WO2007104263A1 - 一种乙型肝炎病毒疫苗增效蛋白及其基因 - Google Patents

一种乙型肝炎病毒疫苗增效蛋白及其基因 Download PDF

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WO2007104263A1
WO2007104263A1 PCT/CN2007/000844 CN2007000844W WO2007104263A1 WO 2007104263 A1 WO2007104263 A1 WO 2007104263A1 CN 2007000844 W CN2007000844 W CN 2007000844W WO 2007104263 A1 WO2007104263 A1 WO 2007104263A1
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hepatitis
protein
vaccine
cdna
virus
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PCT/CN2007/000844
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WO2007104263A8 (zh
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Naishuo Zhu
Huaqing Li
Min Yang
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Fudan University
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Priority to JP2008558624A priority Critical patent/JP5097135B2/ja
Priority to US12/282,754 priority patent/US8895237B2/en
Publication of WO2007104263A1 publication Critical patent/WO2007104263A1/zh
Publication of WO2007104263A8 publication Critical patent/WO2007104263A8/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to a hepatitis B virus vaccine synergistic protein and a gene thereof, and a method and an application for expressing the protein by using genetic engineering principles. Background technique
  • Hepatitis B is a relatively serious infectious disease that threatens human health. It has a high incidence and great harm in China. Hepatitis B vaccination is the most effective measure to prevent hepatitis B infection and reduce the carrier rate of hepatitis B virus. However, the immune effect of hepatitis B vaccine varies greatly among individuals in the population. After routine injection of three needles, many people do not produce enough antibodies to resist hepatitis B virus infection and hepatitis B. Therefore, looking for a way to effectively enhance the immunogenicity of hepatitis B vaccine
  • prokaryotic such as E. coli
  • eukaryotic such as yeast, mammalian cells, etc.
  • the use of prokaryotic (such as E. coli) expression systems or eukaryotic (such as yeast, mammalian cells, etc.) expression systems to clone and express a protein is the most efficient way to obtain large quantities of this protein.
  • the protein encoded by the gene can be obtained, and the animal is immunized together with the hepatitis B vaccine, and the antibody against the hepatitis B surface antigen in the animal is determined by ELISA. Potency, it is convenient to observe whether the protein can enhance the immune effect of hepatitis B vaccine, and thus whether the protein has potential application value in the field of hepatitis B vaccine. Disclosure of invention
  • the object of the present invention is to provide a synergistic protein of hepatitis B virus vaccine and a gene encoding the same, and provide a method and application for expressing the same
  • the synergistic protein of the hepatitis B virus vaccine provided by the present invention and the gene encoding the same have the gene sequence and amino acid sequence of SEQ.ID.NO.1 and SEQ.ID.N0.2, respectively.
  • the present invention screens the above protein gene from a human liver cDNA expression library.
  • the protein is then expressed using genetic engineering techniques. Specific steps are as follows: '
  • the cDNA encoding the protein and the protein were obtained:
  • the hepatitis B virus surface antigen was used as a screening molecule, and the human liver cDNA expression library was screened by imprinting immunoscreening to obtain a positive cDNA clone.
  • the cDNA was sequenced and analyzed. The results showed that the cDNA had an independent open reading frame (OKF) and the entire ORF size was 1035 bp. Further experiments have shown that the protein encoded by it does have specificity for hepatitis B surface antigen.
  • the N-terminus of the cDNA was introduced into the 6xhis purification tag and the enterokinase cleavage site DDDDK, and cloned into the prokaryotic expression vector of pBV220, and subjected to temperature-induced expression in E. coli, and the E. coli was collected and lysed, and the inclusion bodies were extracted. After purification by affinity chromatography with Ni-NTA agarose, the target protein was finally obtained by dialysis and renaturation.
  • mice were immunized with hepatitis B vaccine according to a conventional method, subcutaneously injected, and boosted once every 10 and 17 days of the initial immunization, and a total of three immunizations were performed.
