CN105734066B - 一种含有重组乙肝病毒基因的真核汉逊酵母工程菌的构建及乙肝表面抗原的生产方法 - Google Patents
一种含有重组乙肝病毒基因的真核汉逊酵母工程菌的构建及乙肝表面抗原的生产方法 Download PDFInfo
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Abstract
本发明公开一种含有重组乙肝病毒基因的真核汉逊酵母工程菌的构建及乙肝表面抗原的生产方法，属于生物工程技术领域，构建方法包括：步骤1，构建含有重组乙型肝炎病毒HBsAg基因的质粒pHPZF1.0‑ZS；步骤2，筛选得到表达水平最高的含有重组乙肝病毒HBsAg基因的真核汉逊酵母工程菌。采用本发明所述构建及生产方法，可以获得的乙型肝炎表面抗原汉逊酵母酵母工程菌株能够以甲醇诱导型方式稳定和高效地表达HBsAg重组蛋白，适用于规模化生产HBsAg重组蛋白。
Description
技术领域
本发明涉及一种含有重组乙肝病毒基因的真核汉逊酵母工程菌的构建及乙肝表面抗原的生产方法，属于生物工程技术领域。
背景技术
乙型肝炎(简称乙肝)疫苗已大规模使用愈20年，但乙型肝炎病毒(HepatitisBvirus，HBV)感染仍是最为严重的全球性健康问题之一。据世界卫生组织报道，全世界约20亿人有现状或既往乙肝病毒感染标志，每年约有60万人死于HBV感染引起的肝衰竭、肝硬化和肝细胞癌。我国乙肝病毒携带者超过1亿，现有患者超过2000万，南方流行重于北方，农村重于城市。目前并无特异性治疗乙肝的药物，最有效的手段就是接种乙肝疫苗。
第一代乙肝疫苗为血源疫苗，采用无症状带毒者(HBsAg阳性)血浆为原料制备。由于提取方法不同，所得成分也有差别，但均含提纯的22nm小颗粒乙型肝炎表面抗原。80年代以来开始研制第二代乙肝疫苗，系将S基因编码的226个氨基酸产物装配而成的抗原颗粒，采用基因工程方法在哺乳动物细胞和重组酵母中均能表达。重组乙型肝炎疫苗(汉逊酵母)乙肝疫苗是最新一代基因工程乙肝疫苗，是继血源疫苗、酿酒酵母疫苗之后的第三代疫苗，系国家863重大科技攻关成果，已有2000万名受种者验证了其安全性。对HBsAg阳性母亲所生新生儿，本市已将母婴阻断率较高的重组(汉逊酵母)乙肝疫苗纳入免疫规划，在出生12小时内给予免费接种；在本地的临床观察中，健康成人全程接种后1个月，97.46％的受种者抗体阳转；全程接种后半年，59.8％的受种者抗体水平在1000mIU/mL以上，因而保护期更长。按照世界卫生组织资料，不同乙肝疫苗的相对效力不以HBsAg含量来判断，10ug重组(汉逊酵母)疫苗适用于任意年龄的易感人群，能提供全面高效持久的免疫保护屏障。免疫预防为主，有效遏制乙肝的高流行状态，基因重组(汉逊酵母)技术为乙型肝炎的免疫预防提供了一种全新的选择。
多形汉逊酵母(Hansenula Polymorpha)，又称作Pichia augusta，是当前公认的最为理想的外源基因表达系统之一。汉逊酵母也是一种甲醇营养型酵母，与毕赤酵母类似。汉逊酵 母中甲醇代谢的主要途径有两种(LodeboerA.M.，etal.，1985)：一种是在过氧化物酶体中进行，甲醇在甲醇氧化酶(methanoloxidase，MOX)作用下生成甲醛和H2O2，甲醛再经甲醛脱氢酶(formaldehyde dehydrogenase)和甲酸脱氢酶(formate dehydrogenase，FMD)作用下生成CO2，H2O2在过氧化氢酶(catalase，CAT)的作用下生成H2O和O2；甲醇的另一个代谢途径发生在过氧化物酶体外，甲醇在细胞质中经过一系列酶的催化作用最后转变为糖类，其中二羟丙酮合成酶(dihydroxy acetone synthase，DHAS)是这一过程的关键酶。甲醇代谢过程中的各种关键酶，包括MOX、DHAS和CAT等的表达都是在转录水平进行调解的，它们受甲醇、甘油和山梨醇的诱导，受葡萄糖和乙醇的阻遏，但当葡萄糖浓度低于0.1％时，阻遏作用即被解除。在甲醇完全诱导条件下，过氧化物酶体可占细胞总体积的80％，MOX和DHAS可占细胞总蛋白的15％，它们的启动子具有极强的启动下游基因表达的功能，目前已被用作外源基因在酵母中表达的常用启动子。汉逊酵母作为单细胞真核微生物，既具备原核生物生长快速、易于遗传操作等特点，又有真核细胞翻译后加工和修饰等功能。此外，汉逊酵母还具备安全性好、易于培养、成本低廉、表达量高及遗传稳定等优势，并且能够克服诸如酿酒酵母(Saccharomy cescerevisiae)菌株不稳定、产量低及糖基化侧链过长以及毕赤酵母(Pichia Pastoris)外源基因整合拷贝数较低的问题。目前，应用汉逊酵母表达系统生产的药物(如胰岛素，商品名Wosulin)及HBV疫苗(商品名Hepavax-Gene)均已上市销售。
发明内容
