入源中和性抗甲型肝炎病毒抗体Fab段基因的序列分析及可溶性表达

在用噬菌体表面呈现系统获得人源抗甲型肝炎(甲肝)病毒中和性基因工程Fab抗体的基础上,对所获得的4株中和性Fab抗体轻重链可变区基因进行了序列分析、可溶性表达及生物学特性鉴定.4株Fab抗体重链可变区拥有99%同源的核苷酸序列和相同的CDR区氨基酸序列,属于VHⅢ基因家族.而轻链可变区核苷酸序列同源性为95%和相似的CDR区氨基酸序列,属于VL5基因家族.这些重组抗体都能与人甲肝恢复期血清及具有中和活性的鼠抗甲肝单克隆抗体产生竞争抑制反应,表明其针对甲肝病毒结构蛋白上的主要抗原决定簇.     

一种融合抗体ScFv-Fc通用表达载体的构建

为了构建一个可供自由替换的ScFv区,表达人小分子融合抗体ScFv-Fc的通用载体,利用RT-PCR技术扩增人抗体IgG1的Fc片段克隆至毕赤酵母表达载体pPICZα,将一段人工合成的互补寡核苷酸链插入重组载体pPICZα/Fc中Fc区的上游,引入2个可供小分子抗体ScFv-Fc的ScFv区自由替换的限制性酶切位点。分别扩增人抗狂犬病毒以及抗乙型肝炎表面抗原的ScFv片段,克隆至已构建的通用载体pPICZα/Fc,在毕赤酵母中诱导表达。进一步在1L条件下对活性抗体进行发酵,并利用protein A亲和层析柱进行纯化。应用酵母基因组PCR、ELISA、Western blotting、活性检测等试验对此小分子抗体的表达进行生物学及免疫学分析。结果表明具有狂犬病毒抗原结合活性以及乙肝表面抗原结合活性的人源抗体分子均获得成功表达,1L发酵条件下表达量达到20~30mg/L, protein A亲和层析纯化后纯度＞95%。研究构建了可用于功能性抗体分子ScFv-Fc筛选和表达的通用载体并对其发酵、纯化条件进行了摸索,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。     

人源中和性抗甲型肝炎病毒抗体Fab段基因的序列分析及可溶性表达

在用噬菌体表面呈现系统获得人源抗甲型肝炎（甲肝）病毒中和发生基因工程Fab抗体的基础上,对所获得的4株中和性Fab抗体轻重链可变区进行了序列分析、可溶性表达及生物学特性鉴定。4株Fab抗体重链可变区拥有99％同源的核苷酸序列和相同的CDR区氨基酸序列,属于VHⅢ基因家族,而轻链可变区核苷酸序列同源性为95％和相似的CDR区氨基酸序列,属于VL5基因家族。这些重组抗体都能与人甲肝恢复期血清及具有中和活性的鼠抗甲肝克隆抗体产生竞争抑制反应,表明其针对甲肝癌病毒结构蛋白上的主要抗的决定簇。     

抗A型肉毒毒素人源单链抗体融合蛋白的重组设计

以获得的抗A型肉毒毒素单链抗体为模板,进行融合改构,将人IgGl的Fc片段连接到ScFv的C端,在大肠杆菌中实现抗体融合蛋白ScFv—Fc的表达,表达量30％以上,蛋白以包含体形式存在,经过体外变复性的抗体融合蛋白ScFv．Fc,进行Protein G Sepharose柱亲和层析纯化,纯度达90％-95％。体外活性检测结果表明,重组抗体融合蛋白ScFvFc可以特异结合A型肉毒类毒素抗原,其相对亲和力近似于母本单链抗体,其稳定性高于母本单链抗体。     

抗A型肉毒毒素人源单链抗体融合蛋白的重组设计*

以获得的抗A型肉毒毒素单链抗体为模板,进行融合改构,将人IgG1的Fc片段连接到ScFv的C端,在大肠杆菌中实现抗体融合蛋白ScFv-Fc的表达,表达量30%以上,蛋白以包含体形式存在,经过体外变复性的抗体融合蛋白ScFv-Fc,进行Prote in G Sepharose柱亲和层析纯化,纯度达90%~95%。体外活性检测结果表明,重组抗体融合蛋白ScFv-Fc可以特异结合A型肉毒类毒素抗原,其相对亲和力近似于母本单链抗体,其稳定性高于母本单链抗体。     

人源抗体筛选和表达载体的构建

目的:构建一个可供抗体CDR(决定簇互补区)进行自由替换、筛选、表达的通用载体,并对其生物学功能进行鉴定.方法:在已构建好的具有Fc段的完全人源抗狂犬病病毒抗体表达栽体基础上,利用PCR介导的定点突变技术,引入2个可供CDR3区进行自由替换的限制性酶切位点,构建出通用表达载体.体外合成人源、鼠源抗乳腺癌Her2抗体的CDR3区,克隆至已构建的通用载体,在毕赤酵母中诱导表达.应用ELISA和Western Blotting技术对亲本抗体和新抗体进行生物学及免疫学分析.结果:PCR、Western Blotting等试验表明具有Her2抗原结合活性的人源和鼠源突变型抗体获得成功表达,通过对表达产物的免疫学及功能学检测证明所表达出的抗体具有抗原中和活性,而且鼠源抗体的活性要稍高于人源抗体.结论:成功构建了可用于功能性抗体筛选和表达的通用载体,对抗体的体外亲和力成熟及抗体的人源化有重要意义.     

