CN104151420A - 长效干扰素及其制备方法和用途 - Google Patents
长效干扰素及其制备方法和用途 Download PDFInfo
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Abstract
本发明属生物制药领域，涉及一种长效干扰素及制备方法和用途；所述长效干扰素包括多聚唾液酸修饰的干扰素和长效干扰素重组融合蛋白；其中所述多聚唾液酸采用高碘酸钠进行激活，活化的多聚唾液酸在氰基硼氢化钠存在的情况下与干扰素进行反应形成偶联物；所述重组融合蛋白由N-端的干扰素和C-端的天然存在的亲水性无重复序列的多肽组成，该融合蛋白可在大肠杆菌中高效表达，且制备工艺简单，可用于大规模的药物级融合蛋白的生产制备。本发明的长效干扰素可用于肝炎病毒、SARS、单纯疱疹病毒等病毒感染性疾病的治疗，和用于恶性神经胶质瘤、肺癌等恶性肿瘤及其他细胞增殖性疾病的治疗。
Description
技术领域
本发明属生物制药领域，涉及长效干扰素及制备方法和用途，具体涉及用于抗病毒、抗感染、抗肿瘤和免疫调节的长效干扰素及制备方法和用途。
背景技术
现有技术公开了干扰素（Interferon）是人和动物的细胞在受细菌、病毒感染或核苷酸等刺激物的诱导下分泌的一组具有抗病毒等活性的多功能糖蛋白；是最早用于疾病临床治疗的细胞因子(Pestka S, Langer JA, Fisher PB, Weinstein IB, Ortaldo J, Herberman RB: Symp Fundam Cancer Res 1984, 37:261-281;Gutterman JU: Cytokine therapeutics: Proc Natl Acad Sci U S A 1994, 91(4):1198-1205)。所述干扰素主要有α干扰素、β干扰素、γ干扰素和ω干扰素等类型，根据干扰素产生的细胞来源、受体、氨基酸结构及对酸耐受程度的不同，可将干扰素分为I型干扰素和II型干扰素，其中I型干扰素包括α干扰素、β干扰素和ω干扰素；I型干扰素被称为抗病毒干扰素，其生物学效应主要以抗病毒为主，同时具有抑制细胞生长，抗肿瘤，免疫调节等生物活性。所述γ干扰素属于II型干扰素，抗病毒活性较I型干扰素弱，其生物学活性主要表现为免疫调节和参与组织相容性抗原的表达，又称为免疫干扰素(Stewart WE, 2nd, Wiranowska-Stewart M, Koistinen V, Cantell K: Virology 1979, 97(2):473-476.)。
研究公开了β干扰素是由成纤维细胞产生的，与α干扰素识别相同受体且具有类似生物学效应的蛋白质；天然人β干扰素是由第9号染色体上单一基因编码的分子量约为23千道尔顿的糖蛋白；其前体分子由21个氨基酸的信号肽序列和166个氨基酸的β干扰素分子组成，含有3个半胱氨酸和一个糖基链，其中位于第31、141位的半胱氨酸可形成二硫键，对于β干扰素生物活性的维持具有重要作用(Mark DF, Lu SD, Creasey AA, Yamamoto R, Lin LS: Proc Natl Acad Sci U S A 1984,81(18):5662-5666)。
研究显示，β干扰素具有广谱的抗病毒活性，但其抑制病毒增殖的作用机理因病毒种类的不同而有所差异，如β干扰素可抑制口炎疱疹病毒对细胞的吸附而发挥抗病毒效用；通过抑制病毒脱衣壳和病毒核酸的转录而抑制单纯疱疹病毒和流感病毒的增殖(Kumar M, Liu H, Rice AP: PLoS One 2012, 7(7):e41251)；通过阻断病毒蛋白的合成而抗疱疹病毒；通过抑制逆转录病毒单纯疱疹病毒成熟病毒颗粒的释放而行使抗病毒功能；β干扰素还可通过诱导组织细胞产生蛋白激酶、磷酸二酯酶等抗病毒蛋白而抑制病毒复制和增殖，通过诱导单核巨噬细胞和自然杀伤细胞等免疫细胞的活化来增强对病毒的吞噬作用。有研究发现，β干扰素还具有抗SARS病毒的活性，是唯一一种在感染后仍具有抗病毒效用的干扰素(Subramanian GM, Moore PA, Gowen BB, Olsen AL, Barnard DL, Paragas J, Hogan RJ, Sidwell RW: Chemotherapy 2008, 54(3):176-180)；此外，β干扰素还广泛应用于乙型肝炎病毒和丙型肝炎病毒所引起的病毒性肝炎、流行性病毒性脑炎、单纯疱疹病毒角膜炎和病毒性心肌炎等病毒性感染疾病的治疗(Morikawa H, Kozuka R, Fujii H, Iwai S, Enomoto M, Tamori A, Saito S, Kawada N: Hepatol Res 2013)。
