以Triton X-114液相分离法去除质粒溶液中的内毒素

目的：采用TritonX-114液相分离法去除质粒溶液中的内毒素,以保证实验动物的安全和结果的准确性。方法：通过碱裂解法提取质粒pVAX1和pVAX1-hLDHC,用聚乙二醇6000沉淀对质粒进一步纯化;用TritonX-114抽提的方法,去除质粒溶液中的内毒素。结果：通过3轮TritonX-114抽提,能够将质粒溶液中内毒素水平降至1．95EU／mL,质粒样品的回收率为79．8％,质量保持不变。结论：TritonX-114液相分离法是一种非常有效的去除质粒溶液中内毒素的方法。     

抗淋巴细胞免疫球蛋白制品中热原处理

对热原不合格的抗淋巴细胞免疫球蛋白 (ALG)半制品再制条件进行了研究。将高热原的ALG半制品通过使用碱、酸以及A5 0处理后 ,细菌内毒素的含量明显降低 ,热原质经家兔和鲎试剂检测均合格 ,其生物学效价仍不低于 1 :4 0 0 0 ,而且它们的其它各项主要指标也都达到中国生物制品暂行规程的要求。这一试验结果表明碱、酸处理并辅以A5 0吸附是去除ALG制品中热原的一种有效方法     

重组人BD3-BPI的内毒素中和活性及其在高盐环境中抗菌活性的稳定性

目的：验证重组人BD3-BPI（rhBD3-BPI）是否具有内毒素中和活性,研究其在高盐环境中是否能保持抗菌活性。方法：根据内毒素标准品绘制内毒素活性标准曲线,将100 μL梯度稀释的rhBD3-BPI-与100 μL 10EU／mL脂多糖（LPS）混匀,37℃水浴60min,同时设标准对照（只含10EU／mL LPS的标准品溶液）,并以无热源水作为空白对照,采用基质显色法进行鲎试验测定LPS的活性;将6×10^8 CFU／mL的革兰阳性和阴性标准菌株及临床分离的多药耐药菌株接种于含1mg／mL rhBD3-BPI和0～250mmol／L不同浓度NaCl的液体细菌培养基中,37℃培养3h后用10mmol／L磷酸钠按1：1～1：1000的比例稀释,铺LB培养基平板,37℃过夜培养,观察各平板菌落生长情况,计数并计算杀菌率。结果：在5EU／mL的标准内毒素体系中,当rhBD3-BPI的浓度高于4μg／mL时即开始表现出一定的内毒素中和活性,当rhBD3-BPI的浓度分别为16、32 μg／mL时,其内毒素中和率分别为23％和88％,随后rhBD3-BPI对内毒素的中和活性趋于平稳,50 μg／mL的rhBD3-BPI对所有受检菌均表现出100％的杀伤效应。当NaCl浓度低于150mmol／L时,rhBD3-BPI对各受检菌的杀菌活性均未受明显影响;NaCI浓度升高至150-200mmol/L,rhBD3-BPI对各受检菌的杀菌活性有所下降,但其杀伤率仍在90％以上;当NaCl浓度高于200mmol／L时,盐浓度对rhBD3-BPI杀菌活性的影响才较为明显,但即使NaCl浓度达到250mmol／L,rhBD3-BPI的杀菌活性仍保持在85％以上。结论：rh-BD3-BPI具有内毒素中和活性,在高盐环境中具有良好的抗菌活性稳定性。     

亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

响应面法建立层析纯化去除百日咳丝状血凝素中内毒素工艺

【背景】无细胞组分百日咳疫苗在人群中接种后不良反应发生率大大降低,是未来百日咳疫苗的发展方向,但是新的抗原纯化方式需要工艺中加入内毒素的去除。【目的】使用响应面法优化层析纯化法去除无细胞百日咳疫苗中百日咳丝状血凝素(Filamentous hemagglutinin,FHA)中内毒素的工艺。【方法】通过单因素试验,确定响应面设计范围;根据响应面法设计原理,使用Mini TAB软件,以FHA的回收率和收获的FHA蛋白浓度,同时兼内毒素合格为考察指标,对上样样品量、样品p H、样品电导Cond进行优化,最终确定去除FHA内毒素的层析纯化工艺。【结果】使用目前的层析纯化条件获得的Capto adhere去除FHA的内毒素的最佳工艺条件:p H 5.3,Cond 9.6,Mass 3.0。【结论】用响应面法优化了去除百日咳丝状血凝素中内毒素的层析纯化工艺,这种方法效率高、耗时少,为后续生物制品工艺扩大再生产提供参考。     

