WO2020108427A1 - 一种新型干扰素α及其制备方法、组合物和用途 - Google Patents

一种新型干扰素α及其制备方法、组合物和用途 Download PDF

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WO2020108427A1
WO2020108427A1 PCT/CN2019/120575 CN2019120575W WO2020108427A1 WO 2020108427 A1 WO2020108427 A1 WO 2020108427A1 CN 2019120575 W CN2019120575 W CN 2019120575W WO 2020108427 A1 WO2020108427 A1 WO 2020108427A1
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interferon
ifn
novel interferon
novel
new
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侯云德
张莉
宋瑜丽
唐旭东
张佳莹
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深圳市利云德生物技术有限公司
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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
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    • 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
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of biotechnology medicines, and relates to a new type of interferon alpha and its preparation method, composition and use.
  • Interferon is a class of cytokine protein drugs with broad-spectrum antiviral, antitumor, and immunomodulatory effects, including type I, type II, and type III, which have different receptors and functions, respectively, are A key component of the body's natural immune system. Among them, type I IFN was discovered in 1957. It is an important antiviral substance produced by humans and animals after being infected by viruses. According to the difference of its genes and protein structures, it is divided into IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ , IFN- ⁇ , etc.
  • IFN- ⁇ 1b Three subtypes of IFN- ⁇ have been approved for clinical use, namely IFN- ⁇ 1b, IFN- ⁇ 2a and IFN- ⁇ 2b, all of which are currently genetically recombinant products.
  • the IFN- ⁇ used abroad is mainly IFN- ⁇ 2a and IFN- ⁇ 2b, and its genes are derived from Western white people;
  • the IFN- ⁇ used domestically is mainly IFN- ⁇ 1b, and its genes are used by Chinese academician Hou Yunde in 1982 from healthy Chinese Obtained from cord blood leukocytes.
  • Many years of research by the Institute of Virology, Chinese Academy of Preventive Medicine showed that after being attacked by viruses, Chinese human leukocytes produced IFN- ⁇ 1b interferon as the main type of interferon. Therefore, compared with similar products at home and abroad, genetically engineered IFN- ⁇ 1b has the advantages of significant curative effect (the same obvious efficiency as foreign products), lower side effects, and difficulty in producing neutralizing antibodies. It is more
  • IFN- ⁇ 1b injection is my country's first genetic engineering class I new drug with independent intellectual property rights. It has been widely used in the domestic antiviral field for more than 20 years, and has accumulated a large number of literature reports and clinical experience. Data from multiple multi-center clinical studies have shown that IFN- ⁇ 1b is effective in treating respiratory syncytial virus (RSV) pneumonia, bronchiolitis, hand, foot and mouth disease (HFMD), and viral enteritis, with mild adverse reactions.
  • RSV respiratory syncytial virus
  • HFMD hand, foot and mouth disease
  • viral enteritis with mild adverse reactions.
  • "People's Republic of China Pharmacopoeia 2015 Edition (Part III)" contains recombinant human interferon ⁇ 1b injection. The indications are viral diseases and certain malignant tumors.
  • recombinant human interferon ⁇ 1b is effective against condyloma acuminatum, chronic cervicitis, herpes keratitis, herpes zoster, epidemic hemorrhagic fever, and respiratory syncytial virus pneumonia in children. It is effective against other viral diseases and malignancies Tumors such as chronic myeloid leukemia, melanoma, and lymphoma also have good curative effects.
  • IFN- ⁇ 1b Compared with other types of interferon, IFN- ⁇ 1b has less clinical adverse reactions, so it has achieved good results in the field of pediatric medicine and is one of the few preferred products available in the same category.
