CN1169947C - 必特螺旋霉素的基因工程菌株螺旋霉素链霉菌wsj-195 - Google Patents
必特螺旋霉素的基因工程菌株螺旋霉素链霉菌wsj-195 Download PDFInfo
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- CN1169947C CN1169947C CNB021487715A CN02148771A CN1169947C CN 1169947 C CN1169947 C CN 1169947C CN B021487715 A CNB021487715 A CN B021487715A CN 02148771 A CN02148771 A CN 02148771A CN 1169947 C CN1169947 C CN 1169947C
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- Prior art keywords
- spiramycin
- wsj
- streptomycete
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- bacterial strain
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Abstract
本发明涉及抗生素基因工程菌株螺旋霉素链霉菌WSJ-195，它是在维持4”-异戊酰基转移酶基因整合状态下通过诱变、自然分离等手段提高整合型原始菌株WSJ-1发酵产生必特螺旋霉素的能力，获得高产菌株WSJ-195，其平均发酵效价3020微克/毫升，比质粒型p66B-F21菌株发酵效价高6倍多，比整合型原始菌株WSJ-1的发酵效价高3倍多，不仅发酵单位稳定，所产生的必特螺旋霉素的化学组分也稳定，从而为工业化生产奠定了基础。
Description
技术领域：本发明涉及产生抗生素的基因工程菌菌株。
背景技术：必特螺旋霉素(原名生技霉素)为利用基因重组技术将碳霉素产生菌4”异戊酰基转移酶基因p66B转入螺旋霉素链霉菌获得的克隆菌株(Streptomycesspiramyceticus p66B-F21)的发酵产物，必特螺旋霉素是以异戊酰螺旋霉素III、异戊酰螺旋霉素II、异戊酰螺旋霉素I三个组分为主的混合物，含有一定量的(异)丁酰、丙酰、乙酰螺旋霉素III及(异)丁酰、丙酰、乙酰螺旋霉素II，其化学结构及组分如下：
异戊酰螺旋霉素III R＝COCH2CH3 R’＝COCH2CH(CH3)2
异戊酰螺旋霉素II R＝COCH3 R’＝COCH2CH(CH3)2
异戊酰螺旋霉素I R＝H R’＝COCH2CH(CH3)2
异丁酰螺旋霉素III， R＝COCH2CH3 R’＝COCH(CH3)2
异丁酰螺旋霉素II R＝COCH3 R’＝COCH(CH3)2
丁酰螺旋霉素III R＝COCH2CH3 R’＝COCH2CH2CH3
丁酰螺旋霉素II R＝COCH3 R’＝COCH2CH2CH3
丙酰螺旋霉素III R＝COCH2CH3 R’＝COCH2CH3
丙酰螺旋霉素II R＝COCH3 R’＝COCH2CH3
乙酰螺旋霉素III R＝COCH2CH3 R’＝COCH3
乙酰螺旋霉素II R＝COCH3 R’＝COCH3
其中异戊酰螺旋霉素III应不低于30％；异戊酰螺旋霉素(I+II+III)应不低于65％。