有研究显示，β干扰素具有抗肿瘤的生物学效应；其对恶性神经胶质瘤、乳腺癌、结肠癌、肾癌、非小细胞肺癌、喉癌、胰腺癌、恶性黑色素瘤、多发性骨髓瘤等多种肿瘤具有抑制和杀伤效果。β干扰素抗肿瘤的作用机理主要包括直接抑制肿瘤细胞增殖；作用于机体的免疫系统，提高单核巨噬细胞、自然杀伤细胞等免疫细胞对肿瘤细胞的杀伤能力，诱导肿瘤细胞发生细胞凋亡；抑制肿瘤微环境血管的生成而发挥抗肿瘤的作用等(Okazaki T, Kageji T, Kuwayama K, Kitazato KT, Mure H, Hara K, Morigaki R, Mizobuchi Y, Matsuzaki K, Nagahiro S: Cancer Lett 2012, 323(2):199-207;Park MY, Kim DR, Jung HW, Yoon HI, Lee JH, Lee CT: Cancer Gene Ther 2010, 17(5):356-364;Vitale G, Zappavigna S, Marra M, Dicitore A, Meschini S, Condello M, Arancia G, Castiglioni S, Maroni P, Bendinelli P et al: Biotechnol Adv 2012, 30(1):169-184;Olson MV, Lee J, Zhang F, Wang A, Dong Z: Cancer Gene Ther 2006, 13(7):676-685;van Koetsveld PM, Vitale G, Feelders RA, Waaijers MM, Sprij-Mooij D, de Krijger RR, Speel EJ, Hofland J, Lamberts SW, de Herder WW et al: Endocr Relat Cancer 2013;Roh MR, Zheng Z, Kim HS, Jeung HC, Rha SY, Chung KY: Melanoma Res 2013, 23(2):114-124;Naik S, Nace R, Barber GN, Russell SJ: Cancer Gene Ther 2012, 19(7):443-450)。
1993年β干扰素作为治疗多发性硬化的推荐用药在美国和欧洲被批准上市，至今仍是FDA批准的用于多发性硬化治疗唯一的生物制品类药物，也是目前治疗多发性硬化最有效的药物(Paty DW, Li DK: Neurology 1993, 43(4):662-667)。
目前已上市的β干扰素主要有三种来源，分别为由人成纤维细胞经适度刺激分泌产生的天然人β干扰素、由大肠杆菌表达的非糖基化重组突变型人β干扰素和由中国仓鼠卵巢细胞表达的重组天然人β干扰素(Rodriguez J, Spearman M, Tharmalingam T, Sunley K, Lodewyks C, Huzel N, Butler M: J Biotechnol 2010, 150(4):509-518;Singh AB, Sharma AK, Mukherjee KJ: Mol Biosyst 2012, 8(2):615-628)。从人成纤维细胞中分离的天然人β干扰素十分有限，产量很低，价格昂贵，无法满足日益增长的临床需求；而利用哺乳动物细胞生产的β干扰素虽具有同天然人β干扰素相同的结构功能，但其生产成本高、周期长、条件苛刻且蛋白表达量低，同样严重制约了其在临床治疗中的广泛应用。相比较而言，大肠杆菌作为首个用于生产重组蛋白的工程菌，不仅具有生产成本低、生产周期短、转化效率高、培养条件简单、操作容易等优点，且其遗传背景清楚，表达外源基因产物的水平远高于其他表达系统。研究发现，胞内表达的β干扰素蛋白可占菌体总蛋白的10%~70%,且在大肠杆菌中表达的人β干扰素经纯化、复性后具有与天然β干扰素相同的生物学活性；可以满足β干扰素大量生产的需求。