  • a large amount of pediatric clinical medication experience and multi-center research data show that IFN- ⁇ 1b given by intramuscular injection and inhalation of the respiratory tract has an important role in the treatment of viral infections in children, especially inhalation of IFN- ⁇ 1b to treat viral infections, drugs It can directly act on the respiratory tract mucosa, has the advantages of strong targeting, high efficacy, good safety, easy operation, high compliance of children, etc., and can reach the lungs through bronchi and bronchioles, more concentrated in lung tissue, slow Blood transfusion is more durable than intramuscular administration. Therefore, inhaled IFN- ⁇ 1b has been recommended for use by the National Standard Formulary "Guidelines for Standardized Management of Children's Atomization Center".
  • IFN- ⁇ 1b has low specific activity and poor stability, which limits its further clinical application.
  • the primary objective of the present invention is to provide a new type of interferon ⁇ , labeled as interferon ⁇ 1z (IFN- ⁇ 1z), which is obtained through molecular design and amino acid sequence modification on the basis of interferon ⁇ 1b (IFN- ⁇ 1b), On the basis of maintaining good safety and efficacy of IFN- ⁇ 1b, it has significantly higher biological activity and stability than IFN- ⁇ 1b.
  • IFN- ⁇ 1z interferon ⁇ 1z
  • the present invention provides a novel interferon alpha, said novel interferon alpha having the amino acid sequence shown in SEQ ID NO. 1.
  • the novel interferon ⁇ (IFN- ⁇ 1z) of the present invention has 93% amino acid homology with interferon ⁇ 1b (IFN- ⁇ 1b) and 85% amino acid homology with interferon ⁇ 2b (IFN- ⁇ 2b).
  • the second object of the present invention is to provide a polynucleotide encoding the aforementioned novel interferon ⁇ to better encode the aforementioned novel interferon ⁇ , and the encoded novel novel interferon ⁇ can maintain good IFN- ⁇ 1b On the basis of safety and efficacy, it has significantly higher biological activity and stability than IFN- ⁇ 1b.
  • the present invention provides a polynucleotide encoding the aforementioned novel interferon alpha.
  • the third object of the present invention is to provide a method for preparing the aforementioned new interferon ⁇ , which can better prepare the aforementioned new interferon ⁇ , and the prepared novel interferon ⁇ can maintain good safety of IFN- ⁇ 1b On the basis of its efficacy, it has significantly higher biological activity and stability than IFN- ⁇ 1b.
  • the present invention provides a method for preparing the aforementioned novel interferon alpha, the method includes the following steps:
  • the fourth object of the present invention is to provide a pharmaceutical composition of the aforementioned novel interferon alpha, which can compare with the pharmaceutical composition of IFN- ⁇ 1b on the basis of maintaining the good safety and efficacy of the pharmaceutical composition of IFN- ⁇ 1b It has significantly higher biological activity and stability.
  • the present invention provides a pharmaceutical composition of the aforementioned novel interferon alpha, said pharmaceutical composition containing a therapeutically effective amount and a safe amount of said novel interferon alpha and suitable Amount of pharmaceutically acceptable carrier.
  • the fifth object of the present invention is to provide a use of the aforementioned novel interferon alpha or its pharmaceutical composition in the preparation of a medicament for preventing or treating viral infectious diseases.
  • the present invention provides the use of the aforementioned novel interferon alpha or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating viral infectious diseases.
  • the present invention provides the use of the aforementioned novel interferon alpha or a pharmaceutical composition thereof in the preparation of a medicament for the prevention or treatment of viral infectious diseases, wherein the viral infectious diseases are type B Encephalitis, respiratory syncytial virus infection or viral hepatitis.
  • therapeutically effective amount and safe amount used in the present invention means that when a pharmaceutical active ingredient is administered for the treatment or prevention of a disease, the amount of the pharmaceutical active ingredient is sufficient to achieve treatment or prevention of the disease without causing significant toxic side effects.
  • the therapeutically effective amount and safe amount will vary depending on the active ingredient of the drug, the disease and its severity, and the age and weight of the patient being treated.
