CN115558612A - 重组全长ⅲ型人源化胶原蛋白酵母工程菌株及构建方法 - Google Patents
重组全长ⅲ型人源化胶原蛋白酵母工程菌株及构建方法 Download PDFInfo
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Abstract
本发明公开了一种分泌表达重组化胶原蛋白毕赤酵母工程菌株及构建方法。将CLA4_signal信号肽基因和全长Ⅲ型人胶原蛋白成熟肽基因进行串联,构建重组质粒并经线性化处理后导入毕赤酵母GS115中,筛选得到高表达毕赤酵母工程菌株pPICZA‑CLA4_signal‑COLⅢ/GS11,发酵目的产物产量达15g/L,该工程菌株具有产业化生产潜力。所得Ⅲ型人胶原蛋白成熟肽全长氨基酸序列,其N‑端和C‑端没有多余氨基酸或者纯化标签,可以有效降低感染、免疫原性等潜在风险,具有良好的安全性,产品可以应用于美容医学/化妆品、医疗器械、生物医学材料和营养保健等广泛领域。
Description
技术领域
本发明涉及基因工程技术领域,更具体地,本发明涉及一种重组全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌株及其构建方法。
背景技术
胶原蛋白是广泛分布于动物结缔组织的一组蛋白质家族,主要存在于动物的皮、骨、软骨、肌腱、韧带和血管中,有丰富的多样性和组织分布特异性。胶原蛋白结构特征是3条左手螺旋构型的α多肽链相互缠绕成右手三螺旋结构,形成的分子结构非常稳定。具有低免疫原性和良好的生物相容性,广泛应用于美容保健、生物医学材料和食品等方面。
依据氨基酸序列和功能的差异,胶原蛋白共分为29种亚型,占比最多研究最透彻的为Ⅰ型、Ⅱ型和Ⅲ型胶原蛋白,人体皮肤内胶原蛋白为Ⅰ型胶原和Ⅲ型胶原,而Ⅲ型胶原蛋白主要存在于婴儿皮肤或血管内膜、肠道。正常婴儿皮肤Ⅲ型胶原占80%,婴儿无瘢痕愈合机制是由于婴儿皮肤较强的Ⅲ型胶原的合成能力。成人皮肤中的Ⅲ型胶原蛋白因为年龄的增加而减少,同时成人真皮已经不具有合成Ⅲ型胶原蛋白的能力,只能合成Ⅰ型胶原蛋白,伤口的愈合经常会留下瘢痕。
多项研究表明正确的氨基酸序列对于胶原蛋白发挥生物学功能至关重要。例如,Ⅰ型胶原蛋白的突变影响骨矿化和限制骨骼生长,可能导致成骨不全症(脆性骨病);Ⅱ型胶原蛋白突变会引起海绵状气管发育不良;Ⅲ型胶原蛋白是中空器官(血管、子宫和肠)的主要结构成分,其基因的突变会导致血管型埃勒斯-当洛综合征(Ehlers-Danlos),患者通常由于大动脉或其他中空器官的破裂而突然死亡。奥尔波特(Alport)综合征病理机制的分子基础是编码IV型胶原蛋白的基因发生突变,导致肾小球、眼内及内耳基底膜的Ⅳ型胶原结构和功能损害而致病;Ⅶ型胶原蛋白的突变则与大疱性表皮松解症(Epidermolysisbullosa)密切相关。人自身胶原蛋白还具有细胞信号传导功能,胶原蛋白特异性细胞受体包括整联蛋白 (integrin)、盘状蛋白结构域受体(DDR)、糖蛋白VI(GPVI)和白细胞相关的免疫球蛋白样受体1(LAIR-1)等,它们可以为蛋白聚糖、糖胺聚糖、生长因子等生物大分子提供独特的结合位点,触发胶原分子与细胞之间的相互作用,引起组织特异性细胞应答。
胶原蛋白主要通过两种途径获得,生产胶原蛋白的传统方法是利用酸、碱、酶解法处理猪、牛、鱼等动物来源组织,提取长度不等的混合胶原肽段,产物纯度不佳,常有异味,生物活性差,并且有病毒感染和免疫排斥等潜在风险,一般用于食品保健品,少部分用于化妆品,无法在医疗器械或较精密的组织工程产品应用。利用基因工程技术在宿主细胞表达的重组胶原蛋白具有组分一致、质量稳定、安全性好的优点,可以广泛用于医疗行业,具有广阔的应用前景。综合比较重组表达Ⅲ型胶原蛋白的主要系统,转基因动物、转基因植物、哺乳动物细胞和昆虫细胞表达胶原蛋白规模化生产成本高、难度大;微生物发酵具有技术成熟、产量高、生产周期短、培养简单和成本低等优点,主要包括大肠杆菌表达系统和毕赤酵母表达系统。
Ⅲ型人胶原蛋白成熟肽含有1068个氨基酸,分子量远大于一般的生物活性蛋白,大肠杆菌系统无法表达正常生物学功能的成熟肽。国内研究一般在大肠杆菌表达分子量较小的Ⅲ型人源化胶原蛋白片段或者重组类人胶原蛋白,CN103122027A通过串联重复Ⅲ型胶原肽段并与Ⅱ型胶原肽段融合在大肠杆菌中获得高表达。基本重复单元为GERGAPGFRGPAGPNGIPGEKGPAGERGAP,为人胶原蛋白Ⅲ型肽段,末端序列为GPPGPCCGGG,为人胶原蛋白Ⅱ型肽段,终产品在N端有2个多余氨基酸GP,中间还存在连接氨基酸SR;通过改进设计,CN109593126A仅是III型胶原肽段重复单元GERGAPGFRGPAGPNGIPGEKGPAGERGAP在大肠杆菌的高效表达,并且无冗余氨基酸。CN1371919A利用大肠杆菌高密度发酵得到高表达的类人胶原蛋白,但其设计的部分氨基酸序列并非人源胶原蛋白序列。生产过程中存在的主要问题是大肠杆菌表达的目的蛋白常以包涵体形式表达,致使产物纯化困难;原核表达系统的翻译后修饰加工体系不完善,表达产物的生物活性较低;产品中残留的内毒素会产生致热源。