临床前药理毒理研究表明，必特螺旋霉素对革兰氏阳性菌，尤其是对耐红霉素、耐β内酰胺酶的金葡菌、肺炎链球菌、化脓性链球菌有较好的抗菌活性，对部分阴性菌如卡他球菌、淋球菌、流感杆菌和部分军团菌及厌氧菌有效，尤其对肺炎支原体的活性显著；药代动力学试验表明，必特螺旋霉素口服有较好的生物利用度，组织渗透性好，组织分布广、维持时间长；急、慢性及生殖毒性和致突变等研究显示，在新一代大环内酯类抗生素中必特螺旋霉素的毒性最低。以上特点说明必特螺旋霉素有较好的临床应用前景。必特螺旋霉素于2001年7月已获国家药品监督管理局进行临床试验的批文(批准文号为2001XL0292)。
利用基因工程技术制造生技霉素(必特螺旋霉素)的方法已于2000年获中国发明专利(ZL97104440.6)。该克隆菌株所含有的4”异戊酰基转移酶基因p66B是以质粒状态存在，已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为CGMCC No.0304。质粒型p66B-F21菌种的发酵单位平均为每毫升450微克，为了在螺旋霉素产生菌维持外源4”异戊酰基转移酶基因的稳定性，在菌种传代斜面中必须加入硫链丝菌素以保持质粒的存在，这些缺点影响了基因工程必特螺旋霉素的实际应用。为了获得产生必特螺旋霉素的基因稳定型菌株，本发明人利用基因整合技术，通过基因同源重组将4”异戊酰基转移酶基因整合在螺旋霉素链霉菌染色体上，获得了基因整合型菌株螺旋霉素链霉菌WSJ1，WSJ1菌株在不加压情况下连续传代，发酵效价及所产生的必特螺旋霉素组分稳定，而且发酵效价比质粒型菌株提高1倍以上(生物工程学报15卷2期171-176，1999)，但是这样的发酵水平仍不符合生产的需求。
本发明的目的是提供稳定的必特螺旋霉素高产菌株，以满足制药工业规模化生产的要求。
发明内容：在维持4”异戊酰基转移酶基因整合状态下，通过紫外、微波、甲基磺酸乙酯等化学诱变剂处理、并应用自然分离手段提高WSJ-1发酵产生必特螺旋霉素的能力，获得高产菌株WSJ-195及其突变体，该菌株或其突变体连续多次在黄豆饼粉-淀粉-琼脂孢子斜面培养基传代，经种子培养和在淀粉-鱼粉-玉米浆发酵培养基进行发酵实验验证；以藤黄微球菌为检定菌，用微生物法测定发酵液效价；发酵液在微硷性条件下用乙酸乙酯等溶媒提取，提取物经薄层色谱分析或高压液相色谱进行分析；以遗传标记的方法对4”异戊酰基转移酶基因在染色体的整合状态进行考察。结果显示，WSJ-195及其突变体的发酵水平稳定(平均发酵效价为3020微克/毫升)，所产生必特螺旋霉素组分符合质量标准的要求，可进入试生产工艺的研究。本发明获得的高产菌株WSJ-195已于2002年9月27日送中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为CGMCCNo.0801。本发明将提供高产菌株发酵条件；提供多次传代菌株发酵单位稳定、所产生的必特螺旋霉素组分稳定的试验证明，并对该菌株的形态及生理生化特征进行描述。
发明效果：本发明提供了必特螺旋霉素基因工程高产菌株，其发酵水平较原始质粒型菌株提高了6倍多，比整合型原株提高了3倍多，该菌株发酵效价及所产生的必特螺旋霉素组分稳定，可应用于制药工业规模化生产。
附图说明：
图1 WSJ-1为出发菌株诱变处理获得WSJ-195的流程图
图2 WSJ-195的发酵产物必特螺旋霉素组分的薄层色谱分析
其中：1.为螺旋霉素对照 2.必特螺旋霉素
No 1 WSJ-195第1批发酵
No 2 WSJ-195第3批发酵
No 3 WSJ-195第6批发酵
No 4 WSJ-195第10批发酵
图3 WSJ-195的发酵产物必特螺旋霉素组分的高压液相色谱分析
其中：RT 59.733为异戊酰螺旋霉素III
RT 39.733为异戊酰螺旋霉素II
RT 34.733为异戊酰螺旋霉素I
图4 WSJ-195菌株的孢子丝及孢子形态
以下将用实施例对本发明进行具体说明，但不限制本发明的范围。
实施例1：
高产菌株螺旋霉素链霉菌WSJ-195的获得