多年以前国内诸多研究机构就开始了对β干扰素的研制，陈国友等构建了pLCM182-17Ser重组人β干扰素表达载体，将其转化大肠杆菌MM294，实现了β干扰素的热诱导表达，并探索了一种适于中试生产的发酵和纯化方法(陈国友，蒋应明，张意，黄欣，厉永健，施群英，王全兴，张明徽，何龙，曹雪涛，基因工程人β干扰素的原核表达及纯化研究. 中国肿瘤生物治疗杂志，2001,8(2):130-133)；中国疾病预防控制中心的李武平等使用大肠杆菌偏爱密码子人工合成了β干扰素1b基因，构建pBV220-IFN-β1b表达载体，转化大肠杆菌DH5α，实现了β干扰素的表达并进行了纯化(李武平，吕宏亮，段招军，张丽萍，刘永清，吴斌文，陈勇，张成海，衣作安，魏开坤，侯云德，干扰素-β1b的高效表达、纯化及抗病毒活性研究. 病毒学报，2002,18(3):240-244)；北京生物制品研究所研制的注射用重组人干扰素β1b是国内第一个获得临床研究批件的β干扰素生物制品(刘建源，张振龙，杨云凯，张琰平，邓宛明，樊晓翔，重组人干扰素-β的中试研究.生物技术通讯，2001,12(4):270-272)，目前正在进行临床II期研究，但是到现在为止尚没有任何国产的β干扰素上市。
此外，由于β干扰素的分子量小、易被肾小球的滤过作用清除，在体内的半衰期很短（皮下给药或肌肉注射的半衰期只有4-6小时），为了维持体内有效的血药浓度，需多次用药或采用蛋白质长效修饰技术延长β干扰素的半衰期。蛋白质的长效化修饰手段主要包括利用PEG、葡聚糖等进行化学手段修饰和采用基因工程手段表达融合蛋白进行修饰(Nairn NW, Shanebeck KD, Wang A, Graddis TJ, VanBrunt MP, Thornton KC, Grabstein K: Bioconjug Chem 2012, 23(10):2087-2097)。目前已上市的长效β干扰素主要是PEG化学修饰产物(Hu X, Miller L, Richman S, Hitchman S, Glick G, Liu S, Zhu Y, Crossman M, Nestorov I, Gronke RS et al: J Clin Pharmacol 2012, 52(6):798-808)。尽管PEG修饰能有效延长β干扰素的半衰期，但由于PEG修饰为化学反应，可能导致β干扰素部分活性丧失；据报道PEG在体内不能有效降解，部分人群产生抗PEG抗体，进一步限制了PEG长效修饰β干扰素的应用(Garay RP, El-Gewely R, Armstrong JK, Garratty G, Richette P: Expert Opin Drug Deliv 2012, 9(11):1319-1323)。此外采用重叠PCR技术将β干扰素cDNA与人血清白蛋白cDNA进行连接，得到融合基因整合到酵母表达宿主的染色体进行表达，可得到长效化的融合蛋白；利用β干扰素与人免疫球蛋白IgG的Fc片段进行融合表达，可获得β干扰素-Fc融合蛋白，延长β干扰素的半衰期，但由于人IgG存在同种异型，多次用药可导致患者体内产生抗体而降低融合蛋白的治疗效果(Dumont JA, Low SC, Peters RT, Bitonti AJ: BioDrugs 2006, 20(3):151-160;Vallee S, Rakhe S, Reidy T, Walker S, Lu Q, Sakorafas P, Low S, Bitonti A: J Interferon Cytokine Res 2012, 32(4):178-184;Zhang Q, Lei J, Ding Y, Chen Y, Qu L, Chen S, Jin J: Sheng Wu Gong Cheng Xue Bao 2009, 25(11):1746-1752)。
所述多聚唾液酸是N-乙酰神经氨酸通过N-连接糖苷键多聚形成的独特碳水化合物，研究发现多聚唾液酸化多肽或蛋白质在保持活性的前提下，修饰后可显著降低肽类化合物水解，延长半衰期，降低免疫原性(Gregoriadis G, Fernandes A, Mital M, McCormack B: Cell Mol Life Sci 2000, 57(13-14):1964-1969)。Fernandes等利用多聚唾液酸对L-天冬酰胺酶进行修饰，修饰后酶的抗原性和免疫原性均低于天冬酰胺酶，但修饰后酶抗胰蛋白酶的水解能力和体外半衰期明显增强(Fernandes AI, Gregoriadis G: Int J Pharm 2001, 217(1-2):215-224)；林怡等对铜锌超氧化物歧化酶进行多聚唾液酸修饰，修饰后产物的稳定性实验结果表明，多聚唾液酸修饰可提高铜锌超氧化物歧化酶的酸碱稳定性、热稳定性和抗酶降解能力(Wu JR, Lin Y, Zheng ZY, Lin CC, Zhan XB, Shen YQ: Biotechnol Lett 2010, 32(12):1939-1945)。