毕赤酵母兼有原核生物和高等真核生物的优点,具备培养基简单、适于高密度培养、成本相对较低、能进行蛋白翻译后加工、易获得可溶性活性重组蛋白等特点,是生产纯度高、安全性高、亲水性强、生物相容性好的重组胶原蛋白的理想表达系统。国外主要研究毕赤酵母重组表达人源化Ⅰ型人胶原蛋白,而国内多是利用毕赤酵母重组分泌表达Ⅲ型人胶原蛋白片段。CN101200718A公开了重复数分别是2、3、6的类人胶原蛋白同向串联基因重组质粒的制备方法。CN102443057A公开了6个重复串联Ⅲ型胶原蛋白片段在毕赤酵母的高效分泌表达,其含有599个氨基酸,分子量为55.0kDa,其长度上远少于天然人胶原蛋白基因,与模板人Ⅲ型胶原α1链的同源性仅为47%。CN111363029A公开了一种通过毕赤酵母重组表达498个氨基酸的人Ⅲ型胶原蛋白片段,获得的重组蛋白分子量为54.5kDa。CN103102407A公开在毕赤酵母SMD1168分泌表达的重组人源化Ⅲ型胶原蛋白全长474个氨基酸,由1~229和233~ 461片段串联而成,C端连接6个组氨酸残基作为特异性亲和纯化标记。CN102020716A公开毕赤酵母SMD1168表达839个氨基酸的胶原蛋白片段,其由谷氨酸-苯丙氨酸连接人Ⅲ型胶原蛋白230个氨基酸肽段与人Ⅰ型胶原蛋白608个氨基酸肽段组成。这几项研究涉及的或是人胶原蛋白部分氨基酸序列片段,或是功能片段的组合,或是对功能片段进行了设计修饰,均不同于Ⅲ型人胶原蛋白的全长氨基酸序列。
拥有多种特异性细胞受体结合位点的全长胶原蛋白是一种新型医用生物材料,体内降解较缓慢,适用于制备医用敷料、皮肤及骨组织修复、注射填充剂等医用产品。但目前尚未报道有利用毕赤酵母系统重组表达全长人源化胶原蛋白的产业化,仅有2个国内专利涉及重组全长Ⅲ型人源化胶原蛋白。CN103725623A公开在毕赤酵母SMD1168分泌表达全长Ⅲ型胶原α链的成熟肽,其N端为STE13切割位点Glu-Ala-Glu-Ala*,可能会导致胶原蛋白N端含有冗余氨基酸。CN109988243A重组全长Ⅲ型人源化胶原蛋白α1链及其应用,公开了在毕赤酵母SMD1168分泌表达全长Ⅲ型胶原,从氨基端依次包含:氨基端亲和纯化标签、人源Ⅲ型胶原成熟肽链以及羧基端亲和纯化标签,其在两端设计的特异性亲和纯化标签可能有潜在的安全性风险。这两个专利都没有提供表达水平数据,也未提供发酵罐实验相关数据。
以上国内外研发及上市的多是Ⅲ型重组人源化胶原蛋白片段和重组类胶原蛋白,具有分子量小、利于皮肤渗透吸收等特点,但是缺乏胶原蛋白的关键性及整体性特征,一般适用于护肤美容等美妆领域。前述2个制备重组全长Ⅲ型人源化胶原蛋白的专利或者由于蛋白酶切割不充分致使N端含有冗余氨基酸,或者为了后期纯化方便而在N端/C端增加了纯化亲和标签,在安全性方面都可能有潜在的风险。
此外,巴斯德毕赤酵母高密度发酵一般采用Invitrogen公司提供的BMGY、BMMY或者BSM 无机盐培养基。由于BMGY/BMMY培养基含有蛋白胨、酵母粉等有机成分,放大培养有机氮源单位体积成本过高,存在有机、动物来源成分带来污染的可能。
发明内容
本发明所要解决的技术问题在于针对上述现有重组胶原蛋白制备技术的不足,提供一种重组全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌,实现Ⅲ型人胶原蛋白成熟肽基因在毕赤酵母的高效分泌表达。同时提供该重组人源Ⅲ型胶原蛋白毕赤酵母工程菌的构建方法、发酵培养及分离纯化方法。
为解决上述技术问题,本发明提供如下技术方案:
一种重组全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌:由CLA4_signal或其突变序列引导Ⅲ型人胶原蛋白成熟肽全长氨基酸序列分泌至宿主菌的细胞外。
酿酒酵母的serine/threonine protein kinase CLA4分子量约93Ka,C末端氨基酸为 KR,能被毕赤酵母的Kex2蛋白酶识别并有效切除,分泌的外源蛋白不含任何冗余氨基酸。本发明以CLA4_signal串联目的蛋白基因,实现了在毕赤酵母宿主菌高效分泌表达Ⅲ型人胶原蛋白成熟肽完整序列。
进一步,所述重组全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌由如下方法得到:将CLA4_signal基因、Ⅲ型人胶原蛋白成熟肽基因串联构建至载体质粒pPICZA,导入毕赤酵母GS115,筛选得到高效分泌表达的转基因工程菌。优选为电击转化导入。
进一步,提供了一种分泌表达全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌:所述重组工程菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号是CGMCCNo.21547。
上述工程菌中重组全长Ⅲ型人源化胶原蛋白被毕赤酵母的Kex2蛋白酶切割信号肽后,分泌表达的重组胶原蛋白的氨基端和羧基端没有纯化标签蛋白,所述两端也不含有其它多余的氨基酸。
所述重组Ⅲ型人胶原蛋白成熟肽的氨基酸序列为SEQ ID NO.1。
所述重组Ⅲ型人胶原蛋白成熟肽的核苷酸序列按照毕赤酵母宿主菌密码子使用频率进行优化,核苷酸序列如SEQ ID NO.2所示。
上述分泌表达全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌CLA4_signal的氨基酸序列如 SEQ ID NO.3所示。
上述分泌表达全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌CLA4_signal的核苷酸序列按照宿主菌密码子使用偏好性进行优化,核苷酸序列如SEQ ID NO.4所示。
本发明提供了一种分泌表达全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌的构建方法,利用基因合成、PCR扩增、定向连接或者无缝连接等分子生物学技术构建重组质粒pPICZA-CLA4_signal-COLⅢ,由CLA4_signal或其突变序列引导目的产物分泌至毕赤酵母宿主菌的细胞外。
其中,CLA4_signal基因和Ⅲ型人胶原蛋白成熟肽基因的PCR扩增引物F1、R1、F2和R2 如下所示:
上游引物F1:aaaacaactaattattcgaaactatgagtttgtcagcagcag,酶切位点为BstBI;
下游引物R1:catcataagaatcgtattgtctcttctgccaaataaaactc;