  • the beneficial effect of the present invention is that the novel interferon ⁇ of the present invention can have significantly higher biological activity and stability than IFN- ⁇ 1b on the basis of maintaining good safety and drug efficacy of IFN- ⁇ 1b.
  • the corresponding nucleic acid template is synthesized according to SEQ ID NO.1 of the sequence table, and a sufficient amount of nucleic acid is amplified by PCR.
  • the primers for the amplification reaction system are:
  • the amplification reaction system includes: dNTP 1 ⁇ l, 10X pfu buffer 5 ⁇ l, upstream and downstream primers 2 ⁇ l, customer template 1 ⁇ l, Pfu enzyme 0.4 ⁇ l (5u/ ⁇ l), ddH2O 38.6 ⁇ l.
  • the amplification reaction conditions are: 95°C, 3 minutes, 95°C, 22 seconds, 68°C, 20 seconds, 72°C, 60 seconds, 72°C, 5 minutes. A total of 24 cycles. After the reaction is completed, it is separated by agarose gel electrophoresis, double digested with Nde I/Xho I, and the target DNA fragment of about 500 bp is recovered by electrophoresis.
  • the pET30a plasmid vector was double digested with Nde I/Xho I, recovered by agarose electrophoresis and ligated with the above-mentioned recovered DNA fragment.
  • the ligation reaction conditions were: seamless cloning mixture, 8.5 ⁇ l, digested vector pET30a, 4 ⁇ l, purified PCR product, 7.5 ⁇ l.
  • the above ligation mixture was placed in the PCR instrument at 37°C for 1 h.
  • Escherichia coli BLD21 competent cells were prepared, the above ligation products were transformed, coated with ampicillin plates, and cultured overnight at 37°C.
  • the expression engineering bacteria of the new interferon ⁇ constructed in the foregoing Example 1 were activated by plating, and single colonies were selected and inoculated in LB medium containing kanamycin with a final concentration of 30 ⁇ g/mL (10 g peptone per liter, Yeast powder 5g, NaCl 10g, adjusted pH 7.0), 37 °C, 235 rpm shaker shaker culture to OD600nm 0.6-0.8.
  • the obtained 10g inclusion body was added into the inclusion body dissolution solution (8mol/L urea, 50mmol/L Tris-HCl, 300mmol/L NaCl, pH 8.0) at a mass volume ratio of 1:10 and then denatured for 2 hours under moderate stirring conditions After the inclusion body is completely dissolved, the precipitate is discarded at room temperature 12,000 rpm/separation core for 20 minutes, and the supernatant is renatured by dilution renaturation method.
  • the composition of the renaturation solution is: 0.15mol/L sodium borate buffer, 3mmol/L oxidized Glutathione, 1mmol/L reduced glutathione, adjust the pH to 9.5.
  • the renaturation process is carried out in a low temperature cold storage at 2-8°C. First, the supernatant is diluted 6 times with the renaturation solution, and after standing for 8 hours, it is then diluted 5 times with the renaturation solution to continue the renaturation for 6 hours.
  • the renaturation solution was subjected to 12000 rpm at 4°C for 30 minutes, and then applied to a DEAE Sepharose FF column equilibrated with 25mmol/L Tris-HCl pH 8.0 solution. After loading the sample, first flush the column with equilibration buffer for 2-3 column volumes, and then elute with 25mmol/L Tris-HCl pH8.0 solution containing 0.35mol/L NaCl to collect the elution peak.
  • the linear flow rate during the above loading, rinsing and elution should be controlled between 50-200cm/h.
  • the elution peak of DEAE Sepharose FF was diluted with 50mmol/L acetic acid-sodium acetate pH4.5 buffer at a volume ratio of 1:10, and then was applied to a CM Sepharose FF column equilibrated with the same buffer.
  • the chromatographic column is continuously rinsed with 2-3 column volumes, and then the main impurity peak is eluted with 25mmol/L acetic acid-sodium acetate pH4.5 buffer containing 0.1-0.15mol/L NaCl Then elute with 25mmol/L acetic acid-sodium acetate pH4.5 buffer containing 0.5mol/L NaCl to collect the target peak.
