注射用重组新蛭素的质量控制研究

目的:建立注射用重组新蛭素原液、成品的质量控制方法及内控质量标准。方法:利用纤维蛋白凝块法测定注射用重组新蛭素的生物学活性,非还原SDS-PAGE和RP-HPLC测定纯度,SDS-PAGE和质谱法分析相对分子质量,免疫印迹验证目的蛋白,其余检测项目按《中华人民共和国药典》(2010年版三部)规定进行。结果:用建立的方法对注射用重组新蛭素3批原液和成品进行了检定,原液抗凝比活性均为512 ATU/mg,非还原型SDS-PAGE和HPLC检测3批原液的纯度结果均大于95%;质谱法分析3批原液相对分子质量符合7.3×103±0.7×103,SDS-PAGE分析3批原液相对分子质量符合13.2×103±1.3×103;免疫印迹表明检测条带为目的蛋白,能被抗水蛭素抗体特异性结合;其余各项指标均符合内控质量标准及《中华人民共和国药典》(2010年版三部)的要求。结论:建立的质量控制方法和内控质量标准可以用于注射用重组新蛭素的检定和质量控制。     

重组小鼠血管内皮生长因子在毕赤酵母菌中的表达和纯化

目的：建立毕赤酵母重组小鼠血管内皮生长因子（mVEGF）的制备方法,为研究mVEGF的生物活性、抗原性等提供基础。方法：通过全基因合成方法获得编码mVEGF的基因片段,将其克隆至表达载体pPICZaA上,电转化整合到毕赤酵母GS115基因组中,用甲醇诱导表达目的蛋白,表达上清经硫酸铵沉淀、SephadexG25柱脱盐、阳离子交换层析三步纯化获得目的蛋白;用还原型和非还原型SDS-PAGE检测目的蛋白的聚体状态,用Westelqq印迹验证纯化蛋白;通过PNGaseF酶切分析目的蛋白的N-糖基化修饰;通过人脐静脉内皮细胞（HUVEC）增殖实验检测目的蛋白的生物活性。结果：获得mVEGF的重组毕赤酵母表达菌株,SDS-PAGE分析可见GSll5表达的重组mVEGF在还原状态下表观相对分子质量约为20×10^3,在非还原状态下约为40×10^3;经Western印迹检测,这些条带均为目的蛋白条带,能被兔抗mVEGF抗体特异性结合,PNGase F酶切后相对分子质量降至18×10^3左右,证明目的蛋白发生了Ⅳ-糖基化修饰;细胞测活实验表明,mVEGF具有刺激HUVEC增殖的生物活性。结论：利用毕赤酵母菌制备了具有生物活性的重组mVEGF。     

不同分泌信号对豹蛙酶在巴斯德毕赤酵母中分泌效率的影响

目的：研究不同分泌信号对豹蛙酶（ONC）在巴斯德毕赤酵母中分泌效率的影响。方法：根据已知天然ONC的氨基酸序列,结合酵母偏爱密码子,设计并合成了ONC基因序列,通过融合PCR获得了不同分泌信号与ONC的融合基因,将其克隆至表达载体pPIC9k中,然后将线性化的重组表达载体电击转化GS115毕赤酵母感受态细胞,通过SDS-PAGE检测目的蛋白表达量。结果：糖基化ONC表观相对分子质量为（14～16）×103,且其糖基化不均匀,非糖基化蛋白肽链的相对分子质量约为12×103;采用不含EAEA结构的α交配因子作为分泌信号时ONC没有表达,利用含EAEA结构的α交配因子和α交配因子的pre肽作为分泌信号时均能够显著提高其表达量。结论：采用含EAEA结构的α交配因子和α交配因子的pre肽作为分泌信号均能够显著提高ONC在巴斯德毕赤酵母中的表达量。     

mNUDC基因在巴斯德毕赤酵母中表达载体的构建及表达

目的:探索小鼠核迁移蛋白C(mNUDC)在毕赤酵母分泌表达的方法.方法:应用PCR扩增本实验室所构建的重组质粒PET28b-his-mNUDC中的mNUDC基因,使用基因重组方法构建毕赤酵母真核表达栽体pPICZaA-his-mNUDC,电击转化酵母茵株KM71后,经摇瓶发酵和甲醇诱导,SDS-PAGE和Westernblot分析鉴定上清中重组mNUDC蛋白表达量.结果:经过PCR方法,有效扩增了mNUDC基因,构建了pPICZα A-his-mNUDC酵母表达质粒,序列分析表明所构建的含mNUDC基因的质粒与设计相同,mNUDC蛋白得到正确表达.使用SDS-PAGE和Western blot方法可以检测到mNUDC的稳定、高效地分泌表达.结论:成功地构建了mNUDC基因的毕赤酵母表达载体pPICZα A-his-mNUDC,并在毕赤酵母中实现分泌型离表达,为进一步研究mNUDC蛋白对小鼠的生物活性奠定了实验基础.     