此外，所述多聚唾液酸通过改变神经系统神经黏附分子的黏附性调节神经细胞发育，对神经细胞功能的维持起重要作用(Weber M, Modemann S, Schipper P, Trauer H, Franke H, Illes P, Geiger KD, Hengstler JG, Kleemann WJ: Neuroscience 2006, 138(4):1215-1223;Angata K, Huckaby V, Ranscht B, Terskikh A, Marth JD, Fukuda M: Mol Cell Biol 2007, 27(19):6659-6668)。
目前已知，重组多肽的氨基酸序列由天然存在的亲水性氨基酸组成，因而可生物降解且没有免疫原性，采用分子遗传操作将重组多肽与治疗性目的蛋白进行融合表达，可显著延长治疗性蛋白的半衰期(Schellenberger V, Wang CW, Geething NC, Spink BJ, Campbell A, To W, Scholle MD, Yin Y, Yao Y, Bogin O et al: Nat Biotechnol 2009, 27(12):1186-1190)。目前已有研究证实重组多肽与人生长激素形成的融合蛋白、与胰高血糖素形成的蛋白均能有效延长目的蛋白的半衰期；与胰高血糖素样肽2相比，重组多肽与其融合形成的重组蛋白在小鼠、大鼠及猴子体内的半衰期分别为34h、38h和120h(Cleland JL, Geething NC, Moore JA, Rogers BC, Spink BJ, Wang CW, Alters SE, Stemmer WP, Schellenberger V: J Pharm Sci 2012, 101(8):2744-2754;Alters SE, McLaughlin B, Spink B, Lachinyan T, Wang CW, Podust V, Schellenberger V, Stemmer WP: PLoS One 2012, 7(11):e50630)。此外，重组多肽与治疗性蛋白融合基因可通过大肠杆菌工程菌实现原核表达，与人血清白蛋白和人免疫球蛋白IgG Fc片段真核表达相比，更具有产业化优势；由于其生物相容性及生物可降解性优异，重组多肽有望成为延长药物蛋白半衰期最理想的修饰工具(Geething NC, To W, Spink BJ, Scholle MD, Wang CW, Yin Y, Yao Y, Schellenberger V, Cleland JL, Stemmer WP et al: PLoS One 2010, 5(4):e10175)。
发明内容
本发明的目的是提供一种长效干扰素及制备方法和用途，具体涉及一种用于抗病毒、抗感染、抗肿瘤和免疫调节的长效干扰素及制备方法和用途；所述的长效干扰素能减少干扰素的免疫原性，延长干扰素在体内的半衰期。
本发明所述的长效干扰素包括多聚唾液酸修饰的干扰素和长效干扰素重组融合蛋白，其中，
所述多聚唾液酸修饰的干扰素，由活化的多聚唾液酸末端第7位的单醛基在氰基硼氢化钠存在的情况下与干扰素反应而成的偶联物；其中，所述多聚唾液酸采用高碘酸钠进行激活；所述的干扰素包括但不限于α干扰素、β干扰素等I型干扰素及γ干扰素等II型干扰素及其同源类型；所述β干扰素的氨基酸序列如SEQ ID No.2所示。
所述长效干扰素重组融合蛋白为，由N-端干扰素与C-端天然存在的亲水性非重复序列多肽融合而成的重组蛋白；其中，所述的干扰素包括但不限于α干扰素、β干扰素等I型干扰素及γ干扰素等II型干扰素及其同源类型，优选β干扰素；所述天然存在的亲水性非重复序列多肽的氨基酸序列如SEQ ID No.1所示：
Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu
His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn （SEQ ID No.1）；
所述长效干扰素重组融合蛋白中，β干扰素的氨基酸序列如SEQ ID No.2所示：
MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN （SEQ ID No.2）。