  • the linear flow rate during the above loading, rinsing and elution should be controlled between 50-200cm/h.
  • the new interferon ⁇ purified in the foregoing Example 2 was placed at room temperature (25°C) for 3 months. During this process, regular sampling was performed to observe changes in appearance, and the purity and biological activity were tested (using the Pharmacopoeia of the People’s Republic of China The 2015 version (Part III) "interferon activity measurement method" and other major quality control indicators change, the results are shown in Table 1 below. The corresponding 25°C stability test results of IFN- ⁇ 1b are shown in Table 2.
  • WISH Human Passage Amniotic Membrane
  • Vero green monkey kidney cells, provided by the Institute of Viral Diseases, Chinese Center for Disease Control and Prevention.
  • VSV Follicular Stomatitis Virus Indiana strain was provided by the National Engineering Laboratory of Viral Genes and introduced by the US ATCC;
  • the measles virus MA strain, herpes simplex virus types I and II (HSV-I, HSV-II), adenovirus type 7 (Ad7), Cox B1 and Sind ⁇ is viruses were provided by the Center for Viral Diseases of the Chinese Center for Disease Control and Prevention;
  • JBEV-28 Attenuated Japanese encephalitis virus-28 strain (JBEV-28), which is an adaptive strain of human lung diploid cells, provided by the encephalitis laboratory of the Institute of Virology;
  • Respiratory syncytial virus is isolated from my country.
  • Natural human leukocyte interferon (nIFN-Le) is induced and isolated from human peripheral blood leukocytes by NDV.
  • Example 5 Acute toxicity test of new interferon alpha in mice
  • mice with a body weight of about 20 g were taken and divided into 2 groups of 15 mice in each group.