将在黄豆饼粉-淀粉-琼脂斜面培养基上，37℃生长8-10天的WSJ-1菌株孢子，用15毫升的无菌水洗下，用玻璃珠打30分钟、棉花过滤，取上清液5毫升，用微波处理(30秒)、微波加耐4-重氮-DL-亮氨酸(1000微克/豪升)菌株筛选、甲基磺酸乙酯(3％处理1小时)、紫外处理(45秒或60秒)或紫外(40秒)与氯化锂复合处理(在培养基中加0.85％氯化锂)等诱变方法进行处理，取经处理过的孢子悬液0.1毫升稀释至103-102/ml浓度，涂布至含有每毫升10微克硫链丝菌素的黄豆饼粉-淀粉-琼脂平皿，37℃生长8-10天，挑取单菌落至黄豆饼粉-淀粉-琼脂斜面培养基，37℃生长8-10天，接种于淀粉-鱼粉-玉米浆发酵培养基(50毫升/250毫升或500毫升三角瓶中)，28℃培养96-120小时，取样、离心，上清液用pH8.0磷酸缓冲液稀释后，以藤黄微球菌(CMCC)28001为检定菌、乙酰螺旋霉素为标准品，用微生物法测定发酵液效价(参考中华人民共和国药典，2000年版二部，附录XIA抗生素微生物检定法)。以WSJ-1为出发菌株诱变处理获得高产菌株WSJ-195(图1)。
实施例2：
高产菌株WSJ-195的发酵验证
将在黄豆饼粉-淀粉-琼脂斜面培养基(黄豆饼粉2％，淀粉3％，葡萄糖1％，NaCl0.4％，CaCO30.5％，琼脂1.2％)上，37℃生长8-10天的WSJ-195菌株挖块接种于种子培养基：黄豆饼粉1.5％，淀粉2％，NaCl0.4％，KH2PO40.05％，CaCO30.5％，鱼胨0.3％(50毫升培养基/250毫升三角瓶)，28℃培养63-65小时，以5-10％接种量转接至发酵培养基：淀粉6％，鱼粉2％，玉米浆1-2％，NH4NO30.6％，CaCO30.5％，KH2PO40.05％，MgSO40.1％，NaCl 0.8-1.0％ pH6.5-7.0，28℃培养96小时。发酵液取样、离心，上清液用pH8.0磷酸缓冲液(KH2PO40.041％，K2HPO40.55％)稀释，以藤黄微球菌(CMCC)28001为检定菌、乙酰螺旋霉素为标准品，用微生物法测定发酵液效价(参考中华人民共和国药典，2000年版二部，附录XIA抗生素微生物检定法)。WSJ-195菌株10批发酵效价见表一。
表一 WSJ-195菌株发酵效价
| No | 发酵效价 | No | 发酵效价 |
| (微克/毫升) | (微克/毫升) | ||
| 1 | 3848 | 6 | 2928 |
| 2 | 2872 | 7 | 3640 |
| 3 | 2974 | 8 | 2560 |
| 4 | 2840 | 9 | 2720 |
| 5 | 2640 | 10 | 3180 |
实施例3：
WSJ-195菌株的发酵产物必特螺旋霉素组分的验证
取实施例2中得到的发酵液，用氢氧化钠(1％)调节pH至8.0-9.0，加入1/2体积的乙酸乙酯混合、静止，分离乙酸乙酯层，在用0.1％氢氧化钠处理过的碱性硅胶薄层板上点样后，在苯∶甲醇2∶1中展开，用碘蒸汽显迹，以螺旋霉素为对照，Rf值高于螺旋霉素的显迹点，即为必特螺旋霉素(图2)。利用ShimadzuLC-6A高压液相色谱仪及Shimadzu Shim-pack CLC-ODS Φ6.0×150mm分析柱，进行WSJ-195的发酵产物必特螺旋霉素组分的反相高效液相分析，流动相为CH3OH∶1％NaH2PO4(53∶47)，检测波长231nm，流速1ml/min，必特螺旋霉素的组分分析结果见高压液相色谱(图3)。
实施例4：
WSJ-195菌株中4”异戊酰基转移酶基因整合在染色体的验证