双歧杆菌三联活菌胶囊对化疗相关性腹泻患者肠黏膜屏障功能的保护作用

目的探讨双歧杆菌三联活菌胶囊对化疗相关性腹泻患者肠黏膜屏障功能的保护作用。方法选取78例化疗相关性腹泻患者,采用随机数字表将患者分为观察组（n=39例）和对照组（n=39例）。两组患者均常规予以补液,维持水电解质平衡及口服思密达3g,3次／d。观察组患者在此基础上加用双歧杆菌三联活菌胶囊420mg,3次／d。对照组除不使用双歧杆菌三联活菌胶囊外余治疗同观察组。观察两组患者治疗前和治疗3d后血清内毒素和和D-乳酸水平的变化,并比较其临床效果。结果治疗3d后,两组患者内毒素和和D-乳酸水平均有明显下降（P〈0．05或P〈0．01）,且观察组下降值明显大于对照组（P〈0．05）;同时观察组患者临床总有效率为92．31％,明显优于对照组的74．36％（x^2=4．52,P〈0．05）。结论双歧杆菌三联活菌胶囊治疗化疗相关性腹泻具有较好的临床效果,其作用机制可能是通过降低血清内毒素和D-乳酸水平,保护和改善改善肠黏膜屏障功能,从而恢复患者的肠功能。     

超滤过结合密度梯度离心纯化鲍曼不动杆菌噬菌体方法的建立

目的 探讨获得低浓度内毒素和高滴度鲍曼不动杆菌噬菌体的方法,为制备安全的噬菌体生物制剂提供参考.方法 用可截留100 kD以上分子量的超滤离心管浓缩噬菌体裂解液并滤出分子量约为10 kD的内毒素,然后用蔗糖密度梯度离心纯化噬菌体浓缩液;分别测定超滤前、超滤后和纯化后的噬菌体滴度,采用鲎试验测定超滤前后内毒素的浓度,通过SDS-PAGE分析超滤前后和纯化后噬菌体蛋白的纯度.结果 经超滤离心法噬菌体滴度从3.9×1010 PFU/mL提高至1.68×1012PFU/mL,并可去除99.2％的内毒素;超滤过结合密度梯度离心后的SDS-PAGE可清晰呈现7种蛋白,分子量为29～100 kD.结论 超滤过结合密度梯度离心是一种简便、快速浓缩和纯化噬菌体的方法,并可有效地去除裂解液中的内毒素.     

CHO细胞表达的抗炭疽保护性抗原人源化抗体的纯化及质量控制分析

目的:建立CHO细胞表达的抗炭疽保护性抗原人源化单抗纯化工艺和质量控制方法。方法:收获50 L生物反应器中无血清悬浮培养的CHO工程细胞培养液,通过高速离心去除细胞及碎片后超滤浓缩上清液,经亲和层析、SPFF阳离子交换层析后,将所得目的蛋白质经G25凝胶柱更换缓冲液以完成纯化;对纯化的产品进行单抗鉴别(Western印迹)、相对分子质量(SDS-PAGE和MOLDI-TOF)、纯度(SEC-HPLC)、生物学活性(毒素中和试验)、产品相关杂质(SEC-HPLC检测聚集体、降解产物)、工艺相关杂质(ELISA分析残余宿主蛋白、残余蛋白A)、安全性(凝胶法检测内毒素、薄膜过滤法考察无菌)分析。结果:样品回收率达61.7%;单抗鉴别实验阳性;MOLDI-TOF测定完整分子的相对分子质量为147 995,与预期相符;单体比例为99.25%,二聚体比例为0.75%;EC50值为0.1516μg/m L。残余宿主蛋白、残余蛋白A、内毒素、无菌检查结果符合药典要求。结论:初步建立了纯化工艺和质量控制方法,为抗炭疽人源化单抗药物的研制奠定了基础。     