商陆抗病毒蛋白重组子筛选及发酵条件初探

目的：筛选体外高表达商陆抗病毒蛋白的毕赤酵母重组菌株,并摸索其适合的发酵条件。方法：通过电击转化将含有商陆抗病毒蛋白（pokeweed antiviral proteins,PAP）基因的分泌型表达载体PIC9K-P导入到毕赤酵母（Pachia pastoris）GS115菌株中。利用免疫印迹法筛选表达量较高的转化子。在摇瓶发酵水平对重组菌株产PAP条件进行初步研究。结果：高表达PAP的重组菌株在发酵时间为96h,10g/L的甲醇浓度,培养基pH值为6.0～6.4时PAP的表达量较高。结论：免疫印迹法适合用于毕赤酵母高表达重组菌株的筛选,重组毕赤酵母的PAP表达量可高达30mg/L。     

甲醇-山梨醇混合碳源诱导提高抗HER2抗体在糖基工程毕赤酵母中的表达

目的：以抗HER2抗体为模型,研究抗体在糖基工程酵母菌中的表达及工程菌发酵技术。方法：首先通过摇瓶试验分析诱导用甲醇浓度对抗体表达的影响,并用高表达HER2的SK-BR-3细胞分析抗HER2抗体的抗原结合活性。以此为基础,在5 L发酵罐中研究甲醇-山梨醇混合碳源流加诱导对抗HER2抗体表达水平的影响;收集发酵培养液,采用阳离子交换层析对目标产物进行纯化;利用SDS-PAGE、Western印迹、Lowry法对抗体的相对分子质量、浓度等进行分析。结果：摇瓶试验结果表明,甲醇浓度为0.5%时抗体表达量最高,且糖基工程毕赤酵母菌表达的抗HER2抗体具有与SK-BR-3细胞抗原结合的活性;在5 L发酵罐中,利用甲醇和山梨醇混合诱导方式发酵表达抗体,其表达量可提高至0.6 g/L,比摇瓶诱导表达的抗体产量提高了近10倍;非还原SDS-PAGE及Western印迹表明抗体相对分子质量为1.5×105,与商业化抗体Herceptin的大小一致;经过一步阳离子交换层析纯化,纯化后抗体浓度为0.365 g/L。结论：采用甲醇-山梨醇混合碳源诱导方式在5 L发酵罐中进行发酵表达,能够提高抗HER2抗体在糖基工程酵母菌中的表达量,本研究可为抗体在酵母中的规模发酵技术提供重要参考。     

人血清白蛋白-C肽融合蛋白在毕赤酵母中的分泌表达

目的构建重组表达人血清白蛋白(HSA)-C肽(CP)融合蛋白的毕赤酵母表达菌株.方法根据表达系统的密码子偏好性优化CP基因,酶切连接pBlue-HSA质粒(HSA1 800bp)和CP(100bp)基因,将HSA-CP融合基因双酶切后插入分泌表达栽体pPIC9K中,重组质粒pPIC9K-HSA-CP经SalⅠ线性化后,电击转化毕赤酵母GS115,表型筛选Mut 转化子.PCR鉴定后,用甲醇诱导摇瓶分泌表达.结果融合基因约为1 900bp,序列测定正确.SDS-PAGE分析表明表达融合蛋白的相对分子量约为70kD,摇瓶培养表达量为140mg/L,Western blot鉴定显示表达的融合蛋白为HSA和CP的杂合分子.结论实现了HSA-CP融合蛋白在毕赤酵母中的分泌表达,细胞活性研究显示HSA-CP融合蛋白对人胚肾293细胞的生长具有一定的促进作用.     

L-蛋氨酸及甲醇浓度对毕赤酵母摇瓶发酵生产S-腺苷蛋氨酸的影响

利用甲醇传感器及高效液相色谱检测毕赤酵母摇瓶发酵过程的甲醇浓度及S-腺苷蛋氨酸(SAM)浓度,发现L-蛋氨酸浓度及甲醇浓度对毕赤酵母细胞生长及合成S-腺苷蛋氨酸具有影响,据此对摇瓶发酵过程的L-蛋氨酸浓度及甲醇浓度进行优化。优化结果表明:当L-蛋氨酸浓度为7.5 g/L时,最适于SAM积累,产量达到0.83 g/L;进而利用甲醇传感器对发酵过程的甲醇浓度进行检测及控制,考察不同甲醇浓度对SAM产量的影响,毕赤酵母产SAM的最佳甲醇浓度为15 g/L,在此浓度下SAM的产量达到1.41 g/L,比对照实验增加了21%。     