上游引物F2:ttttatttggcagaagagacaatacgattcttatgatg;
下游引物R2:ttgttctagaaagctggcggccgcttaaccatagtatggagc,酶切位点为NotI。
本发明还提供一种分泌表达全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌的发酵培养方法,利用BSM无机培养基进行发酵培养,不含动物性来源组分、成分明确,能够获得较高的重组蛋白表达量,有利于提升产品安全性和保证产品质量,同时显著降低发酵的生产成本。
本发明还提供一种重组全长Ⅲ型人源化胶原蛋白的纯化方法,将发酵液上清加入饱和硫酸铵浓缩目标蛋白,离心收集沉淀溶解于柠檬酸盐缓冲液中,经低盐洗脱、高盐洗脱和超滤脱盐等处理得到纯度98%以上的重组全长Ⅲ型人源化胶原蛋白。
本发明提供了一种应用重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌制备全长Ⅲ型人源化胶原蛋白的方法,其特征在于包括如下步骤:
1)构建重组质粒:以CLA4_signal基因和Ⅲ型人胶原蛋白成熟肽基因为模板,PCR反应扩增目的基因,分别双酶切(BstBI/NotI)PCR产物与载体质粒pPICZA,胶回收片段进行定向连接并热击转化至大肠杆菌DH10b,涂布于含有Zeocin的LLB平板,挑取单菌落小量提取质粒,酶切鉴定得到阳性重组质粒;
2)电击转化:限制性内切酶PmeI线性化处理的重组质粒,电击转化至毕赤酵母GS115 感受态细胞,涂布含有Zeocin的YPDS平板,得到转化后菌落;
3)筛选高表达菌株:采用YPD种子培养基和BSM无机培养基进行两级培养发酵,甲醇诱导目的产物的表达,摇瓶筛选数十个转化菌落得到高表达菌株pPICZA-CLA4_signal-COLⅢ /GS115,摇瓶复筛验证其表达稳定性;
4)高密度发酵:接种至发酵罐开始甘油培养阶段,PH4.75-5.25,温度28-30℃,罐压 0.2-1.0bar,使溶氧>20%;当碳源耗尽,溶氧上升,进入甘油流加阶段,维持溶氧在20%~ 30%区间,至菌体湿重200-240g/L,停止补加甘油;待甘油耗尽后进行甲醇诱导阶段,使溶氧>20%,诱导80-100小时后结束发酵;
优选的,在甘油流加阶段流加含12mL/L微量元素PTM1的50%甘油;流加含12mL/LPTM1 微量元素的甲醇进行甲醇诱导;通过调节转速、罐压、空气流量或甲醇流速等方法使溶氧>20%。
5)纯化:将发酵液上清加入饱和硫酸铵浓缩目标蛋白,离心收集沉淀溶解于柠檬酸盐缓冲液中,经低盐洗脱、高盐洗脱和超滤脱盐等处理过程,收集目标产品得到纯度98%以上的重组全长Ⅲ型人源化胶原蛋白。
本发明中重组Ⅲ型人源化胶原蛋白的核苷酸序列如SEQ ID NO.2所示,本发明能够获得目的产物与全长Ⅲ型人胶原蛋白氨基酸序列100%相同的完整的III型胶原成熟肽,适用于对产品有均一度要求的医疗器械、组织工程等高端医学领域。本发明制备的重组全长Ⅲ型人源化胶原蛋白可广泛应用于化妆品、医疗器械、医学材料、组织工程、营养保健和细胞培养等领域,尤其可应用于外用或导入性护肤品、敷料贴、医用敷料、人工皮肤、人工血管、人工骨和黏膜保护剂。
本发明与现有技术相比具有以下优点:
(1)现有商业化毕赤酵母表达载体使用的是α-Factor信号肽引导大分子异源蛋白分泌表达不理想,本发明采用酿酒酵母的CLA4_signal串联全长Ⅲ型人胶原蛋白优化密码子基因,能够在毕赤酵母宿主菌高效分泌表达Ⅲ型人胶原蛋白成熟肽。所获得高产菌株高密度发酵液中重组人源化胶原蛋白产量达15g/L,实现了在毕赤酵母宿主菌高效分泌表达全长Ⅲ型人源化胶原蛋白。
(2)本发明在巴斯德毕赤酵母高效分泌表达重组胶原蛋白,无需对原蛋白基因改造,所得重组全长Ⅲ型人源化胶原蛋白具有完整的蛋白质序列,拥有生理功能所必须的稳定性、亲水性、机械强度、细胞粘附活性及组织相容性等生物学活性。胶原蛋白发生基因突变与多种疾病的发生密切相关,正确的氨基酸序列对于胶原蛋白发挥生物学功能至关重要。本发明所得重组蛋白与Ⅲ型人胶原蛋白成熟肽序列100%相同,也没有任何冗余氨基酸,与人体同源,不会有免疫排异反应,安全性高于国内现有的同类产品。
(3)人胶原蛋白包含多种特异性细胞受体,通过与细胞之间的相互作用引起组织特异性细胞应答。与国内研究较多的分子量较小的重组胶原蛋白片段相比,本发明的重组全长Ⅲ型人源化胶原蛋白保留了其特异性细胞受体特征,在生物医学、医疗器械等方面的应用中拥有相对优势。
(4)本发明采用BSM无机培养基,成本低,成分明确,能够实现全长Ⅲ型人源化胶原蛋白的高效分泌表达并且易于纯化,纯化得到纯度98%以上的目标蛋白。该重组蛋白由绿色环保的生物发酵方法制备,组分一致、质量稳定、具有良好的生物相容性和可生物降解性,克服了传统动物胶原蛋白的病毒风险、排异反应风险、结构和功能的不确定性风险。
附图说明
图1为根据本发明实施例构建全长Ⅲ型人源化胶原蛋白重组载体 pPICZA-CLA4_signal-COLⅢ技术路线。
图2是利用重叠延伸PCR技术构建融合基因CLA4_signal-COLⅢ的电泳图。其中泳道M 为DL5000 DNA Marker,1为CLA4_signal-COLⅢ的PCR扩增产物。
图3是重组质粒pPICZA-CLA4_signal-COLⅢ的酶切电泳图。其中泳道M1为DL5000DNA Marker,1和2分别是BstBI和NotI双酶切、PmeI单酶切的结果,M2为1kb DNA Ladder(Dye Plus)。
图4是重组工程菌pPICZA-CLA4_signal-COLⅢ/GS115的PCR鉴定电泳图。其中泳道M为 DL5000 DNA Marker,1为工程菌的PCR扩增产物。