针对上述现有技术存在的问题，本发明提供一种含有重组乙肝病毒基因的真核汉逊酵母工程菌的构建、及重组乙肝表面抗原的生产方法，适用于规模化生产HBsAg重组蛋白。
为了实现上述目的，本发明采用的一种adr型乙型肝炎病毒表面抗原HBsAg基因，具有序列表中SEQ ID No.1所述的氨基酸序列。
本发明还提供了一种优化adr型乙型肝炎病毒表面抗原HBsAg基因，以上述序列表SEQ ID No.1所述氨基酸序列为基础，结合宿主菌汉逊酵母基因组密码子偏好性，合成优化HBsAg基因核苷酸序列，其具有序列表中SEQ ID No.2所述的核苷酸序列。
本发明还提供了一种包含优化adr型乙型肝炎病毒表面抗原HBsAg基因的重组质粒，将优化后基因加入到汉逊酵母表达载体pHPZF1.0中，所述重组质粒为胞内型质粒pHPZF1.0-ZS。
本发明提供了一种包含上述重组质粒的重组菌，所述重组菌的宿主菌为真核汉逊酵母菌株ATCC34438或其衍生菌种。
本发明提供了一种包含adr型乙型肝炎病毒表面抗原HBsAg基因的真核汉逊酵母工程菌，分类命名为HBsAg1.0-38G1Hansenula ploymorpha HBsAg1.0-38G1，并于2016年1月15日保藏于位于中国武汉武汉大学的中国典型培养物保藏中心，保藏号CCTCC No：M2016039。
本发明还提供了一种重组adr型乙肝病毒表面抗原HBsAg基因的真核汉逊酵母工程菌的构建方法，按照下述步骤进行：
步骤1，以SEQ ID No.2所述核苷酸序列人工合成HBsAg基因，将其重组入表达载体pHPZF1.0，构建含有重组乙型肝炎病毒HBsAg基因的质粒pHPZF1.0-ZS；
步骤2，将步骤1制备的质粒pHPZF1.0-ZS转化真核汉逊酵母菌株ATCC34438或其衍生菌种，经筛选得到表达水平最高的含有重组乙肝病毒HBsAg基因的真核汉逊酵母工程菌。
本发明还提供了一种利用真核汉逊酵母工程菌HBsAg1.0-38G1表达和生产adr型乙型肝炎病毒表面抗原HBsAg的方法，包括以下步骤：
1)种子液制备：取1ml真核汉逊酵母工程菌HBsAg1.0-38G1工作种子，转接于1.2LBMGY培养基中，在35℃下振荡培养48小时，待OD600值达到2～6后，将其作为种子液，备用；
2)初培养阶段：取15L基础发酵培养基，用14％氨水调整并维持至pH为5.5，按照每升基础发酵培养基中加4.37mL微量盐溶液PTM1的比例，添加65.55mlPTM1，在35℃下，自动控制溶氧不低于30％；
3)甘油流加培养阶段：待甘油耗尽，溶氧上升时，流加甘油，通过蠕动泵流加50％甘油，其中含有12mL PTM4/L，甘油补加速度为2G/min，至菌体湿重为250g/L后，调节转速和通气量使溶氧不低于20％，此阶段，用14％氨水将pH值缓慢调节至6.0；
4)甲醇诱导阶段：停止甘油流加后，让菌体饥饿30min，通过蠕动泵流加补料液，补料液为100％甲醇，其中含有12mL PTM4/L，甲醇浓度维持在6.0～6.4％(v/v)，pH用14％氨水调整并维持至6.0，调节转速和通气量，控制溶氧量不低于20％至菌体湿重约300g/L，再将转速调到大于1300rpm，空气通量最大，至发酵总时100h下罐；
5)发酵液经5000rpm4℃离心15分钟，留取沉淀。
作为改进，所述步骤2)中基础发酵培养基由4.29％磷酸二氢钾，0.5％硫酸铵，1.43％硫酸钾，1.17％七水硫酸镁，0.10％二水硫酸钙，0.5882％二水柠檬酸钠，3.00％甘油组成。
作为改进，所述步骤2)中PTM1由0.6％硫酸铜，0.008％碘化钠，0.3％硫酸锰，0.02％钼酸钠，0.002％硼酸，0.05％氯化钴，2％氯化锌，6.5％硫酸亚铁，0.025％生物素，0.5％硫酸组成。
作为改进，所述步骤3)和4)中PTM4成分组成为五水硫酸铜，0.008％碘化钠，0.3％一水硫酸锰，0.02％二水钼酸钠，0.002％硼酸，0.05％二水硫酸钙，0.05％氯化钴，0.7％氯化锌，2.20％七水硫酸亚铁，0.02％生物素，0.10％硫酸。
采用本发明所述方法，可以获得的乙型肝炎表面抗原汉逊酵母酵母工程菌株能够以甲醇诱导型方式稳定和高效地表达HBsAg重组蛋白，适用于规模化生产HBsAg重组蛋白。
附图说明
图1为甲醇诱导型汉逊酵母高效表达载体pHPZF1.0的构建全过程；
图2为乙型肝炎病毒表面抗原HBsAg高效表达重组载体pHPZF1.0-ZS的构建全过程；
图3为SDS-PAGE电泳检测不同重组子HBsAg重组蛋白表达情况：
M为标准分子量蛋白(PageRulerTM Plus Prestained Protein Ladder partNo.26616 10～170KDa，Thermo Scientific公司产品)；
1为阴性对照，pHPZF1.0空载体转化的重组酵母菌株培养120小时破碎液；
2～9分别源自pHPZF1.0-ZS转化酵母不同重组子培养120小时破碎液；