EH-L-Fc在CHO细胞中的表达、纯化及活性分析

目的:为延长重组新蛭素(EH)的半衰期,制备通过连接肽连接的重组新蛭素与IgG1Fc的融合蛋白EH-LFc,并对其进行功能分析。方法:采用重叠PCR技术构建Eh-L-Fc融合基因,克隆至表达载体pcDNA3.1,用脂质体将重组表达载体转染至中国仓鼠卵巢细胞(CHO)中,G418抗性筛选稳定克隆株;Western印迹检测培养上清中EH-LFc蛋白的表达,用有限稀释法对G418抗性筛选出的混合克隆单克隆化,通过Protein A亲和层析柱纯化融合蛋白,Lowry法检测蛋白浓度,SDS-PAGE、HPLC法检测目的蛋白纯度,质谱法分析相对分子质量,凝血因子Ⅹa裂解融合蛋白后采用纤维蛋白凝块法测定其抗凝活性。结果:构建了重组表达载体pcDNA3.1-Eh-L-Fc,并获得稳定表达EHL-Fc的细胞株。表达产物的相对分子质量为72 168,HPLC检测亲和层析获得的EH-L-Fc纯度达93.9%。完整的EH-L-Fc无抗凝活性,经凝血因子Ⅹa裂解后其抗凝比活性为96.6 ATU/mg。结论:获得稳定表达EH-L-Fc的CHO细胞株和较高纯度的重组融合蛋白,且该重组融合蛋白经凝血因子Ⅹa裂解后可释放抗凝活性。EH-L-Fc融合蛋白的获得为研究新蛭素的长效剂型奠定了重要基础。     

猪传染性胃肠炎病毒纤突糖蛋白在毕赤酵母中的分泌表达

目的:利用巴斯德毕赤酵母表达系统表达猪传染性胃肠炎病毒(TGEV)纤突糖蛋白S。方法:根据GenBank中猪TGEV纤突糖蛋白S全基因设计一对引物,并在5'引物和3'引物中引入EcoRⅠ、NotⅠ酶切位点,2.2kb的目的基因S经PCR扩增后克隆于pBS-T载体,再将S基因经双酶切从T载体切下并与穿梭质粒pPIC9k连接,SalⅠ线性化重组穿梭质粒pPIC9k-S,电转化于毕赤酵母GS115感受态细胞,G418筛选鉴定阳性重组子,经甲醇诱导,SDS-PAGE检测诱导后上清。结果:对pPIC9k-S重组酵母表达载体的测序证实已成功克隆了猪TGEVS基因;重组酵母菌诱导表达后,SDS-PAGE检测结果显示表达产物的相对分子质量约为82×103,且S蛋白以可溶性形式分泌表达于胞外。结论:利用巴斯德毕赤酵母真核表达系统成功表达了猪传染性胃肠炎病毒(河北分离株)纤突糖蛋白S。     

Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.     

Expression and purification of soluble B lymphocyte stimulator from recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in a shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30-1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pl) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisified, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 187–192. The text was submitted by the authors in English.     

假蜜环菌发酵工艺的优化研究

目前,国内普遍采用亮菌固体发酵工艺生产亮菌制剂。采用小型发酵罐液体深层发酵和液体静置发酵对亮菌发酵工艺进行优化研究。液体深层发酵采用28℃,200 r/min,通气量1∶1 v/v·m,培养7 d,亮菌干重为16.55g/L,其中菌丝体中多糖含量为5.42%,蛋白含量为1.75%;液体静置发酵采用500 mL三角瓶,28℃,150 r/min,摇瓶3 d后静置发酵14 d,亮菌干重可达10.69 g/L,发酵液中多糖含量为1.016 g/L,蛋白含量为0.320 g/L。对液体静置发酵进一步研究发现其发酵液中亮菌甲素含量可达3.118 mg/L。由此可见,两种发酵方式在保证生物量和活性成分的前提下,缩短了发酵周期,均优于传统的固体发酵工艺,值得工业生产借鉴。     

海洋中海藻糖产生菌的筛选及发酵条件优化

从180余份海水、海泥样品中筛选得到60株产海藻糖较高的菌株,编号为2-14的菌株海藻糖产量最高,为127.9mg/g cell。对2-14菌株进行形态特征、培养特征及生理生化试验,鉴定该菌株为红酵母属(Rhodotorula sp.)。研究摇瓶发酵条件对红酵母海藻糖产量的影响,结果为:初始pH5.5,发酵温度28℃,装液量75mL(250mL三角瓶中)。采用优化后发酵条件红酵母海藻糖产量为193.3mg/g cell,优化前对照值为132.1mg/g cell,优化后的结果是优化前的1.46倍。在5L发酵罐中培养得到最佳发酵时间为54h,发酵罐培养发酵液中海藻糖含量最高达2.5g/L,为摇瓶培养的1.6倍。     

