合，其中mRNA与脂质体的电荷比为：5:1～1:10；所述mRNA与阳离子脂质质量比为27:1～1:18。
在本发明的一些实施方式中，所述疫苗组合物以生理可给药的形式提供，并且适合于口 服、肌内、静脉内、皮下或皮肤注射应用。
本发明的第七方面，提供生物材料在制备产品中的应用，所述生物材料为(a1)～(a6)中的任一种：
(a1)本发明第一方面所述mRNA；
(a2)本发明第二方面所述蛋白质；
(a3)本发明第三方面所述DNA；
(a4)本发明第四方面所述表达载体；
(a5)本发明第五方面所述重组细胞；
(a6)本发明第六方面所述mRNA疫苗组合物；
所述产品的功能为(b1)～(b6)中的任一种：
(b1)预防或治疗EB病毒感染相关疾病；
(b2)预防或治疗EB病毒感染相关肿瘤；
(b3)抑制肿瘤生长；
(b4)提高生产时间；
(b5)引起免疫反应；
(b6)制备EB病毒抗体。
在本发明的一些实施方式中，所述产品为药物。
在本发明的一些实施方式中，所述药物优选为疫苗。
本发明的有益效果是：
本发明提供一种EBNA3A截短mRNA，以及基于截短mRNA制备的相关mRNA疫苗。首先申请人分析比较了EBNA3A不同截短序列的表达强度，发现截短体C表达最强；而且相对于EBNA3A全长，截短体C具有更好的免疫效果；并基于截短体C制备mRNA疫苗，发现其可以显著抑制肿瘤发展，提高生存时间、抗体反应和免疫反应，也具有较高的安全性。表明mRNA疫苗可以给小鼠提供良好的保护性，可以用于开发EBV相关肿瘤的治疗疫苗。
附图说明
图1为不同抗原截短体在293T细胞表达情况。
图2为3A-RNP的PCS粒径数据。
图3为3A-RNP的Zeta电位数。
图4为mRNA疫苗骨架序列设计示意图。
图5为酶联免疫斑点试验(ELISPOT)比较编码截短体C与EBNA3A全长mRNA疫苗激 发小鼠细胞免疫反应效果。
图6为表达EBNA3A全长的B16-luc稳株抗原表达检测。
图7为小鼠成瘤实验设计示意图。
图8为注射3A-RNP的小鼠的肿瘤发展情况统计。
图9为注射3A-RNP的小鼠的肿瘤发展情况检测。
图10为注射3A-RNP的小鼠的生存时间统计。
图11为注射3A-RNP的小鼠的抗体反应。
图12为注射3A-RNP的小鼠的细胞免疫反应。
图13为注射3A-RNP的小鼠的安全性评估。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例1 抗原截短体的选择与表达
根据华南地区高发毒株EBV M81(GenBank:AFY97830.1)的EBNA3A序列，本实施例设计了包含EBNA3A抗原富集片段的编码区，并突变了入核序列(K378A，K397A,R379A，R398A)。
由于在EBNA3A中，T细胞表位被认为主要在250～600aa中有更多的富集，因此申请人分析比较了不同截短序列的表达强度，并选择了其中表达最强的片段进行下一步免疫学对照实验。其中A：EBNA3A(280-567aa，DNA序列如SEQ ID NO.1所示；RNA序列如SEQ ID NO.2所示；氨基酸序列如SEQ ID NO.3所示)；B：EBNA3A(280-510aa，DNA序列如SEQ ID NO.4所示；RNA序列如SEQ ID NO.5所示；氨基酸序列如SEQ ID NO.6所示)；C：EBNA3A(320-567aa，DNA序列如SEQ ID NO.7所示；RNA序列如SEQ ID NO.8所示；氨基酸序列如SEQ ID NO.9所示)，c端均带Flag为用于检测的标签(DNA序列如SEQ ID NO.10所示；RNA序列如SEQ ID NO.11所示；氨基酸序列如SEQ ID NO.12所示)。
目标抗原产生检测方法(Western-Blot)如下：
1)将培养24小时以上的293T细胞消化并种入12孔板中，将细胞密度控制在150000个每孔；
2)将六孔板于37℃，5％CO 2孵育16-20小时后利用显微镜观察细胞状态。细胞汇合度 达到60％以上即可进行编码抗原的pcaggs质粒转染。
3)利用PET转染相应质粒到293细胞中(每孔转染1μg的质粒，PEI：质粒质量比为3:1)，并于37℃，5％CO 2条件下继续培养24小时。