图4为Western-Blot电泳检测不同重组子HBsAg重组蛋白表达情况；
M为标准分子量蛋白(PageRulerTM Plus Prestained Protein Ladder partNo.26616 10～170KDa，Thermo Scientific公司产品)；
1为阴性对照，pHPZF1.0空载体转化的重组酵母菌株培养120小时破碎液；
2～9分别源自pHPZF1.0-ZS转化酵母不同重组子培养120小时破碎液；
Western-Blot使用的一抗为抗HBsAg的鼠源原抗(Hep B HBsAg(1023):sc-53299，SANTA CRUZ公司产品)。
图5为发酵过程中菌体湿重和SDS-PAGE电泳检测抗原表达的变化情况：
M为标准分子量蛋白(PierceTM unstained Protein Molecular Weight Markerpart No. 26610 14.4～116KDa，Thermo Scientific公司产品)；
1～10分别为源自工程菌HBsAg1.0-38G1不同发酵时间点菌体的破碎液，对应的时间点分别为24、48、54、60、66、72、78、84、90和96小时，HBsAg重组蛋白的分子量20～25kD；
图6为重组蛋白HBsAg层析纯化后的蛋白检测：
M为标准分子量蛋白(PierceTM unstained Protein Molecular Weight Markerpart No.26610 14.4～116KDa，Thermo Scientific公司产品)；
其中，1为破碎液上清；2为阴离子交换层析产物；3为阳离子交换层析产物；4为300K中空纤维柱浓缩后产物；5为分子筛产物；
图7为纯化后的重组蛋白HBsAg的HPSEC检测图谱。
具体实施方式
下面以汉逊酵母组成型表达HBsAg蛋白为例，描述一些优先的实施方案，但本发明的应用不仅限于此。本发明的进一步描述只是一些特别的优先实施方案，是按照专利申请要求以及为了解释和说明本专利的内容。很显然，在不背离本发明的精神和范围之内，可在此基础上，做进一步的改进和变化。
利用常规的分子生物学技术(内切酶和连接酶处理)，将MOX启动子和终止子插入到pPICZ C中并取代载体原位置上的诱导型启动子-乙醇氧化酶启动子和终止子，得到汉逊酵母表达载体pHPZF1.0；
实施例1：汉逊酵母甲醇氧化酶启动子和终止子的克隆
根据已知序列(GenBank：A11156，AR363832和E00783)，分别合成位于MOX基因启动子两端的引物MOX_P-F和MOX_P-R，
其中MOX_P-F为5′-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3′，
MOX_P-R1为5′-GGAACCTCCACCAACAACAATGATATCGAAT-3′，和合成位于MOX基因终止子两端的引物MOX_TT-F1和MOX_TT-R1，
其中MOX_TT-F1为5′-TCGGAACTTACGAGGAGACCGGACTTGCCAG-3′，
MOX_TT-R1为5′-CTTGTGTCTCACACCCATAATGATCCCGTT-3′，以汉逊酵母基因组DNA为模板(基因组提取方法参见《分子克隆实验指南第三版》485页)，通过PCR，扩增获得MOX启动子和终止子，将扩增片段直接插入到pEASY-Blunt-Zero质粒中，按照该公司所提供的方法，分别得到含有中间质粒载体pMOX-P的细菌克隆和pMOX-TT的细菌克隆(见附图3、图4)，然后，通过核苷酸测序分析，确定MOX启动子(MOX-P)和MOX终止子(MOX-TT)是正确和完整的，分别见SEQ ID NO：3和SEQ ID NO：4。
实施例2：表达载体pHPZF1.0的构建
从pMOX-P质粒上PCR扩增MOX-P启动子，利用BglII-EcoRI位点克隆入载体pPICZC,得到质粒pPICZC-MOXP；根据测序验证得到的MOX启动子序列，重新设计引物MOX_P-R2。以MOX_P-F和MOX_P-R2为引物，
其中MOX_P-F为5′-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3′(划线部分为BglII位点)，MOX_P-R2为5′-CACGTGAATTCCTCGTTTCGAAGCTTTGTTTTTGTACTTTAGATT-3′(划线部分为EcoRI位点)，以中间质粒载体pMOX-P DNA为模板(质粒提取方法参见《分子克隆实验指南第三版》485页)，通过PCR，扩增获得MOX启动子，PCR产物两端引入BglII和EcoRI位点。用限制性内切酶BglII-EcoRI进行双酶切，通过琼脂糖凝胶电泳分离和回收该DNA片段。用同样的限制性内切酶处理质粒pPICZ C(美国Invitrogen公司产品)，通过琼脂糖凝胶电泳分离和回收线性化后的pPICZ C质粒DNA，然后将上述两个DNA片段混合后并用连接酶连接在一起便得到中间质粒载体pPICZC-MOXP(见附图1)，然后用上述质粒载体转化大肠杆菌细胞DH5α(美国Invitrogen公司产品)以方便进行该质粒的复制和保存，最后，通过核苷酸测序分析，确定MOX启动子序列和插入位点的正确和完整的。