  • Groups 1 and 2 were injected with a single subcutaneous injection of the novel interferon ⁇ and IFN- ⁇ 1b purified in Example 2.
  • the two groups did not show any abnormal performance, indicating that the maximum tolerance of mice to the new interferon alpha was above 5.8mg/Kg, which is equivalent to 3480 times the clinical dosage of humans.
  • the new interferon ⁇ and IFN- ⁇ 1b are equally safe.

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Abstract

本发明属于生物技术药物技术领域，提供了一种干扰素α及其制备方法、组合物和用途。所述的干扰素α具有SEQ ID NO.1所示的氨基酸序列，其能够在保持良好的安全性与药效的基础上具有良好的生物学活性和稳定性。

Description

一种新型干扰素α及其制备方法、组合物和用途 技术领域
本发明属于生物技术药物技术领域，涉及一种新型干扰素α及其制备方法、组合物和用途。
背景技术
干扰素(interferon，IFN)是一类具有广谱抗病毒、抗肿瘤和免疫调节作用的细胞因子蛋白质类药物，包括I型、II型和III型，它们分别具有不同的受体和功能，是机体天然免疫系统的关键组成部分。其中I型IFN于1957年发现，是人和动物受病毒感染后产生的重要抗病毒物质，根据其基因和蛋白结构的不同，又分为IFN-α、IFN-β、IFN-ε、IFN-κ、IFN-ω等。IFN-α已有3个亚型被批准为药品在临床使用，分别为IFN-α1b、IFN-α2a和IFN-α2b，目前均为基因重组产品。国外使用的IFN-α主要是IFN-α2a和IFN-α2b，其基因来源于西方白种人；国内使用的IFN-α主要是IFN-α1b，其基因由我国侯云德院士于1982年从健康中国人脐血白细胞中获得。中国预防医学科学院病毒学研究所多年的研究表明，中国人白细胞在受到病毒攻击后，产生的多种干扰素中以IFN-α1b型干扰素为主。因此，基因工程IFN-α1b与国内外同类产品相比，具有疗效显著(显效率与国外产品相同)、副作用更低、不易产生中和抗体等优点，更适合中国人使用。
IFN-α1b注射液是我国第一个具有自主知识产权的基因工程I类新药，已经在国内抗病毒领域广泛应用20余年，积累了大量文献报道和临床使用经验。多个多中心临床研究的资料显示，IFN-α1b治疗呼吸道合胞病毒(RSV)肺炎、毛细支气管炎、手足口病(HFMD)和病毒性肠炎等疗效好，不良反应轻。《中华人民共和国药典2015年版(三部)》收录重组人干扰素α1b注射液，适应症为病毒性疾病和某些恶性肿瘤，主要用于治疗慢性乙型肝炎、慢性丙型肝炎和毛细胞白血病等。此外，重组人干扰素α1b对尖锐湿疣、慢性宫颈炎、疱疹性角膜炎、带状疱疹、流行性出血热和小儿呼吸道合胞病毒 性肺炎等病毒性疾病均有效，对其他病毒性疾病和恶性肿瘤如慢性粒细胞白血病、黑色素瘤、淋巴瘤等也有良好疗效。
由于IFN-α1b与其它型别干扰素相比，其临床不良反应小，因此其在儿科领域用药取得了良好的效果，是同类产品中可用的少有优选产品。大量儿科临床用药经验和多中心研究资料表明，通过肌肉注射和呼吸道雾化吸入等途径给予IFN-α1b，对儿童病毒感染的治疗有重要作用，尤其是雾化吸入IFN-α1b治疗病毒感染，药物能直接作用于呼吸道黏膜，具有靶向性强、疗效高、安全性好、操作简便、儿童依从性高等优点，并可经支气管、细支气管到达肺部，较多地集中在肺部组织，缓慢入血，较肌肉注射给药作用更持久。因此，雾化吸入IFN-α1b已被国家标准处方集《儿童雾化中心规范化管理指南》推荐使用。