抗汉坦病毒NP scFv基因表达及生物活性分析

诱导已构建的重组质粒pGEX-6P—1—scFv原核表达抗汉坦病毒核衣壳蛋白单链抗体,并用酶免疫实验检测单链抗体生物活性。用IPTG诱导重组原核表达质粒pGEX-6P-1-scFv表达抗汉坦病毒NP单链抗体融合蛋白,经亲和层析纯化,并应用SDS—PAGE电泳检测单链抗体融合蛋白,应用酶免疫实验检测抗NP单链抗体生物学活性。SDS—PAGE电泳检测显示,原核重组质粒pGEX-6P-1-scFv已表达分子量约为56ku的单链抗体融合蛋白;酶免疫实验检测显示,单链抗体具有与汉坦病毒NP抗原特异性结合的生物学活性。结果表明,已构建的原核表达重组质粒pGEX-6P-1-scFv,能够成功表达具有与汉坦病毒NP抗原特异性结合生物学活性的单链抗体。     

人源性抗bFGF单链抗体表达载体的构建与酵母表达

为了提高人源性抗bFGF抗体的表达量,从噬菌体抗体库筛选出的人源性抗bFGF抗体基因中亚克隆单链抗体(single chain fragment variable,ScFv)基因,并将其构建到酵母表达载体pPICZαA中。表达载体质粒经线性化后,电转化法转化至毕赤酵母GS115中,甲醇诱导表达。表达产物经镍离子亲和层析和阴离子交换层析纯化,并检测其生物学活性。酶切鉴定结果显示人源性的酵母表达载体构建成功。SDS-PAGE和Western blot结果显示,抗bFGF单链抗体获得了高效表达,表达量可达124mg/L,目的蛋白大小为36 kDa左右。通过两步纯化方案,目的蛋白的纯度可达95%以上。ELISA结果显示纯化的目的蛋白可与bFGF特异性结合。CCK8检测结果显示,纯化的抗bFGF单链抗体可剂量依赖性地抑制人肺腺癌细胞株A549的增殖。研究结果表明在毕赤酵母中可获得人源性抗bFGF单链抗体高效表达,表达产物具有很好的生物学活性。     

抗人P185^erbB2的scFv—Fc融合蛋白的表达及免疫功能分析

为提高鼠源单抗用于体内治疗的效应功能及降低人抗鼠抗体反应 ,将编码抗人P185 erbB2 单抗轻、重链可变区 (VL、VH)融合构建的单链抗体 (scFv)基因片段与人IgG1的Fc区基因片段融合构建了scFv Fc融合基因 ,并将其克隆到哺乳动物细胞表达载体pCIDN中。用重组载体转染CHO细胞 ,通过G4 18筛选获得稳定高表达克隆。更换无血清培养基培养 ,经重组蛋白质A亲和层析柱纯化scFv Fc融合蛋白。融合蛋白质在还原SDS PAGE中表现为5 2kD的条带 ;与SK BR 3细胞裂解液共培育可特异性地沉淀出P185 erbB2 蛋白 ;FACS分析表明融合蛋白质识别P185 erbB2 蛋白的胞外段 ;ELISA测定融合蛋白质对细胞表面抗原P185 erbB2 的亲和常数为 7.5× 10 -10 (mol/L) -1。构建scFv Fc融合基因 ,将其克隆到表达载体 ,进一步稳定表达抗P185 erbB2 的scFv Fc融合蛋白 ,为进一步的体外、体内研究鼠源单抗治疗效果创造了条件。     

人源抗ICAM-1单链抗体的制备及其生物学活性鉴定

利用噬菌体展示技术筛选特异性人源抗ICAM-1单链抗体（Anti-human ICAM-1 scFv）并进行生物学活性鉴定。应用Tomlinson I+J噬菌体抗体库,以P1抗原肽为包被抗原,经过4轮“吸附-洗脱-扩增”进行亲和富集筛选。以PCR反应、ELISA抗原交叉反应和Dot blotting实验进行阳性克隆的鉴定。scFv经原核表达和分离纯化后,以Western blotting实验、竞争ELISA实验和细胞黏附抑制实验对其生物学活性进行初步鉴定。Tomlinson I+J噬菌体抗体库经4轮亲和富集筛选,利用ELISA方法成功筛出4株阳性克隆。通过PCR鉴定反应、ELISA抗原交叉反应和Dot blotting实验,最终获得了1株既能与P1抗原肽特异结合又能与人ICAM-1抗原特异结合的阳性克隆J-A1。对scFv进行原核表达和亲和层析后获得了高纯度的目的蛋白。竞争ELISA实验和细胞黏附抑制实验证实纯化的scFv具有良好的亲和活性和抗细胞黏附活性。文中成功利用噬菌体展示技术筛选到特异性人源抗ICAM-1 scFv,为进一步探索该抗体在炎症相关性疾病治疗中的应用奠定了基础。     

From transcriptional landscapes to the identification of biomarkers for robustness

Background

Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.