从pMOX-TT质粒上PCR扩增MOX-TT终止子，利用SalI-BamHI位点克隆入上面中间质粒pPICZC-MOXP,得到汉逊酵母表达载体pHPZF1.0；根据测序验证得到的MOX终止子序列，重新设计引物MOX_TT-F2和MOX_TT-R2。
其中MOX_TT-F2为5′-CCAATGTCGACCATCATCATCATCATCATTGAGGAGACGTGGAAGGACATAC-3′(划线部分为SalI位点)，
MOX_TT-R2为5′-CAATTAGATCTGCTAGCATTGGGGATCCGGGATATCACCACAACGTCCG-3′(划线部分为BglII位点)，以中间质粒载体pPICZC-MOXP为模板(质粒提取参见质粒小量DNA提取试剂盒AP-MN-P-250，Axygen公司产品)，通过PCR，扩增获得MOX终止子，PCR产物两端引入SalI和BglII位点，其中BglII与BamHI是同尾酶。用限制性内切酶SalI-BglII进行双酶切，通 过琼脂糖凝胶电泳分离和回收该DNA片段。用SalI-BamHI限制性内切酶处理质粒pPICZC-MOXP，通过琼脂糖凝胶电泳分离和回收线性化后的pPICZC-MOXP质粒DNA，然后将上述两个DNA片段混合后并用连接酶连接在一起便得到得到汉逊酵母表达载体pHPZF1.0(见附图1)，然后用上述质粒载体转化大肠杆菌细胞DH5α(美国Invitrogen公司产品)以方便进行该质粒的复制和保存，最后，通过核苷酸测序分析，确定MOX启动子序列和插入位点的正确和完整的。
实施例3：adr亚型HBsAg共有氨基酸序列的分析
由于基因型C包含了所有adr血清型，因此我们以日本报道的乙肝病毒C型标准基因组序列AY123041为检索序列，Blast NCBI核酸数据库(相似性参数设置为93～100％)，共检索HBV基因组序列近2000条。经排查，其中中国报道序列基因型C的HBV基因组序列617条，排除测序错误和HBsAg无义突变序列，共获得中国报道的adr血清型HBsAg蛋白序列479条(其中25条来自香港序列，4条来自台湾地区)。使用BioEdit软件功能进行氨基酸序列比对分析，获得最具代表性的HBsAg共有氨基酸序列(consensus amino acid sequence，即在HBsAg每个氨基酸位置均采用出现概率最高的氨基酸残基的序列)，其序列如SEQ ID NO：1所示。
实施例4：密码子优化后的HBsAg基因的人工合成
HBsAg基因源于乙型肝炎病毒，其密码子为哺乳动物生物所偏好，而汉逊酵母属于真菌，故它们在基因密码子偏好方面存在着一定的差异，而这种差异很可能会影响到HBsAg基因及其转录产物在汉逊酵母细胞中的稳定性和表达效率。为提高HBsAg的生物产量，在不改变其氨基酸序列的前提下(见SEQIDNO：1)，按照酵母所偏爱的密码子(SharpPM，etal.，1986)，人工设计和合成了新的HBsAg蛋白的DNA编码序列HBsAg-ZS(见SEQ ID NO：2)。与此同时，为了便于此后基因克隆和重组方便，其合成的DNA序列需要避免以下酶切位点SphI、ScaI、HindIII、BamHI、NheI、BstBI、EcoRI和KpnI。
实施例5：重组质粒pHPZF1.0-ZS的构建
用限制性内切酶HindIII和SalI进行双酶切，将优化合成得到的新HBsAg基因与载体pHPZF1.0用连接酶连接在一起便得到汉逊酵母表达载体pHPZF1.0-ZS(见附图2)，然后用上述质粒载体转化大肠杆菌细胞JM109(购自美国GIBCO公司)以方便进行该质粒的复制和保存。
实施例6：线性化质粒表达载体DNA的制备
首先用试剂盒提取质粒(详细步骤参见质粒小量DNA提取试剂盒AP-MN-P-250，Axygen公司产品)，从上述的大肠杆菌DH5α(美国Invitrogen公司产品)细胞中制备提取pHPZF1.0-ZS质粒DNA，然后用1～2倍过量的限制性内切酶NsiI进行酶切，使之完全线性化，可利用琼脂糖凝胶电泳检测酶切是否完全；然后回收试剂盒(详细步骤参见胶回收试剂盒Wizard SV Gel and PCR Clean-up System，Promega公司产品)，使用无菌的去离子水洗脱，-20℃保存，备用。
实施例7：酵母细胞的转化