但是，IFN-α1b比活性较低，稳定性欠佳，这都限制了其在临床的进一步推广应用。
发明内容
本发明的首要目的是提供一种新型干扰素α，标记为干扰素α1z(IFN-α1z)，其是在干扰素α1b(IFN-α1b)的基础上经过分子设计和氨基酸序列改造得到，以能够在保持IFN-α1b良好的安全性与药效的基础上较IFN-α1b具有明显更高的生物学活性和稳定性。
为实现此目的，在基础的实施方案中，本发明提供一种新型干扰素α，所述的新型干扰素α具有SEQ ID NO.1所示的氨基酸序列。
本发明的新型干扰素α(IFN-α1z)与干扰素α1b(IFN-α1b)的氨基酸同源性为93％，与干扰素α2b(IFN-α2b)的氨基酸同源性为85％。
本发明的第二个目的是提供一种编码前述新型干扰素α的多核苷酸，以能够更好的编码前述新型干扰素α，编码得到的前述新型干扰素α能够在保持IFN-α1b良好的安全性与药效的基础上较IFN-α1b具有明显更高的生物学活性和稳定性。
为实现此目的，在基础的实施方案中，本发明提供一种编码前述新型干扰素α的多核苷酸。
本发明的第三个目的是提供一种前述新型干扰素α的制备方法，以能够 更好的制备前述新型干扰素α，制备得到的前述新型干扰素α能够在保持IFN-α1b良好的安全性与药效的基础上较IFN-α1b具有明显更高的生物学活性和稳定性。
为实现此目的，在基础的实施方案中，本发明提供一种前述新型干扰素α的制备方法，所述的制备方法包括如下步骤：
(1)新型干扰素α表达工程菌的构建；
(2)新型干扰素α表达工程菌的发酵及色谱(层析)分离纯化。
本发明的第四个目的是提供一种前述新型干扰素α的药物组合物，以能够在保持IFN-α1b的药物组合物良好的安全性与药效的基础上较IFN-α1b的药物组合物具有明显更高的生物学活性和稳定性。
为实现此目的，在基础的实施方案中，本发明提供一种前述新型干扰素α的药物组合物，所述的药物组合物含有治疗有效量且安全量的所述的新型干扰素α和适宜量的可药用载体。
本发明的第五个目的是提供一种前述新型干扰素α或其药物组合物在制备预防或治疗病毒感染性疾病的药物中的用途。
为实现此目的，在基础的实施方案中，本发明提供一种前述新型干扰素α或其药物组合物在制备预防或治疗病毒感染性疾病的药物中的用途。
在一种优选的实施方案中，本发明提供一种前述新型干扰素α或其药物组合物在制备预防或治疗病毒感染性疾病的药物中的用途，其中所述的病毒感染性疾病是乙型脑炎、呼吸道合胞病毒感染或病毒性肝炎。
本发明中使用的术语“治疗有效量且安全量”表示当施用药物活性成分用于治疗或预防疾病时，药物活性成分的量足以实现对疾病的治疗或预防且不引起明显毒副作用。治疗有效量与安全量将依药物活性成分、疾病和它的严重性和所治疗病人的年龄、体重等而异。
本发明的有益效果在于，本发明的新型干扰素α能够在保持IFN-α1b良好的安全性与药效的基础上较IFN-α1b具有明显更高的生物学活性和稳定性。
具体实施方式
通过如下的实施例对本发明的实施作进一步说明，但本发明的实施方式 并不局限于如下的实施例。
实施例1：新型干扰素α表达工程菌的构建与表达序列确认
根据序列表的SEQ ID NO.1合成对应的核酸模板，通过PCR的方法扩增出足够量的核酸。
扩增反应体系引物为：
F：CATATGTGCGACCTGCCACA(SEQ ID NO.2)
R：CTCGAGTTAGTTGGTGCTCAGGC(SEQ ID NO.3)
扩增反应体系包含：dNTP 1μl、10X pfu缓冲液5μl、上下游引物各2μl、客户模板1μl、Pfu酶0.4μl(5u/μl)、ddH2O 38.6μl。
扩增反应条件为：95℃，3分钟、95℃，22秒、68℃，20秒、72℃，60秒、72℃，5分钟。共24个循环。反应完成后用琼脂糖凝胶电泳进行分离，用Nde I/Xho I双酶切，电泳回收500bp左右的目的DNA片段。
将pET30a质粒载体用Nde I/Xho I双酶切，经琼脂糖电泳回收并和上述回收的目的DNA片段连接。连接反应条件为：无缝克隆混合液，8.5μl、酶切载体pET30a，4μl、已纯化好的PCR产物，7.5μl，上述连接混合液放在37℃条件下在PCR仪1h即可。