图5为重组工程菌pPICZA-CLA4_signal-COLⅢ/GS115进行发酵罐培养,甲醇诱导后根据生产工艺留取发酵液,制备蛋白样品进行SDS-PAGE电泳检测。其中泳道M为PageRulerTM Prestained Protein Ladder,1-7为不同诱导时间的发酵液样品。
图6为纯化的重组Ⅲ型胶原蛋白及其胶原蛋白水解酶处理样品进行SDS-PAGE电泳检测。其中泳道M为PageRulerTMPrestained Protein Ladder,1为纯化样品,2为胶原蛋白水解酶(Sigma,C5138-25MG)处理样品。
具体实施方式
下面结合具体实施例解释本发明,参考附图描述的实施例是示例性的,不能理解为对本发明的限制。下述实施例中的实验方法,如无特别说明,均为常规方法。
实施例构建毕赤酵母基因工程菌
本发明中所获得的重组毕赤酵母工程菌pPICZA-CLA4_signal-COLⅢ/GS115,是利用重叠延伸PCR技术得到的融合基因CLA4_signal-COLⅢ与表达载体pPICZA定向连接,将构建的重组质粒电击转化到毕赤酵母细胞GS115,筛选得到高效分泌表达的转基因工程菌。菌株于2020 年12月24日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,分类命名为巴斯德毕赤酵母Pichia pastoris,菌种编号为CGMCC No. 21547。其中所述重组Ⅲ型人胶原蛋白的核苷酸序列如SEQ ID NO.2所示,能够获得重组全长Ⅲ型人源化胶原蛋白,氨基酸序列如SEQ ID NO.1所示,适用于对产品有均一度要求的医疗器械领域。
以下为毕赤酵母基因工程菌的具体构建过程:其中,本发明所选用的毕赤酵母Pichia pastoris GS115菌株、表达载体pPICZA均购自美国Invitrogen公司。本发明应用的培养基配方如下:
LLB培养基(1L):酵母提取物5g,蛋白胨10g,氯化钠10g(固体培养基含2%琼脂粉)。
YPD培养基(1L):酵母提取物10g,蛋白胨20g,葡萄糖20g。
YPDS培养基(1L):酵母提取物10g,蛋白胨20g,葡萄糖20g,1M山梨醇(固体培养基含2%琼脂粉)。
BSM无机培养基配方(1L):85%H3PO426.7 ml,CaSO4·2H2O 0.93g,K2SO418.2 g,MgSO4·7H2O 14.9g,KOH 4.13g,Glycerol 40g,PMT14.35 ml。
PMT1培养基配方(1L):CuSO4·5H2O 6.0g,KI 0.088g,MnSO4·H2O 3.0g,Na2MoO4·2H2O 0.2g, H3BO30.02g,CoCl2·6H2O 0.5g,ZnCl220.0g,FeSO4·7H2O 65.0g,Biotin 0.2g,浓H2SO45.0ml。
1.目的基因的获得
1.1优化密码子合成基因
根据Genebank登录的Ⅲ型人胶原蛋白成熟肽全长氨基酸序列NCBI ReferenceSequence: NP_000081.2,本发明在不改变胶原蛋白原始氨基酸序列的前提下优化其对应的基因序列,以利于在毕赤酵母的分泌表达。其基因序列如SEQ ID NO.2所示,优化后的Ⅲ型胶原基因序列由南京金斯瑞生物科技有限公司合成。
根据Genebank登录的酿酒酵母CLA4氨基酸序列NCBI Reference Sequence:NP_014101.1 对CLA4_signal进行密码子优化,消除毕赤酵母使用率低的密码子,其基因序列如SEQ ID NO.4 所示,优化后由南京金斯瑞生物科技有限公司合成。
1.2引物的设计与合成
根据优化的CLA4_signal基因和Ⅲ型人胶原蛋白成熟肽基因设计PCR寡核苷酸扩增引物。
上游引物F1:aaaacaactaattattcgaaactatgagtttgtcagcagcag,下划线标记部分为酶切位点BstBI;
下游引物R1:catcataagaatcgtattgtctcttctgccaaataaaactc;
上游引物F2:ttttatttggcagaagagacaatacgattcttatgatg;
下游引物R2:ttgttctagaaagctggcggccgcttaaccatagtatggagc,下划线标记部分为酶切位点NotI。
1.3 PCR反应扩增目的基因
以合成的CLA4_signal基因序列和Ⅲ型人胶原蛋白成熟肽基因为模板进行PCR扩增,反应体系如下:
PCR扩增程序如下:
0.8%琼脂糖凝胶电泳检测PCR扩增产物,如图2所示,与预期产物CLA4_signal-COLⅢ长度3503bp大小相符。
2.重组表达质粒的构建
2.1双酶切反应和回收目的片段
PCR扩增产物CLA4_signal-COLⅢ和载体质粒pPICZA分别用BstBI和NotI双酶切(37℃, 2h),酶切体系如下:
酶切产物进行琼脂糖凝胶电泳,切下含目的片段胶块,加入3倍体积溶解液BufferGM,胶块完全溶解后转移至Spin Column中,12,000rpm离心1min,弃滤液。加入漂洗液Buffer WB洗脱2次后,12,000rpm离心1min甩干残液。在Spin Column膜的中央处加入30μL灭菌水,室温静置1min,12,000rpm离心1min收集洗脱DNA。
2.2定向连接及转化
将PCR扩增产物和载体质粒pPICZA的双酶切胶回收片段,利用T4 DNA连接酶体系于16℃保温2h,具体操作如下:
连接产物加入感受态细胞DH10b,冰浴30min,42℃热击90s,冰浴2min,加入LLB培养基37℃摇培1h,涂布Zeocin抗性平板37℃倒置培养过夜。
2.3重组质粒鉴定
挑取单菌落,小量提取质粒进行BstBI和NotI双酶切、PmeI单酶切,于37℃保温2h,酶切产物进行琼脂糖凝胶电泳鉴定,图3结果显示双酶切产物片段分别为3267bp+3466bp、单酶切产物片段是6734bp,说明成功构建阳性表达质粒pPICZA-CLA4_signal-COLⅢ。
3.重组工程菌株的构建