Results

The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.

Conclusions

Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.     

重组人小分子抗体ScFv-Fc发酵条件的优化及纯化

目的：研究重组人小分子抗体ScFv—Fc在毕赤酵母中分泌表达的最佳条件,以及ScFv—Fc的纯化方法。方法：分别从甲醇浓度、pH、诱导时间等方面对毕赤酵母重组菌株产生ScFv-Fc的发酵过程进行了优化;通过硫酸铵沉淀结合proteinA亲和层析柱,对ScFv—Fc的纯化方法进行了研究。结果：确定ScFv—Fc在毕赤酵母中分泌表达的最佳条件为：在pH5．2的条件下,以0．5％甲醇诱导72h。经过proteinA亲和层析柱纯化后,ScFv—Fc纯度可达94％以上。结论：确定了ScFv-Fe在毕赤酵母中分泌表达的最佳条件以及纯化方法,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。     

Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth

Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (V_L and V_H) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the αIGF-IR scFv, including the N-terminal signal peptide, V_L, linker peptide, V_H, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10⁸ M⁻¹, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics. Received: 21 January 2000 / Accepted: 7 March 2000     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

抗人呼吸道合胞病毒融合糖蛋白单克隆抗体的制备及体外鉴定

目的:获得能稳定分泌抗人呼吸道合胞病毒(human respiratory syncytial virus, RSV)融合糖蛋白(fusion glycoprotein, F)单克隆抗体(monoclonal antibody, mAb)的杂交瘤细胞株,以期用于RSV感染的早期诊断和被动免疫治疗研究。方法:通过杂交瘤技术制备可特异性识别RSV F的单抗,体外鉴定生物学特性。结果:获得了可分泌抗RSV F蛋白的杂交瘤细胞株F8,体外连续传代培养2个月,能稳定分泌抗体F8,培养上清效价为1∶1000,亲和常数(Ka)为6.8×10⁸ L/mol。F8属IgG1型抗体,可特异性识别RSV F1亚单位的AA 205-222。免疫酶法蚀斑减少中和实验证实F8具有体外中和活性及融合抑制活性。结论:获得具有中和活性的抗RSV F蛋白的单克隆抗体,为RSV感染的早期诊断及被动免疫治疗等奠定了基础。     

HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

FMDV VP1基因与HSP70基因在毕赤酵母中的融合表达及免疫特性

利用毕赤酵母系统对O型口蹄疫病毒VP1基因与结核杆菌HSP70基因进行融合表达,并检测此融合蛋白对小鼠细胞免疫和体液免疫的影响。将人工合成的O型口蹄疫病毒VP1基因与结核杆菌HSP70基因克隆入酵母表达载体pPICZαA中,以电穿孔法转化酵母菌X-33,用Zeocin YPDS平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物。以皮下接种的方式给小鼠进行3次免疫,同时设两组对照,分别免疫PBS和常规灭活疫苗,然后通过MTT法和ELISA分别检测淋巴细胞的增殖情况和抗体水平。结果表明融合蛋白既能诱导细胞免疫应答又能诱导体液免疫应答,其诱导产生的抗体水平略低于常规灭活疫苗,而细胞免疫水平则高于后者。     

Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.     

人骨保护素在毕赤酵母中的分泌表达及表达产物的生物活性分析(英文)

为了在毕赤酵母表达系统中分泌表达人骨保护素 (osteoprotegerin ,OPG) ,以人骨肉瘤细胞系MG6 3的mRNA为模板 ,采用RT PCR法得到人OPG编码区cDNA ,克隆入毕赤酵母表达载体pPICZ B ,电转化毕赤酵母GS115 (Mut+) ,经 3%甲醇诱导分泌表达人OPG与组氨酸的融合蛋白 .SDS PAGE及Western印迹分析表明 ,有分子量约 6 6kD的目的蛋白表达 .纯化后的表达产物加入体外培养的小鼠骨髓细胞培养基中 ,当浓度为 10 0ng ml时 ,象牙片上骨吸收陷窝的数量及玻片上的TRAP阳性多核细胞的数量均减少 (P <0 0 5 ) .而同时加入人OPG的多克隆抗体后 ,这一抑制作用可被拮抗 ,在浓度为 5 0ng ml时则无此作用 .人OPG蛋白在酵母系统的成功表达 ,为该蛋白的进一步应用研究提供了依据 .