大鼠内毒素血症不同时期血浆CGRP和背根神经节CGRP mRNA水平的变化

本文采用逆转录聚合酶链反应（RT－PCR）方法测定大鼠内毒素血症不同时期胸腰段背根神经节降钙素基因相关肽（CGRP）mRNA水平的改变,结合血浆CGRP水平的改变,以期全面了解大鼠内毒素血症不同时期CGRP释放与合成的变化。结果显示：注射内毒素（5mg／kg）后30min时,大鼠血浆CGRP开始增高,而背根神经节CGRPmRNA水平无明显变化;注射内毒素后3h时,血浆CGRP及背根神经节CGRPmRNA均明显增高．分别为142％和32％,8h时则进一步增高,分别为216％和85％。提示内毒素不仅刺激外周组织释放CGRP,而且还能通过某些机制激活背根神经节CGRPmRNA的转录,使CGRP合成增加,以作为CGRP大量释放的重要补充来源。     

A C群脑脊髓膜炎球菌多糖菌苗生产工艺与内毒素含量研究

A群及C群流脑多糖抗原用 10 0 0 0 0×g离心或不经超速离心处理 ,经用鲎试验法测定内毒素含量 ,2种工艺生产的A群及C群流脑多糖抗原的内毒素含量均很低。用未经超速离心处理的A群及C群流脑多糖抗原制成的A C群流脑多糖菌苗 ,经鲎试验法测定 ,内毒素含量也很低 ;经家兔升温法进行热原质试验 ,家兔体温未明显升高。证明A C群流脑多糖菌苗生产工艺不经 10 0 0 0 0×g离心去除内毒素步骤是可行的。所制备的流脑多糖抗原内毒素含量符合要求。     

抗结核分枝杆菌RpfB结构域单克隆抗体的制备及鉴定

目的:制备抗结核分枝杆菌Rpf B结构域单克隆抗体。方法:将p PRO-EXHT-Rpf B domain原核表达载体接种于大肠杆菌DH5中,用IPTG诱导表达Rpf B结构域蛋白,以纯化的Rpf B结构域蛋白作为免疫原,皮下包埋免疫小鼠3次,每次间隔2周;分离小鼠的脾细胞,与Sp2/0细胞融合,克隆化制备抗Rpf B结构域单抗,ELISA检测其效价,鉴定其特异性和相对亲和力,观察制备的抗Rpf B结构域单抗对Rpf家族其他蛋白的识别能力及其对结核分枝杆菌和藤黄微球菌的生长抑制作用。结果:制备了3株抗Rpf B结构域单抗,特异性高,亲和力较强,均能特异性识别Rpf B结构域。经小鼠腹腔注射制备腹水并纯化,获得了较高纯度的单抗,所制备的抗Rpf B结构域多肽的单克隆抗体可以识别多种Rpf样蛋白及其结构域蛋白。在抗体滴度为1∶1000时可有效抑制Rpf B结构域对结核分枝杆菌H37Ra和藤黄微球菌的生长促进作用,提示抗Rpf B结构域单抗可能会抑制进入机体内生长停滞或潜伏感染的结核分枝杆菌的再次激活,可能具有预防隐性感染复发的作用。结论:抗Rpf B结构域单抗的制备为进一步研究Rpf B结构域的生物学和免疫特性提供了实验工具。     

Endotoxin removing method based on lipopolysaccharide binding protein and polyhydroxyalkanoate binding protein PhaP

Polyhydroxyalkanoates (PHAs) granule associated protein PhaP has a strong affinity to PHA and other hydrophobic polymers. Human lipopolysaccharide binding protein (hLBP) is a natural endotoxin receptor in plasma. In this study, genes encoding hLBP fused with PhaP were expressed in Pichia pastoris GS115 for production of the fusion protein. The purified rhLBP-PhaP fusion protein was immobilized on particles of polyhydroxybutyrate (PHB), which is a member of microbial polyhydroxyalkanoates (PHA). The rhLBP-PhaP-coated PHB particles were added to endotoxin containing water and protein solutions to study their endotoxin removal and protein recovery efficiencies. The influences of ionic strengths and pH on endotoxin removal and protein recovery in different protein solutions were also studied using acidic proteins including bovine serum albumin (BSA), ovalbumin, and basic protein α-chymotrypsinogen as model proteins. The results showed that rhLBP-PhaP particles could remove endotoxin with an efficiency of over 90%. All endotoxin removal and protein recovery efficiencies were only slightly affected by ionic strengths but were drastically affected by pH changes. Our results demonstrated that rhLBP-PhaP particles with their high efficiency, ease of preparation, and nontoxicity will be a suitable system for endotoxin removal in the protein purification industry.     