3.1毕赤酵母GS115的电转化
内切酶PmeI线性化处理重组质粒,取1-10μg线性化质粒加入80μL毕赤酵母GS115感受态细胞中,混合均匀转至2mm电转化杯中,冰浴5min,设置电击仪参数为1.5KV,200 Ω,25μF,电转结束立即往转化杯中加入1mL预冷的1M山梨醇溶液混匀,将混合液转至灭菌的离心管中,30℃度静置培养培养1h。取200μL菌液涂布于Zeocin抗性(100μg/ml) 的YPDS平板,30℃培养2-5天,直到有单菌落出现。
3.2重组子的PCR鉴定
利用酵母基因组试剂盒提取YPDS平板转化子的基因组DNA,PCR反应体系如下:
PCR反应参数如下:
扩增产物经0.8%琼脂糖凝胶电泳检测如图4所示,含目的基因条带约3729bp,AOX1基因条带约2200bp,证明目的基因已整合到该重组工程菌株的染色体上。
3.3摇瓶筛选重组表达菌株
以1%接种量转接保存的甘油菌液到5mL YPD培养基的三角瓶,30℃,200rpm培养至OD=2~ 6。1500g离心5min收集菌体,将其分别悬浮于200mL BSM无机培养基(添加终浓度0.004%的组氨酸)的三角瓶,30℃,200rpm培养20h进入诱导期。诱导温度25℃,每隔24h补加终浓度1.0%甲醇(500ml甲醇加6ml PMT1)进行诱导表达。经多批次初筛、复筛和验证筛选获得高产菌株pPICZA-CLA4_signal-COLⅢ/GS115。
4.重组基因工程菌株的高密度发酵
将保存的工程菌甘油菌液接种于1000mL YPD培养基,30℃,200rpm培养至OD=2~6制备一级种子。按10%接种量将一级种子转接至装有20L BSM无机培养基的30L发酵罐,同时加入终浓度0.004%组氨酸。设定发酵初始温度30℃,转速500rpm,通气20L/min(通气量为 1V/V·min),氨水调节PH5.0,罐压0.2-1.0bar,开始发酵,溶氧大于20%。当溶氧陡然上升时,开始流加含12mL/L微量元素PTM1的50%甘油进入甘油流加阶段,维持溶氧在20%~30%区间。发酵液的湿菌重达到200-240g/L时,停止补料待甘油耗尽开始甲醇诱导阶段,流加含12mL/L PTM1微量元素的甲醇,诱导温度25℃,通过调节转速、罐压和通气量使溶氧大于20%。
按时间点28h、40h、52h、64h、76h、88h和100h分别留取菌液样品进行SDS-PAGE电泳检测,图5所示28-76h目的蛋白随时间延长表达量逐渐增加,超过88h后目的蛋白有降解趋势。考马斯亮蓝染色方法测定高密度发酵液中重组Ⅲ型人源化胶原蛋白产量达到15g/L。
5.重组Ⅲ型人源化胶原蛋白的分离纯化
发酵液离心去除菌体收集上清液,加入饱和硫酸铵溶液至饱和度25%,搅拌充分混匀室温静置1小时,离心收集沉淀。超纯水溶解沉淀后用30KD超滤膜包进行换液,置换到25mM 柠檬酸盐、50mM氯化钠、PH6.2的柠檬酸盐缓冲液中,得到层析上样液。利用25mM柠檬酸盐、 50mM氯化钠、PH6.2的柠檬酸盐缓冲液经过Capto SP ImpRes(Cytiva公司)层析柱进行低盐洗脱,再用25mM柠檬酸盐、250mM氯化钠、pH6.2的柠檬酸缓冲液进行高盐洗脱。将收集的洗脱液用30KD超滤膜包进行换液,置换到20mM PBS(PH7.0)缓冲液中,得到纯度98%以上的重组全长Ⅲ型人源化胶原蛋白。图6所示胶原蛋白水解酶(Sigma,C5138-25MG)可以专一性水解纯化的重组Ⅲ型人源化胶原蛋白。
本发明的内容不限于实施例所列举,本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换,均为本发明的权利要求所涵盖。
序列表
<110> 华北制药集团新药研究开发有限责任公司
<120> 重组全长Ⅲ型人源化胶原蛋白酵母工程菌株及构建方法
<130> 2021
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1068
<212> PRT
<213> 人属(Homo sapiens)
<400> 1
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Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro
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Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser
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Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly
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Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro
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Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly
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Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu
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Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Ser Asp
