炭疽受体CMG2-Fc 融合蛋白在CHO 细胞中的表达、纯化与鉴定

目的:构建炭疽受体CMG2和人IgG1 Fc片段融合基因载体,转染CHO细胞并通过毒素中和试验检测CMG2-Fc拮抗炭疽毒素(PA+LF)的能力。方法:将含有CMG2胞外区1-217AA片度基因和人IgG1的Fc片段基因共同连接入pcDNA3.1载体转染CHO细胞并筛选高表达CMG2-Fc的CHO细胞系,通过小鼠RAW264.7巨噬细胞保护试验检测CMG2-Fc拮抗炭疽毒素的能力。结果:获得了表达CMG2-Fc的细胞株,毒素中和实验显示该蛋白可以有效抑制炭疽毒素引起的细胞损伤。结论:CMG2-Fc能够保护小鼠巨噬细胞免受炭疽毒素攻击,提示其可以作为抗毒素治疗炭疽感染。     

CMG2-Fc 融合蛋白突变体筛选及中和炭疽毒素活性分析

目的:筛选能有效中和炭疽毒素和抵抗炭疽毒素损伤细胞的CMG2-Fc(炭疽毒素受体II-人免疫球蛋白Fc段融合蛋白)突变体。方法:运用FoldX等计算软件分析CMG2与PA晶体学结构,设计能提高CMG2-PA亲和力的突变体分子,并与人IgG1Fc片段构成融合基因,转染CHO-S细胞并通过亲和层析获得CMG2-Fc突变体蛋白,通过亲和力检测和细胞保护实验分析各突变体中和炭疽毒素能力。结果:筛选并表达了8个CMG2-Fc突变体分子,亲和力实验显示其中E117Q突变可明显提高CMG2-Fc与PA的亲和力(KD=1.35×10-11 mol/L),细胞保护实验提示E117Q突变能有效提高CMG2-Fc中和炭疽毒素能力(CMG2-Fc(E117Q)的IC50为15 ng/μL,而wt CMG2-Fc的IC50为50ng/μL)。结论:CMG2-Fc(E117Q)突变体分子可作为拮抗炭疽毒素损伤的炭疽治疗药物分子,进行进一步研究。     

PA domain4-Fc融合蛋白抗血清可以保护小鼠巨噬细胞免受炭疽毒素损害

目的:探讨炭疽杆菌保护性抗原PA(protective antigen)domain4能否作为炭疽疫苗和炭疽感染时紧急预防用药.方法:构建含有PA domain4和人IgG Fc片段的表达载体,通过免疫大耳白兔获得针对该融合蛋白的免疫血清,通过小鼠巨噬细胞保护试验验证PA domain4-Fc是否具有疫苗和紧急预防用功能.结果:获得了表达PA domain4-Fc融合蛋白的CHO细胞株和针对PA domain4-Fc的兔抗血清,细胞保护试验证实PA domain4-Fc抗血清能够保护小鼠巨噬细胞免受炭疽毒素的攻击,但PA domain4-Fc蛋白本身并不能直接拮抗炭疽毒素对细胞的损害.结论:PA domain4-Fc抗血清可以保护小鼠巨噬细胞免受炭疽毒素损害,表明PA domain4-Fe具有作为炭疽疫苗的可能,但PA domain4-Fc蛋白不能直接竞争性拮抗炭疽毒素损害细胞.     

炭疽毒素受体与人IgG1的Fc段融合蛋白ATR_Fc在CHO细胞中的表达

ATR-Fc是人炭疽毒素受体（ATR）的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1-227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3-1的HindⅢ和NotⅠ位点得到表达ATR-Fc融合蛋白的真核表达载体pcDNA31/ATR9Fc,并用脂质体方法将该载体转染至CHO-K1细胞中,用G418筛选并获得ATR-Fc表达水平为10～15μg/(106cells·d)的基因工程CHO细胞系ATR-Fc-1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR-Fc与PA的亲和性,表明ATR-Fc可与PA特异性结合。     