将-80℃保存的汉逊酵母(购自美国ATCC菌种保藏中心)接种到5mLYPD中(1％酵母提取物，2％胰蛋白胨，2％葡萄糖)，37℃震荡培养1天左右，将培养好的菌液以1％接种量重新接种于100mLYPD中，37℃震荡过夜培养(8h)至OD600为1.3～1.5，4000rpm离心5分钟，倒掉上清，将沉淀的酵母细胞重悬在40mL溶液A(50mM磷酸钾缓冲液，pH7.5，25mM DTT)中，37℃温浴15min，4000rpm离心5分钟，倒掉上清，将沉淀的酵母细胞重悬在200mL冰预冷的溶液B(270mM蔗糖，10mMTris-HCl，pH7.5，1mMMgCl2)中，4000rpm离心5分钟，倒掉上清，将沉淀的酵母细胞重悬在100mL预冷的溶液B中，4000rpm离心5分钟，倒掉上清，将沉淀的酵母细胞重悬在1mL预冷的溶液B中，吸取80uL于1.5mL离心管中，与上述线性化表达载体pHPZF1.0-ZS质粒DNA(4～5ug)分别充分混匀，然后转移到冰浴后的0.2cm无菌电击杯中，利用电击仪(Bio-Rad公司产品)将线性化的表达载体质粒DNA导入酵母感受态细胞中，使用的电击参数为电压1.5kV，电容50uF，电阻125Ω。电击完成后，立即向电击杯中加入1mL室温的YPD培养基(1％酵母提取物，2％酪蛋白胨，2％葡萄糖)，充分混匀后，35℃静置1小时，然后涂布于固体YPD培养基(液体培养基中加入了2％琼脂粉，0.1mg/mL Zeocin)上，平板倒置于35℃恒温培养箱中2～3天，至转化重组子出现。
实施例8：高表达酵母菌株的筛选
将在YPD培养基(0.1mg/mL Zeocin)上生长的酵母单菌落(转化子)用无菌牙签逐一挑取到含有梯度Zeocin的YPD培养基(分别含有1mg/mL、2mg/mL、4mg/mL、6mg/mL Zeocin)上，平板倒置于35℃恒温培养箱中1～2天。随着携带有Zeocin抗性基因的外源质粒整合到酵母基因组中的拷贝数的增加，转化子对Zeocin的抗性增强。挑取能够在含有6mg/mLZeocin的YPD平板上生长的转化子，接种于100mLBMMY培养基[1％酵母提取物，2％酪蛋白胨，100mM磷酸钾缓冲液(pH7.0)，1.34％YNB，0.00004％Biotin，0.64％甲醇(v/v)]中，35℃震荡培养120小时，每隔24小时补充0.64ml(v/v)甲醇一次，发酵液10000rmp离心5分钟，收集菌 体，破碎后，进行SDS-PAGE电泳检测。
从附图3、图4可知，在经表达载体pHPZF1.0-ZS转化所获得的重组酵母菌株培养上清液泳道中含有目标蛋白带，而在经表达载体pHPZF1.0转化所获得的重组酵母菌株培养上清液泳道中则没有目标蛋白带。该结果表明：HBsAg基因被按照酵母所偏爱的密码子所优化被置于一个汉逊酵母MOX启动子操纵之下，连同MOX终止子，一起被整合到汉逊酵母基因组中，随着重组酵母工程菌的生长，HBsAg基因在MOX启动子的作用下能够在甲醇的诱导下得到高效表达。从中挑选出一株作为工程菌加以保存，并将其命名为汉逊酵母HBsAg1.0-38G1Hansenula ploymorpha HBsAg1.0-38G1，并于2016年1月15日保藏于位于中国武汉武汉大学的中国典型培养物保藏中心，保藏号CCTCC No：M2016039。
实施例9：重组酵母在30L发酵罐中的高密度发酵
1)种子液制备：取1管真核汉逊酵母工程菌HBsAg1.0-38G1工作种子(1ml/管),尽数转接于1.2LBMGY培养基中，35℃振荡培养48小时，OD600值达到2～6，然后将其作为种子液；
2)初培养阶段：在30L发酵罐中盛放了15L基础发酵培养基(4.29％磷酸二氢钾，0.5％硫酸铵，1.43％硫酸钾，1.17％七水硫酸镁，0.10％二水硫酸钙，0.5882％二水柠檬酸钠，3.00％甘油)，pH用14％氨水调整并维持至5.5。再按照下述比例，在每升基础发酵培养基中加入4.37mL微量盐溶液PTM1(0.6％硫酸铜，0.008％碘化钠，0.3％硫酸锰，0.02％钼酸钠，0.002％硼酸，0.05％氯化钴，2％氯化锌，6.5％硫酸亚铁，0.025％生物素，0.5％硫酸)，在35℃条件下，自动控制溶氧不低于30％；
3)甘油流加培养阶段：当甘油耗尽后,溶氧(DO)陡然上升,此时，流加甘油。通过蠕动泵流加50％甘油(其中含有12mLPTM1/L)，甘油补加速度为7G/min，至菌体湿重约250～300g/L，调节转速和通气量，溶氧不低于20％，此阶段，用14％氨水将pH值缓慢调节至6.0。
4)甲醇诱导阶段：停止甘油流加后让菌体饥饿30min后，通过蠕动泵流加补料液，补料液为100％甲醇(其中含有12mLPTM1/L)，甲醇浓度在线控制，浓度维持在6.0～6.4％(v/v)。pH用14％氨水调整并维持至6.0。调节转速和通气量，控制溶氧量不低于20％至菌体湿重约300g/L，然后，将转速调到大于1300rpm，空气通量最大，溶氧量不于控制，至发酵总时100h下罐；
5)每隔24小时取数毫升发酵液经5000rpm4℃下离心10分钟，取破碎液上清液进行SDS-PAGE检测，发现目的蛋白条带随着甲醇诱导时间的延长而浓度显著增加，分子量约为24kDa，与推测中分子量基本吻合。此外，从电泳图中发现，在培养100小时后，重组蛋白表达量达到最高峰(见附图5)。