  • the test group was injected subcutaneously every 3 days, and the control group was not injected.
  • Blood was taken from the tail vein of the mouse on the 20th day after the initial immunization, and serum was separated.
  • the titer of anti-hepatitis B surface antigen antibody in mice was determined by ELISA. SPSS software analyzed the differences between the experimental group and the control group. It was found that the protein can significantly enhance the antibody titer in mice, that is, the protein can enhance the immune effect of the hepatitis B vaccine.
  • the use of the protein can enhance the immunogenicity of the hepatitis B virus vaccine and increase the anti-hepatitis B surface antigen antibody titer.
  • the protein in the field of hepatitis B vaccine, on the one hand, can be combined with hepatitis B vaccine, which can enhance the immune effect of hepatitis B vaccine, and can reduce the dose and frequency of hepatitis B vaccine injection; on the other hand, the specificity of the protein on hepatitis B surface antigen and It can promote the anti-hepatitis B surface antigen antibody production in vivo, and can be used in the field of hepatitis B virus carrier and hepatitis B patient therapeutic synergist and therapeutic hepatitis B vaccine development research.
  • Figure 2 Effect of the protein on anti-HBsAg antibody titer in Balb/c mice II, with untreated Balb/c mouse serum as a negative control, set 10 times higher than the negative control OD 495 (0. ⁇ ) The above is a positive value, and the anti-HBsAg antibody titer of each group of mice was calculated by the one-dimensional regression equation of the OD value and the serum dilution multiple.
  • Independent-Sample T test, P ⁇ 0.04 which was statistically significant, so it is considered that this protein can up-regulate the titer of mouse HBsAg antibody. .
  • the best way to implement the invention is the best way to implement the invention
  • the gene for obtaining the protein The whole hepatitis B surface antigen was used as a screening molecule, and the human liver cDNA expression library was screened by imprinting immunoscreening method. After screening a certain number of clones, a positive cDNA clone was finally obtained.
  • the cDNA was sequenced and found to have an independent open reading frame (ORF). The entire O JF size was 1035 bp (SEQ.ID.N0.1), and the encoded amino acid sequence of the protein was SEQ.ID.N0.2. .
  • Genetic engineering to express and purify the protein The gene can be cloned into a eukaryotic or prokaryotic expression vector system for expression and purification.
  • a 6xhis purification tag and a enterokinase cleavage site DDDDK are introduced at the N-terminus of the cDNA, and the 6xhis purification tag facilitates affinity purification of the fusion protein of interest, and the enterokinase cleavage site enables the purified fusion protein in enterokinase. Cut off the 6xhis tag and DDDDK. This intact DNA was cloned into the pBV220 prokaryotic expression vector, transformed into E. coli, and positive transformants were screened.
  • mice 7-week-old Balb/c mice, male and female, were divided into two groups, the experimental and control groups.
  • the mice were immunized with hepatitis B vaccine according to the conventional method, and the immunization protocol was as follows: number of immunizations (subcutaneous injection) interval time test group control group
  • the protein was taken from the tail vein of the mouse for 17 days, and serum was separated.
  • the titer of anti-hepatitis B surface antigen antibody in mice was determined by ELISA: the isolated serum was diluted by dilution, the ELISA plate was coated with hepatitis B surface antigen, and the diluted serum was used as the primary antibody, and the HRP-labeled anti-mouse IgG antibody was used as the primary antibody. Secondary Antibodies. The OD 495 value was measured after color development. The differences between the experimental group and the control group were analyzed by SPSS software.
  • mice immunized with hepatitis B vaccine showed that the titer of anti-hepatitis B surface antigen antibody in the test group was 9.24 ⁇ 10 4 after 20 days of initial immunization, which was significantly higher than the antibody titer of the control group. (2.68X10 4 ), indicating that the protein has the ability to up-regulate antibodies against hepatitis B surface antigen in mice, and can enhance the immune effect of hepatitis B vaccine.