制备大肠杆菌BLD21感受态细胞，转化上述连接产物，涂布氨苄平板，37℃过夜培养。
挑取单菌落作为模板，用上述设计的引物F、R进行扩增，产物经琼脂糖电泳检测，阳性克隆在474bp左右出现特异条带；取阳性克隆小量培养，提取质粒用Nde I/Xho I双酶切。分别在3kb左右和500bp左右出现特异性的条带，与预计情况相符，初步说明重组质粒构建成功。为进一步确认其序列，以T7通用引物对ABI377测序仪进行全自动序列测定。
实施例2：新型干扰素α的发酵、纯化与检测
将前述实施例1构建得到的新型干扰素α的表达工程菌经涂平板活化后挑选单菌落接种于含有终浓度为30μg/mL的卡那霉素的LB培养基中(每升用蛋白胨10g、酵母粉5g、NaCl 10g配制，调节pH为7.0)，37℃、235转/分摇床摇瓶培养至OD600nm为0.6-0.8。然后以5％的体积接种量接种于50L的LB培养基中在80L发酵罐中进行发酵培养(含30μg/mL的卡那霉素)， 培养温度为37℃，培养过程中用氨水调节pH在6.5-7.5之间，用转速控制溶氧值在3-5％之间。在OD600nm达到1.0后按质量体积比1:5000的比例加入IPTG 10g继续诱导培养4.5小时，诱导培养温度为37℃，并在此过程中向发酵罐中适当的补加LB培养基。诱导培养时间到后放罐室温5000转/分离心20分钟收集菌体，所得菌体用TE缓冲液(50mmol/L Tris-HCl,5mmol/L EDTA,pH8.0)洗涤离心两次以除去发酵液中的主要杂质。
取上述处理得到的菌体40g以1:15的质量体积比加入600ml TE缓冲液置超声波破碎仪上进行超声破碎，条件为打5秒，歇5秒，共60分钟，所得破碎液室温12000转/分离心20分钟弃上清，沉淀以1:10的质量体积比加入TE缓冲液洗涤离心两次得分离包涵体。
所得10g包涵体以1:10的质量体积比加入包涵体溶解液(8mol/L尿素，50mmol/L Tris-HCl，300mmol/L NaCl，pH8.0)溶解后在适度搅拌条件下变性处理2小时，待包涵体完全溶解后室温12000转/分离心20分钟弃沉淀，上清以稀释复性法进行复性处理，复性液组成为：0.15mol/L硼酸钠缓冲液，3mmol/L氧化型谷胱甘肽，1mmol/L还原型谷胱甘肽，调节pH为9.5。复性过程在2-8℃低温冷库中进行，首先用复性液将上清稀释6倍，放置8小时后再用复性液稀释5倍继续复性6小时。
透析后的复性液经4℃12000转/分离心30分钟后上用25mmol/L Tris-HCl pH8.0溶液平衡的DEAE Sepharose FF色谱柱。上样完成后先用平衡缓冲液继续冲洗色谱柱2-3个柱体积，然后以含有0.35mol/L NaCl的25mmol/L Tris-HCl pH8.0溶液进行洗脱收集洗脱峰。上述上样、冲洗与洗脱过程中的线性流速应控制在50-200cm/h之间。
DEAE Sepharose FF洗脱峰用50mmol/L醋酸-醋酸钠pH4.5缓冲液以1:10的体积比稀释后上用同样缓冲液平衡的CM Sepharose FF色谱柱。上样完成后先用平衡缓冲液继续冲洗色谱柱2-3个柱体积，然后在用含有0.1-0.15mol/L NaCl的25mmol/L醋酸-醋酸钠pH4.5缓冲液洗脱除去主要杂质峰后用含有0.5mol/L NaCl的25mmol/L醋酸-醋酸钠pH4.5缓冲液洗脱收集目标峰。上述上样、冲洗与洗脱过程中的线性流速应控制在50-200cm/h之间。
用SDS-PAGE电泳加考马斯亮蓝染色法和分子排阻HPLC法分别检测上述CM Sepharose FF色谱柱分离得到的新型干扰素α的纯度，结果均在97％以上。用《中华人民共和国药典2015年版(三部)》规定的“干扰素活性测定法”和“蛋白质含量测定法”确定其比活性为3.37×10 8IU/mg，对照IFN-α1b的比活性为1.32×10 7IU/mg，前者为后者的25.5倍左右。
实施例3：新型干扰素α的稳定性试验
将前述实施例2纯化得到的新型干扰素α在室温(25℃)环境下放置3个月，在此过程中定期取样观察外观的变化，并检测纯度、生物学活性(用《中华人民共和国药典2015年版(三部)》规定的“干扰素活性测定法”)等主要质量控制指标的变化，结果如下表1所示。对应的IFN-α1b的25℃稳定性试验结果如表2所示。