本发明还提供了编码所述长效干扰素重组融合蛋白的亲水性非重复序列多肽的核苷酸序列，如SEQ ID No.3所示：
Atgagctacaacttgcttggattcctacaaagaagcagcaattttcagagtcagaagctcctgtggcaattgaatgggaggcttgaatattgcctcaaggacaggatgaactttgacatccctgaggagattaagcagctgcagcagttccagaaggaggacgccgcattgaccatctatgagatgctccagaacatctttgctattttcagacaagattcatctagcactggctggaatgagactattgttgagaacctcctggctaatgtctatcatcagataaaccatctgaagacagtcctggaagaaaaactggagaaagaagatttcaccaggggaaaactcatgagcagtctgcacctgaaaagatattatgggaggattctgcattacctgaaggccaaggagtacagtcactgtgcctggaccatagtcagagtggaaatcctaaggaacttttacttcattaacagacttacaggttacctccgaaac （SEQ ID No.3）；
本发明中，编码所述长效干扰素重组融合蛋白的核苷酸序列如SEQ ID No.4所示：
atgagctacaacttgcttggattcctacaaagaagcagcaattttcagagtcagaagctcctgtggcaattgaatgggaggcttgaatattgcctcaaggacaggatgaactttgacatccctgaggagattaagcagctgcagcagttccagaaggaggacgccgcattgaccatctatgagatgctccagaacatctttgctattttcagacaagattcatctagcactggctggaatgagactattgttgagaacctcctggctaatgtctatcatcagataaaccatctgaagacagtcctggaagaaaaactggagaaagaagatttcaccaggggaaaactcatgagcagtctgcacctgaaaagatattatgggaggattctgcattacctgaaggccaaggagtacagtcactgtgcctggaccatagtcagagtggaaatcctaaggaacttttacttcattaacagacttacaggttacctccgaaacGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCATGActcgag （SEQ ID No.4）。
本发明所述长效干扰素重组融合蛋白中，所述多肽以SEQ ID No.1所示的氨基酸序列为单位进行重复组合，与干扰素的N-端或C-端进行融合；其组合方式包括但不限于N-端干扰素和C-端亲水性无重复序列多肽的组合。
本发明还提供了一种含有如SEQ ID No.3或SEQ ID No.4所示核苷酸序列的载体；一种含有如SEQ ID No.3或SEQ ID No.4所示核苷酸序列的宿主细胞。
本发明所述长效干扰素能延长干扰素的体内半衰期、同时保持干扰素的活性、降低其免疫原性；
本发明的长效干扰素进一步可制备治疗抗病毒药物、抗感染药物、抗肿瘤药物或免疫调节性的药物；用于肝炎病毒、SARS、单纯疱疹病毒等病毒感染性疾病的治疗，同时也可用于恶性神经胶质瘤、肺癌等恶性肿瘤及其他细胞增殖性疾病的治疗。
本发明的长效干扰素重组融合蛋白可在大肠杆菌中高效表达，且制备工艺简单，可用于大规模的药物级融合蛋白的生产制备。
附图说明
图1为β干扰素的多聚唾液酸修饰SDS-PAGE图谱，其中，
M Marker; 1-2 β干扰素 interferon-β; 3 PSA 修饰后的β干扰素 interferon-β modified by PSA。
图2为β干扰素-XTEN目的基因的PCR产物凝胶电泳图谱，其中，
1 marker； 2-3 PCR产物 PCR product；4 marker。
图3为本发明中所述亲水性非重复序列多肽与β干扰素融合蛋白编码基因序列。
图4为本发明中所述重组质粒的酶切鉴定图谱，其中，
1 marker；2 pMD19-T-IFNβ-XTEN NcoI+XhoI双酶切 pMD19-T-IFNβ-XTEN NcoI+XhoI digestion；3 pET22b NcoI+XhoI双酶切 pET24b NcoI+XhoI digestion；4 marker 。