丙酮丁醇梭菌ldhL基因的克隆及其在大肠杆菌中的表达

首次从丙酮丁醇梭菌(Clostridium acetobutylicum ATCC824)中克隆得到L-乳酸脱氢酶(L-lactate dehydrogenase,ldhL)基因,并将其连接到pSE380表达载体上,得到重组质粒pSE380ldhL,将重组质粒转化到乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJl44大肠杆菌中进行表达。SDS-PAGE分析表达产物的分子量约为34kD,摇瓶发酵后用HPLC检测分析L-乳酸产量为2.4g/L,纯度达到99.9%,不需要再进行手性分离,为以后在工业上生物法生产高纯度的L-乳酸打下基础。     

毕赤酵母发酵生产T4溶菌酶的中试研究

研究了T4溶菌酶的中试发酵放大生产。在200L发酵罐中毕赤酵母诱导表达T4溶菌酶蛋白,发酵液蛋白表达量达到1.69g/L,酶活达到192U/mg;制得冻干酶粉211.1g。经测定粗酶粉中T4溶菌酶的含量达到68.9%,酶活为182U/mg,总收率达到66%,成功建立了一条T4溶菌酶的中试发酵生产工艺线路。     

Optimization of ectoine synthesis through fed-batch fermentation of Brevibacterium epidermis

A production process for ectoine has been developed, using Brevibacterium epidermis DSM20659 as the producer strain. First, the optimal conditions for intracellular synthesis of ectoine were determined. The size of the intracellular ectoine pool is shown to be dependent on the external salt concentration, type of carbon source, and yeast extract concentration. Under the optimized conditions of 1 M NaCl, 50 g/L monosodium glutamate, and 2.5 g/L yeast extract, a maximum concentration of intracellular ectoine of 0.9 g/L was obtained in shake flask cultures. After optimizing the batch fermentation parameters of temperature, pH, agitation, and aeration, the yield could be further increased by applying the fed-batch fermentation principle in 1.5- to 2-L fermentors. Glutamate and yeast extract were fed to the bacterial cells such that the total glutamate concentration in the broth remained constant. A total yield of 8 g ectoine/L fermentation broth was obtained with a productivity of 2 g ectoine/L/day. After the bacterial cells were harvested from the culture broth, the ectoine was recovered from them by a two-step extraction with water and ethanol. Crystallization of the product was obtained after concentration of the extract via evaporation under reduced pressure. After this downstream process, 55% of the ectoine produced in the fermentor could be crystallized in four fractions. The first fractions were of very high purity (98%). This production process can compete with other described production processes for ectoine in productivity and simplicity. Further advantages are the relatively low amounts of NaCl needed and the absence of hydroxyectoine, often a byproduct, in the final product.     

毕赤氏酵母工程菌高密度发酵表达恶性疟原虫融合抗原的研究

疟疾目前仍是危害人类健康的主要传染病,现全球每年新增疟疾病例3～5亿人,其中约300万人死于该病.特别是病原虫抗药性的产生和扩散已给疟疾防治工作带来极大困难,因此研制新的预防措施已成为当务之急.研制有效的疟疾疫苗被认为是人类控制乃至消灭疟疾的重要途径,已越来越受到重视,并已构建和鉴定多个疫苗候选抗原[1,2].本研究进行高密度优化表达的融合抗原就是一个疫苗候选抗原.     

重组巴斯德毕赤酵母产米根霉α-淀粉酶发酵条件的研究

为了提高重组菌的淀粉酶表达量,以可分泌表达米根霉α-淀粉酶的甲醇快速利用型巴斯德毕赤酵母重组菌为基础,采用摇瓶发酵方式对影响重组菌表达淀粉酶的多个因素进行了研究和优化。摇瓶发酵条件确定为:温度为30℃,pH值为6.0,接种量为2.0(OD_(600)),甲醇补加方式采用前72 h发酵时间内每隔12h添加至终浓度为1.0%,72 h以后每隔24 h添加至终浓度为1.0%,在此条件下获得的淀粉酶最高表达量为47.5 U/mL,且在无机盐培养基中和有机氮源培养基中获得的淀粉酶发酵单位相当。以摇瓶发酵数据为基础确定15 L发酵罐放大实验条件为:无机盐培养基,温度为30℃,pH值为6.0,接种量为10%,甲醇流加方式采用DO—Start法控制,在此发酵条件下获得的淀粉酶表达量为440 U/mL,约为摇瓶发酵方式获得的淀粉酶表达量的9倍。