  • the immune effect of traditional hepatitis B vaccine varies greatly among individuals in the population. After routine injection of three needles, many people do not produce enough antibodies to resist hepatitis B virus infection and hepatitis B.
  • the hepatitis B vaccine-enhancing protein involved in the specification can significantly enhance the immune effect of the hepatitis B vaccine, and therefore can be used for the development of hepatitis B vaccine adjuvant.
  • a certain amount of the protein is injected to enhance the immunity of the hepatitis B vaccine.
  • Originality increase the body's anti-hepatitis B surface antigen antibody titer, thereby increasing the immune efficiency of the epidemic, and reducing the amount of hepatitis B infection and the number of injections.
  • the protein has specificity for hepatitis B surface antigen and can promote the production of anti-hepatitis B surface antigen antibodies in vivo. Therefore, for the treatment of hepatitis B virus carriers and hepatitis B patients, the protein can be used as a synergist. In this field, at the same time, the development of therapeutic hepatitis B vaccine has certain application value.

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Description

一种乙型肝炎病毒疫苗增效蛋白及其基因 技术领域
本发明涉及一种乙型肝炎病毒疫苗增效蛋白及其基因， 以及利用基因工程原理表达该 蛋白的方法和应用。 背景技术
乙型肝炎是当前威胁人类健康的一种比较严重的传染病，在我国发病率高、 危害性大。 接种乙肝疫苗是预防感染乙型肝炎和降确低乙肝病毒携带率的最有效的措施。 但是乙肝疫苗 的免疫效果在人群当中的个体差异很大， 常规注射三针以后很多人体内并不能产生足够的 抗体以抵抗乙肝'病毒的感染和乙肝发病。 因此， 寻找能够有效增强乙肝疫苗免疫原性的分 木
子是解决该问题的一个重要途径。
采用基因工程原理， 应用原核 （如大肠杆菌）表达系统或真核（如酵母、 哺乳动物细 胞等）表达系统克隆表达某种蛋白是获取大量该蛋白的最为有效的方法。 从 cDNA表达库 中筛选得到的某个基因经基因工程克隆表达纯化后， 可以获得由该基因编码的蛋白， 将其 与乙肝疫苗一同免疫动物，再用 ELISA方法测定动物体内抗乙肝表面抗原的抗体效价，可 以很方便地观察到该蛋白是否能够增强乙肝疫苗的免疫效果， 从而推断该蛋白在乙肝疫苗 领域是否具有潜在的应用价值。 发明的公开