炭疽毒素受体与人IgG1的Fc段融合蛋白ATR_Fc在CHO细胞中的表达

ATR_Fc是人炭疽毒素受体(ATR)的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1_227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3.1的HindⅢ和NotⅠ位点得到表达ATR_Fc融合蛋白的真核表达载体pcDNA3.1ATR_Fc,并用脂质体方法将该载体转染至CHO_K1细胞中,用G418筛选并获得ATR_Fc表达水平为10～15μg(106cells·d)的基因工程CHO细胞系ATR_Fc_1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR_Fc与PA的亲和性,表明ATR_Fc可与PA特异性结合。     

Vsig4 和免疫球蛋白Fc段融合蛋白的真核表达纯化

目的:本项目将通过构建中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)真核表达系统获取小鼠Vsig4膜外端和免疫球蛋白Ig G3a-Fc段的融合蛋白,鉴定Vsig4-Fc和Vsig4纳米抗体的相互作用。方法:采用重合延伸PCR法融合小鼠Ig G3a-Fc和Vsig4胞外段的基因序列,将该融合基因插入真核表达载体中并转染CHO细胞。Western blotting鉴定转染细胞上清中的目标蛋白,通过连续两次亚克隆筛选,获得高表达小鼠Vsig4-Fc融合蛋白的单克隆,之后大量培养增殖转染细胞并收集细胞培养上清,选择Protein A柱纯化方法纯化Vsig4-Fc蛋白,最后经ELISA法鉴定Vsig4-Fc和纳米抗体的结合能力。结果:在CHO细胞中成功构建了小鼠Vsig4-Fc真核表达稳转系,并且在真核表达体系中获得可表达15 mg/L的双分子结构Vsig4-Fc的稳定转染细胞系。经鉴定小鼠Vsig4-Fc融合蛋白能与Vsig4纳米抗体结合。结论:重合延伸PCR法使得Vsig4和Fc基因片段的融合更为高效,两次亚克隆筛选优势细胞系大幅提高了真核蛋白的表达量,为进一步研究Vsig4的生物学功能奠定重要基础。     

炭疽毒素受体ATRcDNA的合成和克隆及ATR-Fc融合蛋白的真核表达载体的构建

可溶性炭疽毒素受体(sATR)可以特异性结合炭疽毒素保护抗原(PA),为获得用于中和炭疽毒素以防治炭疽感染的候选抗毒素药物,构建了表达ATRFc抗体样分子融合蛋白的真核表达载体。将全长为681bp的编码炭疽毒素受体N端1～227氨基酸的基因分成长约50～60碱基的18个寡核苷酸片段,相邻片段重叠部分为20～22个碱基,利用重叠延伸PCR和引物PCR法,将合成的片段组装与扩增,得到了含有ATR_1～227的全部编码区和HindIII、BamHI位点在内的DNA片段。回收的基因片段经BamHI/HindIII双酶切连接到pUC19质粒中,挑选阳性克隆进行酶切鉴定和双向序列测定,获得了全序列正确的克隆。将ATR基因与Fc基因连接后插入pcDNA3.1载体多克隆位点HindIII和NotI之间,得到表达ATRFc融合蛋白的真核表达载体pcDNA3.1/ATRFc,为利用CHO哺乳动物细胞表达ATRFc并研究其生物学性质奠定了基础。     

炭疽毒素受体胞外区在毕赤酵母中分泌表达、纯化与活性鉴定

使用分泌型表达载体,实现了重组炭疽毒素受体胞外区 (rATR(CMG2)-EXCELL) 在毕赤酵母 KM71H 培养物上清中的分泌表达 . 表达量约占培养物上清总蛋白质的 20%. 经过螯合柱初步纯化,每升诱导培养物可获得约 1 mg 电泳纯的 rATR(CMG2)-EXCELL. 体外与配基 PA 结合试验和细胞保护试验显示, rATR(CMG2)-EXCELL 具有很好的生物活性 . rATR(CMG2)-EXCELL 的成功表达为今后研究炭疽毒素受体的作用机理、发展新型炭疽治疗药物打下基础 .     