4)待质粒持续表达24小时后，此时将细胞上清液除去，加入100μl/孔RIPA细胞裂解液，冰上反应10min后转移至1.5mlEP管。
5)加入25μl 5X SDS loading buffer(Genstar)，混匀后95度变性5min后准备跑胶。
6)蛋白样品冷却到室温后，直接上样到10.5％SDS-PAGE十孔胶加样孔内，每孔上样量为35μl，80V恒压电泳跑120min，然后进行转膜。
7)先将PVDF膜(0.22μm,5.5*8.5cm)用无水甲醇进行活化(1min)，然后浸入转膜液中，使用Bio-Rad的标准湿式转膜装置，转膜电流为250mA，转膜时间为120分钟。
8)转膜完毕后，立即把蛋白膜放置到预先加入PBST(0.1％tween20)的Western洗膜盒中，漂洗1-2分钟，以洗去膜上的转膜液。加入适量的5％脱脂牛奶，在摇床上缓慢摇动，室温封闭45分钟。
9)一抗孵育：加入1:1000用5％BSA稀释好的一抗(anti-Flag,sigma或者anti-beta-actin，CST)，4℃缓慢摇动孵育过夜。去除一抗后加入适量PBST(0.1％tween20)在侧摆摇床上洗涤10分钟；共洗涤3次。
10)二抗孵育：加入1:3000用5％BSA稀释好的二抗(anti-mouse,CST，或者anti-rabbit，CST)，4℃缓慢摇动孵育过夜。去除一抗后加入适量PBST(0.1％tween20)在侧摆摇床上洗涤10分钟；共洗涤3次。
11)蛋白检测：将显色液A，B液混合，将上述PVDF膜浸入显色液内，1分钟后放入化学发光仪(Biorad)内进行成像；结果见图1，其中截短体C(EBNA3A(320-567aa))是表达最强，并富集了较多的T细胞表位的抗原序列。
实施例2 细胞免疫反应的检测
比较体外表达效果最好的截短体C(EBNA3A(320-567aa))是否能在小鼠体内激发起更有效的细胞免疫反应，申请人制备编码融合截短体C、MHCI跨膜区以及5'-UTR元件、3-UTR元件、polyA尾部元件的mRNA，其DNA序列如SEQ ID NO.13所示，RNA序列如SEQ ID NO.14所示，氨基酸序列如SEQ ID NO.15所示；并制备mRNA脂质体混合物，和编码融合EBNA3A全长和MHCI跨膜区、以及5'-UTR元件、3-UTR元件、polyA尾部元件和信号肽的mRNA(RNA序列如SEQ ID NO.16所示，氨基酸序列如SEQ ID NO.17所示)；并制备mRNA脂质体混合物(DOTMA:DOPA＝2:1)，其中mRNA脂质体混合物的PCS粒径数据见图2；可以 看出包装前粒径约为180nm；截短体C或EBNA3A全长包装后粒径约为350nm；mRNA脂质体混合物的Zeta电位数见图3。
其中脂质体制备方法为：将DOTMA和DOPE以2:1摩尔比溶解于无水乙醇中(100mg DOTMA和40.92mg DOPE溶解于680μl无水乙醇中)，然后将乙醇溶液缓慢滴加于34ml蒸馏水中，以200rpm的转速不断搅拌1小时后通过0.45μm滤膜过滤，4度存放。
RNA-脂质体复合物制备方法为：先将mRNA用PBS稀释至200μl，加入脂质体悬浮液，混匀静置10分钟。RNA与脂质体电荷比为2:1.3(每40μg mRNA溶液添加17.6μl上述脂质体悬浮液)形成的RNA-脂质体复合物的颗粒zeta电位为-30mV左右(带负电)，大小为300-400nm左右。
其中mRNA疫苗骨架序列设计示意图见图4。
通过尾静脉注射的方式使用截短体C与EBNA3A全长mRNA疫苗免疫C57小鼠(每次40μg，分别在3，6，10，15天共4次给药)，并在最后一针7天后取脾对比免疫反应。
酶联免疫斑点试验(ELISPOT)检测方法如下：
1)最后一次免疫7天后取实验组和对照组的小鼠脾脏，研磨后收集细胞悬液，离心后用1640(sigma)培养基洗一遍。
2)裂红：每只小鼠的脾细胞样品用2ml红细胞裂解液(biosharp)裂解2分钟，然后用10mlPBS终止反应，离心，用适量含10％FBS的1640重悬后进行细胞计数。
3)将无菌DPBS(sigma)加入预包被IFN-r的ELISPOT板(Mabtech)，200μl/孔，拍净，重复4次。然后每孔加入200μl含10％FBS的1640室温封闭30分钟，拍净。