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Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly Thr Ala Gly Phe Pro Gly
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Ser Pro Gly Ala Lys Gly Glu Val Gly Pro Ala Gly Ser Pro Gly Ser
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Asn Gly Ala Pro Gly Gln Arg Gly Glu Pro Gly Pro Gln Gly His Ala
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Gly Ala Gln Gly Pro Pro Gly Pro Pro Gly Ile Asn Gly Ser Pro Gly
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Gly Lys Gly Glu Met Gly Pro Ala Gly Ile Pro Gly Ala Pro Gly Leu
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Gly Glu Pro Gly Arg Asp Gly Val Pro Gly Gly Pro Gly Met Arg Gly
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<210> 2
<211> 3207
<212> DNA
<213> 巴斯德毕赤酵母(Pichia pastoris)
<400> 2
caatacgatt cttatgatgt taagtctggt gttgctgttg gtggtttggc tggttaccca 60
ggtcctgcag gtccacctgg tccacctggt ccacctggga cttctggtca tccaggttct 120
cctggttctc caggttatca aggtccacct ggtgaaccag gtcaagctgg tccttccggt 180
ccacctggtc cacctggagc cattggtcct tctggtccag ctggtaaaga tggtgaatct 240
ggtagacctg gtagaccagg agagagaggt ttgccaggtc cacctggtat taagggtcct 300
gctggtattc ctggttttcc tggtatgaaa ggtcacagag gtttcgatgg tagaaacggt 360
gaaaagggag agactggtgc tccaggtttg aaaggtgaaa acggtttgcc tggagagaat 420
ggtgctccag gtcctatggg tccaagaggt gctcctggtg aaagaggtag acctggtttg 480
ccaggtgctg ctggtgctag aggtaacgat ggtgctagag gttctgacgg acaaccaggt 540
ccacctggtc cacctggcac tgctggtttt cctggttctc caggtgctaa gggtgaagtt 600
ggtccagctg gttctcctgg ttctaacggt gctccaggtc aaagaggtga gccaggtcct 660
caaggtcatg ctggtgctca aggtccacct ggtccacctg gtattaatgg ttctccaggt 720
ggtaaaggtg aaatgggacc agctggtatt cctggtgctc caggtttgat gggtgctaga 780
ggtccacctg gtccagctgg tgctaacggt gctcctggtt tgagaggtgg tgctggtgaa 840
ccaggtaaaa acggtgctaa aggagagcca ggtcctagag gtgaaagagg tgaggctggt 900
attcctggtg tccctggtgc taagggtgaa gatggtaaag atggttctcc tggagagcca 960
ggtgctaacg gacttccagg tgctgcagga gagagaggtg ctcctggttt tagaggtcct 1020
gctggtccaa atggtattcc aggtgaaaag ggtccagccg gagagagagg tgctccagga 1080
ccagctggtc caagaggtgc tgctggtgag ccaggtagag atggtgttcc tggtggtcct 1140
ggtatgcgtg gtatgcctgg ttctccaggt ggtcctggtt ctgatggtaa accaggtcca 1200
cctggttctc aaggagagtc tggtagacca ggtccacctg gaccttctgg tcctagaggt 1260
caaccaggtg ttatgggttt cccaggtcct aagggtaacg atggtgctcc aggtaaaaat 1320
ggtgaaagag gtggtcctgg tggtcctggt cctcaaggtc cacctggtaa aaacggagag 1380
actggtccac agggtccacc tggacctacc ggaccaggtg gagataaagg agatacaggt 1440