Endotoxin detection and elimination in biotechnology

Endotoxins liberated by gram-negative bacteria are frequent contaminants of aqueous and physiological solutions. Because of their potent biological effects in vivo and in vitro, their detection and removal are essential for the safe parenteral administration of products produced from natural sources, as well as those produced by recombinant DNA technology. Traditional methods of endotoxin detection include the U.S. Pharmacopeia rabbit test and the Limulus amebocyte lysate test. Elimination of endotoxins, however, continues to be a problem. Standard methods of sterilization, such as autoclaving or sterile filtration, have little effect on endotoxin levels. Various techniques for the prevention of endotoxin contamination and endotoxin removal have been discussed. The overall role of endotoxin prevention, detection, and elimination in biotechnology is emphasized.     

重症急性胰腺炎患者血清IL-6水平与内毒素移位及肠黏膜紧密连接蛋白表达关系的研究

目的：大量研究表明重症急性胰腺炎（SAP）患者血清中高浓度IL-6 和肠黏膜低表达的紧密连接蛋白可促进内毒素移位的发生。本文主要研究重症胰腺炎患者血清IL-6 水平对内毒素移位和肠黏膜紧密连接蛋白表达的影响。方法：50 例重症胰腺炎患者,其中12 例在患病早期因结肠受累合并腹胀,对12 例结肠受累患者应用结肠镜行结肠灌洗进行腹腔减压,同时取结肠黏膜进行活组织检查。所有病人在治疗的第3 天,第7天,第10 天,第14 天抽取外周静脉血。40 例健康志愿者作为对照组。应用ELISA方法检测血清IL-6 水平,鲎试验（LAL）方法检测血清内毒素含量,应用免疫荧光和Western blotting 方法检测肠黏膜紧密连接蛋白表达水平。结果：SAP 患者血清IL-6 和内毒素含量明显高于健康对照组,而结肠黏膜紧密连接蛋白表达低于对照组;在临床治疗过程中,早期SAP 患者血清IL-6 和内毒素水平高于晚期（P 值均＜0.05）。SAP 早期血清高浓度的IL-6 与结肠黏膜紧密连接蛋白的低表达具有相关性,差异有统计学意义（r=0.735,P＜0.05）。结论：血清IL-6 水平可作为早期评价重症急性胰腺炎严重程度的一项指标,IL-6 水平与重症急性胰腺炎临床病程有相关性,可能导致肠道内毒素移位。     

Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.     

Endotoxin removal by anion-exchange polymeric matrix.

The effects of anion-exchange polymeric matrices on endotoxin removal from albumin and gamma-globulin solutions are evaluated. The positively charged cellulose acrylic media carrying DEAE or QAE functional groups remove significant amounts of endotoxin from tap water, but are less effective in protein solutions. With properly controlled pH levels and salt concentrations, the endotoxin level in a protein solution can be reduced; however, low endotoxin concentrations, less than 100 pg/ml, are more difficult to remove. The endotoxin removal capacity depends on the number of functional groups existing in the matrix, expressed as the number of milliequivalents (meq), and on the pH operable range, which is directly related to the pK alpha value of the matrix. The effects of pH and salt on endotoxin removal from albumin and gamma-globulin solutions by an anion-exchange polymeric matrix were evaluated statically in test tubes. In addition, a dynamic flow was performed under statically defined conditions on a 250-ml DEAE cartridge for the removal of endotoxin from albumin at a flow rate of 40 ml/min. A greater than 75% reduction in the endotoxin can be achieved, with protein loss occurring only in the early stage of removal. Such processes are useful for the reduction of endotoxin from biological solutions produced by natural sources or recombinant DNA technology.     