实施例10：发酵后HBsAg重组蛋白表达检测
1)三批连续发酵，每批取500mL发酵液经离心和洗涤，然后高压匀破碎，在相同条件下破碎细胞。向破碎液中表面活性剂吐温20，调节pH值，2～8℃搅拌过夜；将破碎后细胞12000g的转速离心除去细胞碎片，上清经过0.45μm和0.2μm微滤。
2)采用ELISA方法测定HBsAg，HBsAg酶联免疫检测试剂盒购自科华公司，HBsAg标准品(纯度99％)购自中国药品生物制品检定院，详细步骤参见试剂盒说明书。
3)结果发现，重组蛋白的平均表达量约1.5g/L，菌体的生物量、生长速度以及重组蛋白的表达量在三批连续发酵过程中基本保持稳定，证实重组汉逊酵母菌株和发酵工艺均具有很好的稳定性，具体参见表1。
表1.HBsAg1.0-38G1发酵过程中菌体浓度和外源蛋白表达测定
| 发酵批次 | 菌体浓度(湿重) | 菌体总重量 | 抗原表达量 |
| 20150119 | 466.7g/ml | 7.686kg | 1.4±0.8g/L |
| 20150130 | 488.0g/ml | 7.325kg | 1.5±0.6g/L |
| 20150403 | 455.0g/ml | 8.035kg | 1.5±0.7g/L |
实施例11：重组蛋白的纯化
在一个发酵周期结束之后，除留下500mL发酵液作为种子液外，其余的发酵液用于重组蛋白的纯化。发酵液经离心并洗涤，然后置于高压匀破碎直至细胞破碎率达到90％。向破碎液中0.05～1.0％(v/v)的表面活性剂吐温20，调节pH值至7.0～9.0，2～8℃搅拌4～16小时；将破碎后细胞12000g的转速离心除去细胞碎片，用1.0MNaOH和1.0MHCI调节pH值至7.5～9.0，且电导率小于5.0mS/cm，0.45μm或0.2μm微滤，然后进行阴离子交换层析，层析介质为DEAE sepherose FF，收集含汉逊酵母表达的重组乙肝表面抗原活性的流穿；将流穿液进行阳离子离子交换层析，层析介质为POROS 50HS，收集含汉逊酵母表达的重组乙肝表面抗原活性的流穿，收集含汉逊酵母表达的重组乙肝表面抗原活性回收率大于20％以上的紫外吸收峰的洗脱液；对疏水层析所得组分，采用截留分子量为100～500KD的超滤膜将样品一次或多次重复浓缩，直至样品中蛋白质浓度为1.0～2.0mg/ml；得到的浓缩液使用凝胶过滤柱进行凝胶过滤，去除部分乙肝表面抗原病毒样颗粒形成的聚集物，在SDS-PAGE电泳和HPLC检测重组蛋白回收纯度，纯度99％以上(见附图6和7)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110>安徽智飞龙科马生物制药有限公司
<120>一种含有重组乙肝病毒基因的真核汉逊酵母工程菌的构建及乙肝表面抗原的生产方法
<160> 4
<210>1
<211>226
<212>PRT
<213>乙型肝炎病毒
<400>1
Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln
1 5 10 15
Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu
20 25 30
Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys
35 40 45
Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser
50 55 60
Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe
65 70 75 80
Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val
85 90 95
Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly
100 105 110
Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala
115 120 125
Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp
130 135 140
Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg
145 150 155 160
Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu
165 170 175
Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu
180 185 190
Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile
195 200 205
Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val
210 215 220
Tyr Ile
225
<210>2
<211>684
<212>DNA
<213>人工序列
<400>2
atggagaaca ccacttcggg attcctgggt cctttgctgg ttctccaggc cggattcttc 60
ctgttgacca gaatcctcac tattcctcag tctctggact cgtggtggac gtccttgaac 120
ttcctcggag gtgctccaac ctgccctggc cagaactcgc aatctccaac ctccaatcac 180
tctcctacct cgtgcccacc tatctgccca ggctacagat ggatgtgcct gagaagattc 240
atcattttcc tgtttatctt gctgctctgc ctgatcttct tgctggtcct cctggactac 300
cagggtatgc tgcctgtttg tccattgctg cctggaacct ccactacttc taccggtcca 360
tgcaagacgt gtaccatccc tgcccagggc acttcgatgt tcccatcctg ctgttgcacc 420
aagccttctg acggcaactg cacctgtatc cctattccat cgtcctgggc tttcgccaga 480
tttctgtggg agtgggcctc ggtgagattc tcctggttgt cgctgctcgt tccattcgtc 540
cagtggtttg tgggattgtc ccctaccgtt tggctgtcgg tcatctggat gatgtggtat 600
tggggtcctt ctctgtacaa catcttgtcc ccattcctgc ctctcttgcc aatcttcttt 660
tgcctgtggg tttacatcta atag 684
<210>3
<211>1511
<212> DNA
<213>人工序列
<400>3
tcgacgcgga gaacgatctc ctcgagctgc tcgcggatca gcttgtggcc cggtaatgga 60
accaggccga cggcacgctc cttgcggacc acggtggctg gcgagcccag tttgtgaacg 120
aggtcgttta gaacgtcctg cgcaaagtcc agtgtcagat gaatgtcctc ctcggaccaa 180
ttcagcatgt tctcgagcag ccatctgtct ttggagtaga agcgtaatct ctgctcctcg 240
ttactgtacc ggaagaggta gtttgcctcg ccgcccataa tgaacaggtt ctctttctgg 300
tggcctgtga gcagcgggga cgtctggacg gcgtcgatga ggcccttgag gcgctcgtag 360
tacttgttcg cgtcgctgta gccggccgcg gtgacgatac ccacatagag gtccttggcc 420
attagtttga tgaggtgggg caggatgggc gactcggcat cgaaattttt gccgtcgtcg 480
tacagtgtga tgtcaccatc gaatgtaatg agctgcagct tgcgatctcg gatggttttg 540
gaatggaaga accgcgacat ctccaacagc tgggccgtgt tgagaatgag ccggacgtcg 600
ttgaacgagg gggccacaag ccggcgtttg ctgatggcgc ggcgctcgtc ctcgatgtag 660
aaggcctttt ccagaggcag tctcgtgaag aagctgccaa cgctcggaac cagctgcacg 720
agccgagaca attcgggggt gccggctttg gtcatttcaa tgttgtcgtc gatgaggagt 780
tcgaggtcgt ggaagatttc cgcgtagcgg cgttttgcct cagagtttac catgaggtcg 840
tccactgcag agatgccgtt gctcttcacc gcgtacagga cgaacggcgt ggccagcagg 900
cccttgatcc attctatgag gccatctcga cggtgttcct tgagtgcgta ctccactctg 960