ccacctggac ctcaaggttt gcaaggtttg cctggtactg gtggtccacc tggagaaaat 1500
ggtaaaccag gagagccagg tcctaaagga gatgctggtg ctccaggtgc tccaggtggt 1560
aaaggagatg ccggtgctcc aggtgaaaga ggtccacctg gtttggccgg tgctccaggt 1620
ttgagaggtg gtgctggtcc acctggacct gagggtggta aaggtgccgc cggtccacct 1680
ggtccacctg gggctgctgg tactccaggt ttgcagggta tgcctggtga aagaggtggt 1740
ttgggttctc caggtcctaa gggagataaa ggagagccag gtggtcctgg tgctgatggt 1800
gttcctggta aagatggtcc aagaggtcct actggtccaa tcggtccacc tggacctgct 1860
ggtcaacctg gagataaagg tgaaggtggt gctcctggtt tgccaggtat tgctggtcca 1920
agaggttctc ctggtgaaag aggtgagacc ggtccacctg gaccagccgg ttttcctggt 1980
gctccaggtc aaaacggtga accaggtggt aaaggagaaa gaggtgctcc tggagagaaa 2040
ggagagggtg gtccacctgg tgttgccggt ccacctggtg gttctggtcc agctggtcca 2100
cctggtcctc aaggtgttaa gggtgaaaga ggttctcctg gtggtccagg tgctgctggt 2160
ttcccaggtg ctagaggttt gcctggtcca cctggttcta acggtaaccc tggtccacct 2220
ggaccttccg gttctccagg taaagacggt ccacctggac ctgccggtaa cactggtgct 2280
cctggttctc caggtgtttc tggtccaaag ggagatgctg gtcaaccagg agagaaaggt 2340
tctccaggtg ctcaaggtcc acctggtgct ccaggtccat tgggtattgc tggtattact 2400
ggtgctagag gtttggctgg tccacctggt atgccaggtc ctagaggttc tccaggacca 2460
cagggagtta agggtgaatc tggtaaacca ggtgctaatg gtttgtctgg agagcgtggt 2520
ccacctggac ctcaaggttt gcctggtttg gctggtactg ctggtgaacc aggtagagat 2580
ggtaaccctg gttctgatgg tttgccaggt agagacggtt ctccaggtgg taaaggagat 2640
agaggtgaga atggttctcc cggtgctcca ggtgctcctg gtcatcctgg tccacctggt 2700
cctgttggtc cagctggtaa atccggagat agaggtgaat ctggtcctgc tggtccagct 2760
ggtgctccag gtcctgctgg ttctagaggt gctccaggac ctcagggtcc aagaggagat 2820
aagggtgaaa ctggagagag aggtgctgct ggtattaaag gtcacagagg ttttcctggt 2880
aacccaggtg ctcctggttc tcctggtcct gctggtcaac aaggtgctat tggttctcca 2940
ggacctgccg gtccaagagg tcctgttggt ccatctggtc cacctggtaa agatggtacc 3000
tccggtcatc caggtcctat tggtccacct ggtccaagag gtaatagagg tgaaagaggt 3060
tctgagggtt ctcctggtca cccaggtcag ccaggtccac ctggtccacc tggtgctcca 3120
gggccttgtt gtggtggtgt tggtgctgct gctattgctg gtattggtgg tgaaaaagct 3180
ggtggtttcg ctccatacta tggttaa 3207
<210> 3
<211> 83
<212> PRT
<213> 人属(Homo sapiens)
<400> 3
Met Ser Leu Ser Ala Ala Ala Asn Lys Ile Ser Asp Asn Asp Phe Gln
1 5 10 15
Asn Ile Gly Pro Ala Pro Arg Pro Pro Ser Ser Asn Ser Gln Gly Arg
20 25 30
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35 40 45
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50 55 60
Ser Gly Trp Val Ser Tyr Lys Asp Asp Gly Ile Leu Ser Phe Ile Trp