抗GPC3 C端抗体辅助杀伤肝癌细胞HepG2的活性检测及抗原表位鉴定

目的：检测制备的7株抗磷脂酰肌醇蛋白聚糖3（GPC3）蛋白C端单克隆抗体是否具有辅助杀伤肝癌细胞的活性,并研究其识别的抗原表位。方法：用细胞增殖法检测制备的抗体是否具有抗体依赖细胞介导的细胞毒性（ADCC）活性;用生物信息软件分析GPC3蛋白C端（359～580残基）的结构及抗原特征,并据此将其分为4个截短片段,将克隆的各基因片段分别连接到原核表达载体pGEX-4T-1中,进行蛋白表达和纯化,用间接ELISA和Western印迹分析GPC3C端单克隆抗体的表位识别情况。结果与结论：制备的7株单克隆抗体对肝癌细胞HepG2均具有不同程度的辅助杀伤作用,其中5号单克隆抗体的辅助杀伤效果最好;表达并纯化了GPC3C端4个截短片段的重组蛋白;间接ELISA和Western印迹检测结果表明,7株抗体均特异性结合GPC3蛋白的473～525残基区段。     

Potentiation of transmembrane signaling by cross-linking of antibodies against the beta chain of the T cell antigen receptor of JURKAT T cells.

Three monoclonal antibodies (mAb) 2D1, 3B9, and 3B12 were produced by immunizing BALB/c mice with JURKAT cells. These mAb induce comodulation of the TCR/CD3 complex expressed on JURKAT cells, but do not react with the CD3- JURKAT variant, J.RT3.T3.1. Immunoprecipitation studies with detergent-solubilized JURKAT cell lystes indicate that these mAb react with proteins having characteristics of the TCR molecules. Their low reactivity with peripheral blood mononuclear cells (PBMC) and lack of reactivity with other CD3+ T cell lines suggest that they may be anti-idiotypic mAb. Results from binding inhibition assays, reactivity with PBMC, and generation of transmembrane signals suggest that these three anti-TCR mAb recognized different epitopes on the TCR beta chain of JURKAT cells. Although the three mAb are capable of inducing the production of inositol phosphates and cytosolic free Ca2+ increase in JURKAT cells, their stimulatory capacities vary and are lower than that observed by anti-CD3 antibody (OKT3) stimulation. However, crosslinking these mAb with rabbit antimouse immunoglobulins potentiates the stimulatory response to comparable levels induced by OKT3. These mAb could be useful as tools to study V beta 8+ T cells in relation to antigen-specific activation.     

抗内毒素Fab’对严重烧伤早期肠源性内毒素血症小鼠NO、MDA与iNOS的影响及意义

目的：观察抗内毒素Fab’对严重烧伤早期肠源性内毒素血症小鼠肠组织中NO、iNOS、MDA水平的影响,探索防治烧伤脓毒症的新措施。方法：采用严重烧伤早期肠源性内毒素血症小鼠模型,分为烧伤组、治疗组及对照组,分别于6、12、24、48h4个时相点测定肠组织中NO、iNOS、MDA的浓度：结果：烧伤后肠组织中NO、iNOS、MDA水平均比正常对照组显著增高;治疗组肠组织中NO、iNOS、MDA水平较烧伤组显著降低。结论：抗内毒素Fab’能减轻内毒素对机体的损害,从而起到对严重烧伤早期肠源性脓毒症的防治作用。     

Endotoxin removal using a synthetic adsorbent of crystalline calcium silicate hydrate

A synthetic adsorbent of crystalline calcium silicate hydrate, the product LRA by Advanced Minerals Corp., has been studied for endotoxin removal from aqueous solutions. This adsorbent removes endotoxin effectively, and the removal is greatly enhanced by the presence of an electrolyte such as NaCl, Tris-HCl, or Na2HPO4. It has an endotoxin removal capacity as high as 6 million endotoxin units (EU) per gram. Its endotoxin removal kinetics is fast, and for instance, over 99.9% endotoxin in a 5000 EU/mL solution was removed by mixing for 2 min at an adsorbent usage of 10 g/L. Using the chromatographic column method to treat a 5000 EU/mL solution, an endotoxin log-reduction factor of 6.2 was achieved with a single pass. This adsorbent also demonstrated significantly better performance when compared to many commonly used endotoxin removal agents, such as ActiClean Etox Endotoxin Removal Resin, Affi-Prep Polymyxin Support, Detroxi-Gel Endotoxin Removing Gel, Q Sepharose Fast Flow Media, and Sigma Endotoxin Removal Solution. Furthermore, it demonstrated a high selective removal of endotoxin from a solution of lambda DNA. This adsorbent provides opportunities for developing disposable, scaleable, and cost-effective methods for endotoxin reduction in many biotechnological and pharmaceutical processes.