4)每孔加入2*10 5个脾细胞，其中EBNA3A多肽组加入EBNA3A多肽(终浓度20μg/ml)，空白对照组只用含10％FBS的1640，阳性对照组加入PMA(终浓度50ng/ml)和Ionomycin(终浓度1μg/ml)，在37摄氏度，5％CO 2条件下培养16-20小时后进行斑点检测。
5)弃掉细胞内容物，按照Mabtech试剂盒厂家说明书的步骤检测斑点。
结果如图5所述，可以看出截短体C的免疫效果显著优于EBNA3A全长。
实施例3 小鼠成瘤实验效果检测
为了检测编码融合截短体C和MHCI跨膜区的mRNA疫苗(简称3A-RNP)是否具有治疗/缓解肿瘤发生的作用，申请人建立了表达EBNA3A全长的B16-luc单克隆细胞稳株，然后通过尾静脉注射2*10 5个肿瘤细胞建立荷瘤小鼠模型，其中小鼠成瘤实验设计示意图见图7。对细胞株中的EBN3A蛋白表达情况进行检测，结果见图6。可以看到其建立稳株成功。
然后在第3，6，10，15天分别注射3A-RNP或者空白脂质体对照，并观察肿瘤生长情况。 可以看到注射了3A-RNP的小鼠的肿瘤发展相比于对照组明显受到抑制(图8&图9)；而且注射了3A-RNP的小鼠的生存时间(图10)、抗体反应(图11)和免疫反应(图12)明显优于对照组。
而且检测到注射了3A-RNP的小鼠体重相比于注射空白脂质体对照没有明显降低，但由于肿瘤生长会导致小鼠状态变差，体重降低，后期空白对照组的小鼠体重低于3A-RNP实验组(图13)，说明了3A-RNP的安全性较高。
综上所述可以看出，通过截短体C制备的mRNA疫苗具有很好的肿瘤抑制效果和免疫效果。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 一种mRNA，其特征在于，所述mRNA的核苷酸序列如SEQ ID NO.8或SEQ ID NO.14所示。
  2. 一种蛋白质，所述蛋白质的氨基酸序列为a)或b)：
    a)如SEQ ID NO.9或SEQ ID NO.15所示；
    b)a)所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列；
    c)与a)或b)所限定的氨基酸序列具有99％以上、95％以上或者90％以上同源性且具有相同功能的氨基酸序列。
  3. 一种DNA分子，所述DNA分子的核苷酸序列如SEQ ID NO.7或SEQ ID NO.13所示。
  4. 一种表达载体，含有权利要求3所述DNA分子。
  5. 一种重组细胞，含有权利要求4所述表达载体。
  6. 一种mRNA疫苗组合物，所述mRNA疫苗组合物包括权利要求1所述mRNA、权利要求2所述蛋白质、权利要求3所述DNA、权利要求4所述表达载体或权利要求5所述重组细胞。
  7. 根据权利要求6所述的mRNA疫苗组合物，其特征在于，所述疫苗组合物还包括药学上可接受的佐剂、载体、稀释剂或赋形剂，优选地，所述载体为脂质体。
  8. 根据权利要求6所述的mRNA疫苗组合物，其特征在于，所述疫苗组合物以生理可给药的形式提供，并且适合于口服、肌内、静脉内、皮下或皮肤注射应用。
  9. 生物材料在制备产品中的应用，所述生物材料为(a1)～(a6)中的任一种；
    (a1)权利要求1所述mRNA；
    (a2)权利要求2所述蛋白质；
    (a3)权利要求3所述DNA；
    (a4)权利要求4所述表达载体；
    (a5)权利要求5所述重组细胞；
    (a6)权利要求6～8任一项所述mRNA疫苗组合物；
    所述产品的功能为(b1)～(b6)中的任一种：
    (b1)预防或治疗EB病毒感染相关疾病；
    (b2)预防或治疗肿瘤；
    (b3)抑制肿瘤生长；
    (b4)提高生产时间；
    (b5)引起免疫反应；
    (b6)制备EB病毒抗体。
  10. 根据权利要求9所述的应用，其特征在于，所述产品为药物；所述药物优选为疫苗。
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