tagcgactgg acatctcgag actgggcttg ctgtgctgga tgcaccaatt aattgttgcc 1020
gcatgcatcc ttgcaccgca agtttttaaa acccactcgc tttagccgtc gcgtaaaact 1080
tgtgaatctg gcaactgagg gggttctgca gccgcaaccg aacttttcgc ttcgaggacg 1140
cagctggatg gtgtcatgtg aggctctgtt tgctggcgta gcctacaacg tgaccttgcc 1200
taaccggacg gcgctaccca ctgctgtctg tgcctgctac cagaaaatca ccagagcagc 1260
agagggccga tgtggcaact ggtggggtgt cggacaggct gtttctccac agtgcaaatg 1320
cgggtgaacc ggccagaaag taaattctta tgctaccgtg cagtgactcc gacatcccca 1380
gtttttgccc tacttgatca cagatggggt cagcgctgcc gctaagtgta cccaaccgtc 1440
cccacacggt ccatctataa atactgctgc cagtgcacgg tggtgacatc aatctaaagt 1500
acaaaaacaa a 1511
<210>4
<211>335
<212> DNA
<213>人工序列
<400>4
gagacgtgga aggacatacc gcttttgaga agcgtgtttg aaaatagttc tttttctggt 60
ttatatcgtt tatgaagtga tgagatgaaa agctgaaata gcgagtatag gaaaatttaa 120
tgaaaattaa attaaatatt ttcttaggct attagtcacc ttcaaaatgc cggccgcttc 180
taagaacgtt gtcatgatcg acaactacga ctcgtttacc tggaacctgt acgagtacct 240
gtgtcaggag ggagccaatg tcgaggtttt caggaacgat cagatcacca ttccggagat 300
tgagcagctc aagccggacg ttgtggtgat atccc 335
Claims (2)
1.一种真核汉逊酵母工程菌，其特征在于，分类命名为HBsAg1.0-38G1，并于2016年1月15日保藏于位于中国武汉武汉大学的中国典型培养物保藏中心，保藏号CCTCC No：M2016039。
2.一种采用权利要求1所述的真核汉逊酵母工程菌HBsAg1.0-38G1表达和生产adr型乙型肝炎病毒表面抗原HBsAg的方法，其特征在于，包括以下步骤：
1)种子液制备：取1mL真核汉逊酵母工程菌HBsAg1.0-38G1工作种子，转接于1.2LBMGY培养基中，在35℃下振荡培养48小时，待OD600值达到2～6后，将其作为种子液，备用；
2)初培养阶段：在30L发酵罐中盛放15L基础发酵培养基，用14％氨水调整并维持至pH为5.5，按照每升基础发酵培养基中加4.37mL微量盐溶液PTM1的比例，添加65.55mL PTM1，在35℃下，自动控制溶氧不低于30％；
3)甘油流加培养阶段：待甘油耗尽，溶氧上升时，流加甘油，通过蠕动泵流加50％甘油，其中含有12mL/L的PTM1，甘油补加速度为7g/min，至菌体湿重为250g/L后，调节转速和通气量使溶氧不低于20％，此阶段，用14％氨水将pH值调节至6.0；
4)甲醇诱导阶段：停止甘油流加后，让菌体饥饿30min，通过蠕动泵流加补料液，补料液为100％甲醇，其中含有12mL/L的PTM1，甲醇体积浓度维持在6.0～6.4％，pH用14％氨水调整并维持至6.0，调节转速和通气量，控制溶氧量不低于20％至菌体湿重300g/L，再将转速调到大于1300rpm，空气通量最大，至发酵总时100h下罐；
5)发酵液经5000rpm4℃离心10分钟，留取沉淀；
所述步骤2)中基础发酵培养基由4.29％磷酸二氢钾，0.5％硫酸铵，1.43％硫酸钾，1.17％七水硫酸镁，0.10％二水硫酸钙，0.5882％二水柠檬酸钠，3.00％甘油组成；
所述步骤2)、步骤3)和步骤4)中PTM1由0.6％硫酸铜，0.008％碘化钠，0.3％硫酸锰，0.02％钼酸钠，0.002％硼酸，0.05％氯化钴，2％氯化锌，6.5％硫酸亚铁，0.025％生物素，0.5％硫酸组成。
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