本发明的目的是提供一种乙型肝炎病毒疫苗的增效蛋白及编码该蛋白的基因， 并提供 表达该蛋白的方法和应用
本发明提供的乙型肝炎病毒疫苗的增效蛋白及编码该蛋白基因， 其基因序列和氨基酸 序列分别为 SEQ.ID.NO.1和 SEQ.ID.N0.2。
本发明从人肝 cDNA表达库中筛选到上述蛋白基因。再利用基因工程技术表达该蛋白。 具体步骤如下： '
编码该蛋白的 cDNA及该蛋白的获得： 以乙型肝炎病毒表面抗原为筛选分子， 利用印 迹免疫筛选的方法对人肝 cDNA表达库进行筛选，得到一株阳性的 cDNA克隆。对该 cDNA 进行测序分析， 结果显示该 cDNA具有独立的开放阅读框架（OKF), 整个 ORF大小为 1035bp。 进一步的实验证明其编码的蛋白确实对乙肝表面抗原有特异性。 将该 cDNA的 N端引入 6xhis purification tag和肠激酶酶切位点 DDDDK, 并克隆到 pBV220原核表达载体中， 在大肠杆菌中进行温度诱导表达， 收集并裂解大肠杆菌， 提取 包涵体，经粗纯后再用 Ni-NTA agarose进行亲和层析纯化，经透析复性最终得到目的蛋白。
该蛋白对乙肝疫苗免疫效果的影响： 将实验动物分为试验组和对照组。 以小鼠为例， 按常规方法用乙肝疫苗免疫小鼠， 皮下注射， 并于初次免疫的第 10天、 17天各加强免疫 一次， 共计免疫三次。 试验组每 3天皮下注射该蛋白， 对照组不注射。 在初次免疫后的第 20天从小鼠尾静脉取血， 分离血清。 用 ELISA方法测定小鼠体内抗乙肝表面抗原抗体的 滴度。 SPSS软件分析试验组和对照组的差异。结果发现该蛋白可以明显增强小鼠体内抗体 的滴度， 即该蛋白可以增强乙肝疫苗的免疫效果。
由此可见， 利用该蛋白可以增强乙型肝炎病毒疫苗的免疫原性、 增加抗乙肝表面抗原 抗体滴度这个特点， 我们可将其作为一种乙肝疫亩的增效分子即佐剂而应用于乙肝疫苗领 域， 一方面可以将该蛋白与乙肝疫苗联用， 既能增强乙肝疫苗的免疫效果， 又可以减少乙 肝疫苗的注射剂量和次数； 另一方面利用该蛋白对乙肝表面抗原的特异性及其可促进体内 抗乙肝表面抗原抗体产生的特性， 可将其用于乙型肝炎病毒携带者和乙肝病人治疗的增效 剂以及治疗型乙肝疫苗的开发研究等领域。 附图的简要说明
图 1 该蛋白对 Balb/c小鼠抗 HBsAg抗体效价的影响 I
图 2该蛋白对 Balb/c小鼠抗 HBsAg抗体效价的影响 II， 以未经处理的同龄 Balb/c小 鼠血清为阴性对照， 设高于阴性对照 OD495值 10倍（0.Π) 以上的为阳性值， 通过 OD值 和血清稀释倍数对数值的一元回归方程的计算各组小鼠的抗 HBsAg抗体效价， 试验组小 鼠 （n=5) 的 HBsAg抗体效价为 9.24><104, 对照组小鼠 （n=5) 的 HBsAg抗体效价为 2.68 104,Independent-Sample T test, P<0.04，有统计学意义， 因此认为该蛋白可以上调小鼠 HBsAg抗体的效价。 实现本发明的最佳方式
获得该蛋白的基因： 用完整的乙肝表面抗原作为筛选分子， 利用印迹免疫筛选的方法 对人肝 cDNA表达库进行筛选， 筛选一定数量的克隆以后， 最终得到一株阳性的 cDNA克 隆。对该 cDNA进行测序分析，发现该 cDNA具有独立的开放阅读框架（ORF)，整个 O JF 大小为 1035bp (SEQ.ID.N0.1 ), 其编码的蛋白氨基酸序列为 SEQ.ID.N0.2。 基因工程表达和纯化该蛋白： 可将该基因克隆到真核或原核表达载体系统进行表达和 纯化。例如，在该 cDNA的 N端引入 6xhis purification tag和肠激酶酶切位点 DDDDK, 6xhis purification tag有利于目的融合蛋白的亲和纯化，肠激酶酶切位点可以使纯化的融合蛋白在 肠激酶的作用下切除掉 6xhis tag和 DDDDK。 将这一完整的 DNA克隆到 pBV220原核表 达载体中， 转化大肠杆菌， 筛选阳性转化子。 挑取阳性单克隆于液体培养基中培养并进行 温度诱导发酵： 首先在 37°C， 250rpm培养转化的大肠杆菌至 OD6(K)=0.5~0.6， 然后迅速升 温至 42Ό开始诱导发酵 5小时。发酵结束后离心收集菌体， 裂解菌体以提取包涵体， 包涵 体粗纯后用 Ni-NTA agarose柱亲和纯化， 经透析复性后最终得到目的蛋白。 用 Western和 ELISA实验验证所获得的蛋白正是所需要的目的蛋白。