65 70 75 80
Gln Lys Arg
<210> 4
<211> 249
<212> DNA
<213> 巴斯德毕赤酵母(Pichia pastoris)
<400> 4
atgagtttgt cagcagcagc caataagatt tcagataacg acttccagaa catcggacca 60
gcaccaagac ctccttcctc taattctcaa ggtagaactt gttacaacca aactcaacca 120
atcactaagt tgatgtctca attggatttg acttctgctt ctcatttggg tacttctact 180
tctaagaaaa agagtggatg ggtttcctac aaggatgatg gtattttgag ttttatttgg 240
cagaagaga 249
Claims (10)
1.一种重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:由CLA4_signal或其突变序列引导Ⅲ型人源化胶原蛋白成熟肽分泌至宿主菌的细胞外。
2.根据权利要求1所述的一种重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:所述的工程菌株由如下方法得到:将CLA4_signal基因、Ⅲ型人胶原蛋白成熟肽基因串联构建至载体质粒pPICZA,导入毕赤酵母GS115,筛选得到转基因工程菌。
3.根据权利要求1所述的一种重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:所述重组工程菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号是CGMCC No.21547。
4.根据权利要求1所述的重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:所述Ⅲ型人胶原蛋白成熟肽的氨基酸序列为SEQ ID NO.1;所述Ⅲ型人胶原蛋白成熟肽的核苷酸序列按照毕赤酵母宿主菌密码子使用频率进行优化,核苷酸序列如SEQ ID NO.2所示。
5.根据权利要求4所述的重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:所述氨基酸序列的氨基端和羧基端没有纯化标签蛋白,两端也不含有其它多余的氨基酸。
6.根据权利要求1所述的一种重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:所述CLA4_signal的氨基酸序列如SEQ ID NO.3所示;所述CLA4_signal的核苷酸序列按照宿主菌密码子使用偏好性进行优化,核苷酸序列如SEQ ID NO.4所示。
7.根据权利要求1-6所述的一种重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌,其特征在于:所述CLA4_signal基因和Ⅲ型人胶原蛋白成熟肽基因的PCR扩增引物F1、R1 、F2和R2如下所示:
F1:aaaacaactaattattcgaaactatgagtttgtcagcagcag,酶切位点为BstBI;
R1:catcataagaatcgtattgtctcttctgccaaataaaactc;
F2:ttttatttggcagaagagacaatacgattcttatgatg;
R2:ttgttctagaaagctggcggccgcttaaccatagtatggagc,酶切位点为NotI。
8.一种重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌的构建方法,其特征在于:将CLA4_signal基因、Ⅲ型人胶原蛋白成熟肽基因串联构建至载体质粒pPICZA,导入毕赤酵母GS115,筛选得到转基因工程菌。
9.一种应用重组全长Ⅲ型人源化胶原蛋白酵母基因工程菌制备全长Ⅲ型人源化胶原蛋白的方法,其特征在于包括如下步骤:
1)构建重组质粒:以CLA4_signal基因和Ⅲ型人胶原蛋白成熟肽基因为模板,PCR反应扩增目的基因,分别双酶切(BstBI/NotI)PCR产物与载体质粒pPICZA,胶回收片段进行定向连接并热击转化至大肠杆菌DH10b,涂布于含有Zeocin的LLB平板,挑取单菌落小量提取质粒,酶切鉴定得到阳性重组质粒;
2)电击转化:限制性内切酶PmeI线性化处理的重组质粒,电击转化至毕赤酵母GS115感受态细胞,涂布含有Zeocin的YPDS平板,得到转化后菌落;
3)筛选高表达菌株:采用YPD种子培养基和BSM无机培养基进行两级培养发酵,甲醇诱导目的产物的表达,摇瓶筛选数十个转化菌落得到高表达菌株pPICZA-CLA4_signal-COLⅢ/GS115,摇瓶复筛验证其表达稳定性;
4)高密度发酵:接种至发酵罐开始甘油培养阶段, PH4.75-5.25,温度28-30℃,罐压0.2-1.0bar,使溶氧> 20%;当碳源耗尽,溶氧上升,进入甘油流加阶段,维持溶氧在20%~30%区间,至菌体湿重200-240g/L,停止补加甘油;待甘油耗尽后进行甲醇诱导阶段,使溶氧> 20%,诱导80-100小时后结束发酵;
5)纯化:将发酵液上清加入饱和硫酸铵浓缩目标蛋白,离心收集沉淀溶解于柠檬酸盐缓冲液中,经低盐洗脱、高盐洗脱和超滤脱盐等处理过程,收集目标产品得到纯度98%以上的重组全长Ⅲ型人源化胶原蛋白。
10.一种重组全长Ⅲ型人源化胶原蛋白毕赤酵母工程菌的应用,其特征在于:所述重组全长Ⅲ型人源化胶原蛋白用于制备外用或导入性护肤品、敷料贴、医用敷料、人工皮肤、人工血管、人工骨和黏膜保护剂。
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