由于在构建基因整合型菌株中采用了以阿普霉素抗性为标记的链霉菌/大肠杆菌穿梭质粒pKC1139，而在基因同源重组双交换的标记中采用了硫链丝菌素抗性标记，整合型菌株只能在含硫链丝菌素的培养基中生长而不能在含阿普霉素的培养基中生长，因此，以这两种抗性基因为标记即可判断基因的整合状态。将WSJ-195孢子适当稀释后，等量分别涂布在含10微克/毫升硫链丝菌素和含25微克/毫升阿普霉素的黄豆饼粉-淀粉-琼脂平皿上，28-37℃培养8-10天，分别记录生长菌落数，结果表明，在含硫链丝菌素培养基中的菌落数平均为1020个，而在含阿普霉素培养基上只有9-10个菌落生长，说明4”异戊酰基转移酶基因基本上是以整合于染色体的形式存在。
实施例5：
WSJ-195菌株的形态特征
1)形态学特征
于高氏1号琼脂或葡萄糖天门冬素琼脂上，28℃培养7-10天后，在显微镜下观察基内菌丝无横隔，不断裂，气生菌丝多分枝；在电镜下观察孢子丝直，孢子卵圆形，表面光滑(图4)。
1.培养学特征
在高氏1号琼脂或葡萄糖天门冬素琼脂上培养，基内菌丝为黄或灰黄色，无可溶性色素，气生菌丝灰色或灰黄绿。
2.生理特性
参照“Bergey‘s Manual of Systematic Bacteriology”Vol.IV及常用的”细菌系统鉴定手册”的相关方法进行生理生化的测定。
表二 螺旋霉素链霉菌WSJ-195生理生化特征
WSJ-195显示能降解黄嘌呤、水解淀粉、在0.1％酚中生长，并能还原硝酸盐，在3-7％的氯化钠中生长良好。WSJ-195能利用多种碳源，仅在棉籽糖、木糖和D-Saccharic acid上不生长。WSJ-195菌种生理生化特征见表二。
| 生理特征 | 结果 | 碳源利用 | 结果 |
| 黄嘌呤降解 | + | 棉籽糖 | - |
| 淀粉水解 | + | a-甲基-D-葡萄糖 | + |
| 木聚糖分解 | - | 阿拉伯糖 | + |
| 0.1％酚生长 | + | 肌醇 | + |
| 0.01％叠氮钠生长 | - | 甘露醇 | + |
| 3％NaCl中生长 | + | 丙二酸钠 | + |
| 5％NaCl中生长 | + | 松三糖 | + |
| 7％NaCl中生长 | + | 山梨糖 | + |
| 硝酸盐还原 | + | 鼠李糖 | + |
| 甘露糖 | + | ||
| 半乳糖 | + | ||
| D-Saccharicacid | - | ||
| 乳糖 | + | ||
| 木糖 | - | ||
| 蔗糖 | + | ||
| 果糖 | + |
本发明WSJ-195菌株与螺旋霉素链霉菌原株(微生物学报22(1)：13-16，1982)及文献报道的螺旋霉素产生菌-生二素链霉菌(Pinnert-Sindico，S：Ann.Inst.Pasteur.，87：703-709，1954)形态学及生理生化差异见表三、四。
表三 WSJ-195和螺旋霉素链霉菌原株及生二素链霉菌
形态学差异
| 菌种 | 孢子丝及孢子形态 | 气生菌丝体 | 基内菌丝体 | 可溶性色素 |
| WSJ-195 | 孢子丝直，孢子卵圆形，表面光滑 | 灰色或灰黄绿 | 黄或灰黄色 | 无 |
| 螺旋霉素链霉菌 | 孢子丝螺旋，孢子长方形，表面有疣状凸起并带粗短小刺 | 淡紫灰 | 深葡萄酱紫 | 浅栗紫 |
| 生二素链霉菌 | 孢子丝螺旋，孢子卵圆形，表面光滑或略带疣 | 白至灰色 | 无至灰黄 | 无 |
表四 WSJ-195和螺旋霉素链霉菌原株及生二素链霉菌
生理生化学差异
| 生理生化 | WSJ-195 | 螺旋霉素链霉菌 | 生二素链霉菌 |
| 淀粉水解 | + | + | |
| 硝酸盐还原 | + | - | - |
| 葡萄糖 | + | + | + |
| 阿拉伯糖 | + | - | + |
| 肌醇 | + | + | + |
| 甘露糖 | + | + | + |
| 棉籽糖 | - | - | - |
| 山梨糖 | + | - | - |
| 鼠李糖 | + | - | + |
| 半乳糖 | + | + | + |