该蛋白对乙肝疫苗免疫效果的影响评价： 以小鼠为例， 选取 7周龄 Balb/c小鼠， 雌雄 各半， 分为试验和对照两组。按常规方法用乙肝疫苗免疫小鼠， 皮下注射， 免疫方案如下： 免疫次数（皮下注射） 间隔时间 试验组 对照组
第一次免疫 HBsAg 80/ g , 该蛋白 HBsAg
80/xg
3天 该蛋白 6 g
6天 该蛋白 9.5/xg
第二次免疫 9天 HBsAg 120^g , 该蛋白 HBsAg
120 ig
12天 该蛋白 65/ig
15天 该蛋白 75[ig
第三次免疫 16天 HBsAg 120 ig HBsAg
120/ g
17天 该蛋白 75 g 于初次免疫后的第 20天从小鼠尾静脉取血， 分离血清。 ELISA法测定小鼠体内抗乙 肝表面抗原抗体的滴度： 将分离到的血清进行倍比稀释， 用乙肝表面抗原包被 ELISA板， 稀释的血清作为一抗， HRP标记的抗小鼠 IgG抗体作为二抗。显色后测 OD495值。用 SPSS 软件分析试验组和对照组的差异。
结果 （见图 1， 图 2)发现， 用乙肝疫苗免疫小鼠， 试验组小鼠在初次免疫 20天后抗 乙肝表面抗原抗体的效价为 9.24x l04， 明显高于对照组的抗体效价（2.68X104), 说明该蛋 白具有上调小鼠体内抗乙肝表面抗原抗体的能力， 可以增强乙肝疫苗的免疫效果。
具体应用： 传统乙肝疫苗的免疫效果在人群当中的个体差异很大， 常规注射三针以后 很多人体内并不能产生足够的抗体以抵抗乙肝病毒的感染和乙肝发病。 而该说明书中涉及 到的这种乙肝疫苗增效蛋白能够明显增强乙肝疫苗的免疫效果， 因此， 可将其用于乙肝疫 苗佐剂的开发。 在注射乙肝疫苗的同时， 注射一定量的该蛋白， 能够增强乙肝疫苗的免疫 原性， 增加体内抗乙肝表面抗原抗体的效价， 从而提高疫亩的免疫效率， 并减少乙肝疫亩 用量和注射次数。
此外， 该蛋白具有对乙肝表面抗原的特异性， 并能够促进体内抗乙肝表面抗原抗体的 产生， 因此， 对于乙型肝炎病毒携带者和乙肝病人的治疗， 该蛋白可以作为一种增效剂应 用于该领域， 同时， 对治疗性乙肝疫苗的开发研究也具有一定的应用价值。

Claims

权利要求书
1. 一种乙型肝炎病毒疫苗增效蛋白， 其特征在于氨基酸序列为 SEQ.ID.N0.2。
2. 一种编码乙型肝炎病毒疫苗增效蛋白的基因， 其特征在于核苷酸序列为 SEQ.ID.N0.1。
3. 一种如权利要求 1 蛋白的筛选方法， 其特征在于具体步骤为： 以乙型肝炎病毒表面抗 原为筛选分子， 利用印迹免疫筛选的方法对人肝 cDNA表达库进行筛选， 得到一株阳 性的 cDNA克隆；对该 cDNA进行测序分析，显示该 cDNA具有独立的开放阅读框架， 其大小为 1035b。
4. 一种表达如权利要求 1 所述的乙型肝炎病毒疫亩增效蛋白的方法， 其特征在于将该蛋 白基因的 N端引入 6xhis tag和肠激酶酶切位点， 克隆到 pBV220表达载体中， 在大肠 杆菌中表达， 收集并裂解大肠杆菌， 提取包涵体， 经粗纯后再用 Ni-NTA agarose进行 亲和层析纯化， 经透析复性最终得到目的蛋白。
5. 如权利要求 1所述蛋白作为乙型肝炎疫苗佐剂的应用。
PCT/CN2007/000844 2006-03-16 2007-03-16 一种乙型肝炎病毒疫苗增效蛋白及其基因 WO2007104263A1 (zh)

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