图5为本发明中所述重组表达载体的酶切鉴定图谱，其中，
1 marker；2-5 pET22b-IFNβ-XTEN NcoI+XhoI双酶切 pET22b-IFNβ-XTEN NcoI+XhoI digestion；6 marker。
图6为本发明中采用PCR方法克隆含有NcoI和XhoI酶切位点的融合蛋白编码基因序列，并用1%琼脂糖凝胶电泳检测图谱，其中，
M为DL5000 ；1为pET24b双酶切回收产物；2为融合蛋白编码基因双酶切回收产物。
图7为本发明实施例3中酶切产物观察图，其中，
M为DL5000 ；1为pET24b-融合蛋白编码基因双酶切产物。
图8图8 β干扰素-XTEN融合蛋白诱导表达的SDS-PAGE图谱，其中，
M marker；1 未诱导的对照 untreated control；2-5 诱导产物 induced product。
具体实施方式
以下结合附图通过具体实施例进一步说明但不限定本发明。
实施例1 多聚唾液酸对重组人β干扰素的化学修饰
（1）多聚唾液酸的活化
①将多聚唾液酸按10mg多聚唾液酸/ml高碘酸钠的比例与新鲜配制的100mM 高碘酸钠混合，置于20℃暗处搅拌5min；
②加入两倍体积的乙二醇中和过量的高碘酸钠，置于20℃暗处继续搅拌30min；
③将氧化的多聚唾液酸置于0.01%碳酸铵缓冲液（pH7.4）中4℃透析24h，透析袋应能截留3.5KDa的分子；
④以分子量为8KDa的干燥聚乙二醇覆盖反相透析浓缩多聚唾液酸溶液；
⑤将透析液冻干，置于-40℃保存备用；
（2）多聚唾液酸修饰β干扰素的反应
①β干扰素采用含0.1%SDS pH7.4 10mM的磷酸盐缓冲液稀释成浓度为0.2mg/ml的溶液；
②在4mg/ml氰基硼氢化钠存在的情况下，活化的多聚唾液酸与β干扰素摩尔比按50:1进行混合；
③将反应容器置于35℃的环境中密闭搅拌反应48h；
④反应结束后用70%硫酸铵沉淀蛋白，4℃搅拌1h后离心收集沉淀；
⑤将沉淀用pH7.4 10mM的磷酸盐缓冲液溶解并在相同的缓冲液中4℃透析24h。
取活化的多聚唾液酸与纯化的β干扰素按摩尔比50:1进行反应,控制氰基硼氢化钠的用量为4mg/ml,蛋白浓度为0.2mg/ml，反应体系为5ml，35℃密闭搅拌反应48h。对反应后的产物与10mM PBS pH7.4溶液中透析24h，浓缩反应产物并进行SDS-PAGE凝胶电泳分析；实验结果如图1所示，与未反应的β干扰素相比，经多聚唾液酸修饰的β干扰素分子量明显增加，一单位的多聚唾液酸的分子量为30KDa，反应后产物的分子量约为50KDa，与预期分子量大小一致。
实施例2 获得亲水性非重复序列多肽与β干扰素融合蛋白编码基因及构建含有信号肽表达载体
人工化学合成亲水性非重复序列多肽与β干扰素融合蛋白编码基因序列，并将其克隆至pUC57 simple质粒载体中，得到含有融合蛋白编码基因的质粒；
首先以该质粒中的融合蛋白编码基因序列为模板，采用Primer设计引物，上游引物为：5’ actgCCATGGCCAGCTACAACTTGCTTGGAT 3’，其中加下划线处为NcoI酶切位点；下游引物：5’ agtCTCGAGTCATGGTGCGGTAGAAGAT 3’,其中加下划线处为XhoI酶切位点；引物由上海生工生物工程公司合成；
采用PCR方法克隆含有NcoI和XhoI酶切位点的融合蛋白编码基因序列，并用1%琼脂糖凝胶电泳检测，实验结果如图2所示，PCR产物在1000bp左右，与β干扰素-XTEN目的基因预期大小（942bp）相符。
PCR反应I：在0.2ml的PCR管中加入以下成分并混合均匀：
2X Taq Mix 25μl；上游引物 2μl；下游引物 2μl；pUC57-融合蛋白编码基因质粒 1μl；超纯水 20μl。
PCR反应程序为：94℃，5min；94℃，1min；55℃，1min；72℃，2min；72℃，30min。一共进行30个循环反应。
将克隆到的PCR产物连接pMD19-T simple载体，挑取阳性克隆，送至上海英骏贸易有限公司进行测序，测序结果如图3所示，经比对与人工化学合成的核苷酸序列一致。
取pET22b表达载体及pMD19-融合蛋白编码基因质粒，采用NcoI及XhoI限制性核酸内切酶进行双酶切。双酶切的反应体系为20ul，其中质粒10ul，NcoI1ul，XhoI1ul，10X buffer H 2ul,无菌水6ul。37℃酶切5h。酶切产物采用1%琼脂糖凝胶电泳分离，实验结果如图4所示。采用胶回收试剂盒回收目的片段，其中pET22b表达载体回收5000bp左右的片段，pMD19-IFNβ回收1000p的片段。