| 乳糖 | + | - | + |
| D-木糖 | - | - | + |
| 蔗糖 | + | ± | ± |
| D-果糖 | + | - | + |
| 甘露醇 | + | - | + |
Claims (2)
1.一株基因工程螺旋霉素链霉菌(Streptomycesspiramyceticus)WSJ-195 CGMCC No.0801。
2.基因工程螺旋霉素链霉菌(Streptomycesspiramyceticus)WSJ-195 CGMCC No.0801在发酵产生抗感染抗生素必特螺旋霉素中的应用。
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| CN100465283C (zh) * | 2003-12-12 | 2009-03-04 | 华东理工大学 | 一种添加金属离子提高必特螺旋霉素产量的发酵工艺 |
| WO2011110084A1 (zh) * | 2010-03-09 | 2011-09-15 | 沈阳同联集团有限公司 | 异戊酰螺旋霉素ⅰ、ⅱ或ⅲ的分离制备方法、及含有它们的药用组合物及其应用 |
| WO2011147313A1 (zh) * | 2010-05-25 | 2011-12-01 | 沈阳同联集团有限公司 | 左旋异戊酰螺旋霉素i、ii或iii，及其制剂、制备方法及应用 |
| CN102229634B (zh) * | 2010-05-25 | 2014-05-28 | 沈阳同联集团有限公司 | 左旋异戊酰螺旋霉素i、其制剂、制备方法及应用 |
| BR112012029905B1 (pt) * | 2010-05-25 | 2022-01-11 | Shenyang Fuyang Pharmaceutical Technology Co., Ltd | Levocarrimicina, composições farmacêutica, métodos de preparação e uso dos mesmos |
| CN102311471B (zh) * | 2010-05-25 | 2013-10-16 | 沈阳同联集团有限公司 | 左旋异戊酰螺旋霉素ii、其制剂、制备方法及应用 |
| CN101914482B (zh) | 2010-07-23 | 2012-07-04 | 中国医学科学院医药生物技术研究所 | 一株异戊酰螺旋霉素i组分高含量、高产量基因工程菌 |
| CN102329839B (zh) * | 2011-09-08 | 2014-05-14 | 上海同联医药技术有限公司 | 一种高效价可利霉素的发酵生产方法及其所用的培养基 |
| CN107868789B (zh) | 2015-12-31 | 2020-11-17 | 沈阳福洋医药科技有限公司 | 可利霉素生物合成基因簇 |
| RU2746047C1 (ru) | 2017-04-06 | 2021-04-06 | Шенянг Фуянг Фармасьютекал Технолоджи Ко., Лтд. | Применение карримицина и его фармацевтически приемлемой соли для производства медикаментов для лечения и/или профилактики опухоли |
| CN111349595A (zh) * | 2018-12-20 | 2020-06-30 | 沈阳福洋医药科技有限公司 | 一种螺旋霉素产生菌、可利霉素产生菌、构建方法、应用及提高产物产量的方法 |
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