表1新型干扰素α的25℃稳定性试验结果
Figure PCTCN2019120575-appb-000001
表2 IFN-α1b的25℃稳定性试验结果
Figure PCTCN2019120575-appb-000002
Figure PCTCN2019120575-appb-000003
实施例4：新型干扰素α的细胞药效试验
(一)材料
1、细胞
WISH(人传代羊膜)细胞，由纽约大学医学院微生物教研室提供；
Vero(绿猴肾)细胞，由中国疾病预防控制中心病毒病研究所提供。
所有传代细胞均按常规方法传代培养。
2、病毒
滤泡性口膜炎病毒(VSV)Indiana株由病毒基因国家工程实验室提供，美国ATCC引进；
麻疹病毒MA株、单纯疱疹病毒I、II型(HSV-I、HSV-II)、7型腺病毒(Ad7)、Cox Β1和Sindβis病毒由中国疾病预防控制中心病毒病研究所毒种中心提供；
流行性乙型脑炎病毒-28减毒株(JΒEV-28)，为人肺二倍体细胞适应株，由病毒学研究所脑炎研究室提供；
呼吸道合胞病毒(RSV)均由我国分离。
以上病毒均按常规在各自的敏感细胞上传代。
3、干扰素
自然人白细胞干扰素(nIFN-Le)由人周围血白细胞经NDV诱导产生并分离。
各类干扰素的比活性均＞10 6IU/mg。
(二)方法：干扰素抗病毒活性的测定
上述细胞形成单层后，加不同浓度的干扰素处理，对照组用营养液代替干扰素。37℃培养24小时后，倾去干扰素液，用l00 TCID50的上述病毒攻击，37℃继续培养，待对照组细胞完全病变后，判断结果，从所加干扰素的浓度算出相对抗病毒活性。在同一细胞培养上比较干扰素对不同病毒的敏感 性时，以VSV为对照，均采用病变抑制法测定干扰素的抗病毒活性。所得结果如下表3-5所示。
表3新型干扰素α与其它干扰素对不同病毒感染的相对抗病毒活性的比较
Figure PCTCN2019120575-appb-000004
表4新型干扰素α与其它干扰素在人二倍体细胞上对乙型脑炎的抑制作用
Figure PCTCN2019120575-appb-000005
表5新型干扰素α与其它干扰素在HeLa细胞上对呼吸道合胞病毒的抑制作用
Figure PCTCN2019120575-appb-000006
Figure PCTCN2019120575-appb-000007
上述结果表明，新型干扰素α在不同细胞株上对不同病毒的抗病毒滴度均小于IFN-α1b，说明新型干扰素α的抗病毒效果在相同条件下明显优于IFN-α1b。
实施例5：新型干扰素α的小鼠急性毒性试验
取体重为20g左右的小鼠30只，均分为2组，每组15只。组1和组2分别皮下单次注射实施例2纯化得到的新型干扰素α和IFN-α1b。注射后连续观察14天，结果两组均未见任何异常表现，表明小鼠对新型干扰素α的最大耐受量均在5.8mg/Kg以上，相当于人临床用量的3480倍。新型干扰素α和IFN-α1b安全性相当。

Claims (6)

  1. 一种新型干扰素α，其特征在于：所述的新型干扰素α具有SEQ ID NO.1所示的氨基酸序列。
  2. 一种编码权利要求1所述的新型干扰素α的多核苷酸。
  3. 一种根据权利要求1所述的新型干扰素α的制备方法，其特征在于，所述的制备方法包括如下步骤：
    (1)新型干扰素α表达工程菌的构建；
    (2)新型干扰素α表达工程菌的发酵及色谱分离纯化。
  4. 一种根据权利要求1所述的新型干扰素α的药物组合物，其特征在于：所述的药物组合物含有治疗有效量且安全量的所述的新型干扰素α和适宜量的可药用载体。
  5. 一种根据权利要求1所述的新型干扰素α或根据权利要求4所述的药物组合物在制备预防或治疗病毒感染性疾病的药物中的用途。
  6. 根据权利要求5所述的用途，其特征在于：所述的病毒感染性疾病是乙型脑炎、呼吸道合胞病毒感染或病毒性肝炎。
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