连接反应在T4 DNA连接酶催化下进行，pET22b表达载体酶切片段与IFNβ目的片段的摩尔数比约为1:3。连接反应体系为25ul，其中T4 连接酶1ul，10X T4 DNA 连接酶缓冲液2.5ul，16℃连接过夜。
连接产物转化感受态大肠杆菌DH5α，涂布于氨苄青霉素抗性LB平板上，37℃培养过夜，挑取单克隆，扩大培养后抽提质粒，采用NcoI及XhoI双酶切。酶切产物采用1%琼脂糖凝胶电泳分离，观察酶切产物的大小，实验结果如图5所示。将扩大培养的菌液采用pET载体通用引物测序，测序反应由上海英潍捷基贸易有限公司完成。
实施例3 构建不含信号肽的亲水性非重复序列多肽与β干扰素融合蛋白编码基因表达载体
人工化学合成亲水性非重复序列多肽与β干扰素融合蛋白编码基因序列，并将其克隆至pUC57 simple质粒载体中，得到含有融合蛋白编码基因的质粒。
首先以该质粒中的融合蛋白编码基因序列为模板，采用Primer设计引物，上游引物为：5’ CATATGAGCTACAACTTGCTTGGAT 3’，其中加下划线处为NdeI酶切位点；下游引物：5’ agtCTCGAGTCATGGTGCGGTAGAAGAT 3’,其中加下划线处为XhoI酶切位点；引物由上海生工生物工程公司合成。
采用PCR方法克隆含有NcoI和XhoI酶切位点的融合蛋白编码基因序列，并用1%琼脂糖凝胶电泳检测，实验结果如图6所示：PCR产物在1000bp左右，与β干扰素-XTEN目的基因预期大小（942bp）相符。
PCR反应I：在0.2ml的PCR管中加入以下成分并混合均匀：
2X Taq Mix 25μl；上游引物 2μl；下游引物 2μl；pUC57-融合蛋白编码基因质粒 1μl；超纯水 20μl。
PCR反应程序为：94℃，5min；94℃，1min；55℃，1min；72℃，2min；72℃，30min。一共进行30个循环反应。
将克隆到的PCR产物连接pMD19-T simple载体，挑取阳性克隆，送至上海英骏贸易有限公司进行测序，测序结果经比对与人工化学合成的核苷酸序列一致。
取pET24b表达载体及pMD19-融合蛋白编码基因质粒，采用NdeI及XhoI限制性核酸内切酶进行双酶切。双酶切的反应体系为20ul，其中质粒10ul，NdeI1ul，XhoI1ul，10X buffer H 2ul,无菌水6ul。37℃酶切5h。酶切产物采用1%琼脂糖凝胶电泳分离，采用胶回收试剂盒回收目的片段，其中pET24b表达载体回收5000bp左右的片段，pMD19-IFNβ回收1000p的片段。
连接反应在T4 DNA连接酶催化下进行，pET24b表达载体酶切片段与IFNβ目的片段的摩尔数比约为1:3。连接反应体系为25ul，其中T4 连接酶1ul，10X T4 DNA 连接酶缓冲液2.5ul，16℃连接过夜。
连接产物转化感受态大肠杆菌DH5α，涂布于氨苄青霉素抗性LB平板上，37℃培养过夜，挑取单克隆，扩大培养后抽提质粒，采用NdeI及XhoI双酶切。酶切产物采用1%琼脂糖凝胶电泳分离，观察酶切产物的大小，实验结果如图7所示。将扩大培养的菌液采用pET载体通用引物测序，测序反应由上海英潍捷基贸易有限公司完成。
实施例4 亲水性非重复序列多肽与β干扰素融合蛋白的表达
将测序正确的pET22b-融合蛋白编码基因质粒转化感受态大肠杆菌TransB （DE3)，涂布于氨苄青霉素、硫酸卡那霉素和四环霉素三抗性LB平板，37℃培养过夜。随机挑取平板上4个单菌落，扩大培养，按1%的比例转接至3ml 新鲜LB试管中，37℃震荡培养至OD600≈0.6时，分别取样加入终浓度为0.5mM和1mM的IPTG，继续震荡培养3h，取1ml菌液离心收集菌体，进行聚丙烯酰胺凝胶电泳检测分析；实验结果如图8所示，与未经IPTG诱导对照组相比，IPTG诱导的实验组在30KDa附近有目的蛋白表达，大小与预期相符。
SEQUENCE LISTING
<110> 复旦大学
<120> 长效干扰素及其制备方法和用途
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 166
<212> PRT
<213> 亲水性非重复多肽
<400> 1
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gaatcttcta ctgctccagg tacctctcct agcggcgaat cttctaccgc tccaggtacc 900
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Claims (12)
1.一种长效干扰素，其特征在于，其为多聚唾液酸修饰的干扰素或长效干扰素重组融合蛋白，其中，
所述的多聚唾液酸修饰的干扰素，由活化的多聚唾液酸末端第7位的单醛基在氰基硼氢化钠存在的情况下与干扰素反应而成；
所述的长效干扰素重组融合蛋白，由N-端干扰素与C-端天然存在的亲水性非重复序列多肽融合而成。
2.按权利要求1所述的长效干扰素，其特征在于，所述的干扰素选自α干扰素、β干扰素或γ干扰素及同源类型干扰素。
3.按权利要求2所述的长效干扰素，其特征在于，所述β干扰素的氨基酸序列如SEQ ID No.2所示。
4.按权利要求1所述的长效干扰素，其特征在于，所述多聚唾液酸采用高碘酸钠进行激活。
5.按权利要求1所述长效干扰素，其特征在于，所述长效干扰素重组融合蛋白中，天然存在的亲水性非重复序列多肽的氨基酸序列如SEQ ID No.1所示。
6.按权利要求1所述长效干扰素，其特征在于，编码所述长效干扰素重组融合蛋白的亲水性非重复序列多肽的核苷酸序列如SEQ ID No.3所示。
7.按权利要求1所述长效干扰素，其特征在于，编码所述长效干扰素重组融合蛋白的核苷酸序列如SEQ ID No.4所示。
8.按权利要求1所述长效干扰素，其特征在于，所述多肽以SEQ ID No.1所示的氨基酸序列为单位进行重复组合，与干扰素的N-端或C-端进行融合；其组合方式为N-端干扰素和C-端亲水性无重复序列多肽的组合。
9.一种载体，含有如SEQ ID No.3或SEQ ID No.4所示核苷酸序列。
10.一种宿主细胞，含有如SEQ ID No.3或SEQ ID No.4所示核苷酸序列。
11.权利要求1~8中任一项所述的长效干扰素在制备治疗抗病毒药物、抗感染药物、抗肿瘤药物或免疫调节性药物中的用途。
12.按权利要求11所述的用途，其特征在于，所述药物为治疗肝炎病毒药物、治疗SARS药物、治疗单纯疱疹病毒药物、治疗恶性神经胶质瘤药物、治疗肺癌药物或治疗其他细胞增殖性疾病的药物。
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| CN106146668A (zh) * | 2015-04-01 | 2016-11-23 | 中国人民解放军第二军医大学 | 长效白细胞介素22的制备方法及其用途 |
| CN106832004A (zh) * | 2017-02-20 | 2017-06-13 | 中国科学院微生物研究所 | 一种口服禽干扰素融合蛋白及其作为免疫增强剂的应用 |
| CN107099569A (zh) * | 2017-06-30 | 2017-08-29 | 北京北生研生物制品有限公司 | 一种规模化发酵生产重组人干扰素β1b蛋白的方法 |
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| US20080003202A1 (en) * | 2006-03-28 | 2008-01-03 | Thierry Guyon | Modified interferon-beta (IFN-beta) polypeptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106146668A (zh) * | 2015-04-01 | 2016-11-23 | 中国人民解放军第二军医大学 | 长效白细胞介素22的制备方法及其用途 |
| CN106832004A (zh) * | 2017-02-20 | 2017-06-13 | 中国科学院微生物研究所 | 一种口服禽干扰素融合蛋白及其作为免疫增强剂的应用 |
| CN106832004B (zh) * | 2017-02-20 | 2019-09-24 | 中国科学院微生物研究所 | 一种口服禽干扰素融合蛋白及其作为免疫增强剂的应用 |
| CN107099569A (zh) * | 2017-06-30 | 2017-08-29 | 北京北生研生物制品有限公司 | 一种规模化发酵生产重组人干扰素β1b蛋白的方法 |
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