Toll样受体5激动剂CBLB502-Fc融合蛋白的表达纯化及初步鉴定

目的:构建Toll样受体5激动剂CBLB502-Fc融合蛋白的真核表达载体,并在CHO细胞中表达,纯化得到具有生物学活性的目的蛋白。方法:首先从合成的pUC57-CBLB502质粒中扩增出CBLB502基因片段,酶切后克隆至pUC57-Fc质粒中,然后双酶切pc DNA3.1和pUC57-CBLB502-Fc,将CBLB502-Fc基因片段克隆至载体pc DNA3.1,挑选阳性克隆并测序,再将重组质粒转染CHO细胞,筛选高表达细胞系,用免疫印迹鉴定表达情况,Protein A亲和柱纯化目的蛋白,SDS-PAGE鉴定目的蛋白,用小鼠辐射试验初步验证目的蛋白的生物学活性。结果:构建了pc DNA3.1-CBLB502-Fc真核表达载体;转染CHO细胞,筛选得到CBLB502-Fc高表达细胞系,并经免疫印迹鉴定培养上清;纯化得到较高纯度的融合蛋白并经SDS-PAGE鉴定;小鼠辐射实验表明CBLB502-Fc具有抗辐射作用。结论:真核表达并纯化了具有抗辐射作用CBLB502-Fc融合蛋白,为后续研究CBLB502-Fc的生物学功能奠定了重要基础。     

细胞自噬与炭疽致死毒素的相互作用

目的：研究炭疽致死毒素在巨噬细胞中引起细胞自噬现象以及细胞自噬对炭疽致死毒素毒性的影响。方法：采用电子显微镜观察、单丹磺酰尸胺（MDC）荧光染色、Western印迹检测研究炭疽致死毒素作用后的巨噬细胞;采用MTT法检测细胞自噬对炭疽致死毒素毒性的影响。结果：采用以上3种方法,在巨噬细胞J774A.1中均可检测到细胞自噬现象;通过诱导或抑制细胞自噬,分别提高或降低了炭疽致死毒素的半数致死浓度。结论：炭疽致死毒素在巨噬细胞内能引起细胞自噬现象;细胞自噬能减弱炭疽致死毒素对巨噬细胞的毒性。     

Improving the Anti-Toxin Abilities of the CMG2-Fc Fusion Protein with the Aid of Computational Design

CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.     

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.     

重组腺相关病毒2型/人肿瘤坏死因子受体:Fc(rAAV2/hTNFR:Fc)的构建和生物学活性研究

为研究腺相关病毒2型载体应用于类风湿性关节炎进行基因治疗的可行性,首先构建携带人肿瘤坏死因子Ⅱ型受体胞外区和人免疫球蛋白IgG1Fc段融合基因的重组2型腺相关病毒(rAAV2／hTNFR：Fc),并对其生物学活性进行研究。以RT—PCR分别从U937和人淋巴细胞总RNA中扩增人肿瘤坏死因子Ⅱ型受体胞外区和人免疫球蛋白IgG1Fc段基因,并以重叠延伸PCR的方法将二者融合后克隆入pSNAV1载体质粒进行重组病毒生产,在进行重组病毒理化分析后,以TNFa细胞毒中和试验来研究该重组病毒的生物学活性。结果显示：所构建的重组病毒rAAV2／hTNFR：Fc基因结构与预期一致;病毒在体外感染BHK-21细胞后,含TNFR：Fc融合蛋白的表达上清可以有效中和人、大鼠、小鼠TNFα对L929的细胞毒性。所研究构建的重组腺相关病毒可以用来作为阻断TNFα的手段,进行类风湿性关节炎的基因治疗研究。     

尘螨变应原Derf1真核表达载体的构建及转染CHO细胞

目的：构建尘螨变应原Der f1真核表达载体,转染真核细胞并进行蛋白表达。方法：根据Genebank中Der f1基因的核酸序列（AB034946）,设计引物,采用PCR法,从保存的JM109工程菌中扩增Der f1编码基因,克隆到真核表达质粒pcDNA3.1/myc-his A上,以脂质体法转染CHO细胞,经G418筛选,进行稳定表达细胞株的筛选和鉴定。结果：将目的基因Der f1成功连接到pcDNA3.1/myc-hisA-Derf1并转染CHO细胞,获得稳定表达的CHO细胞株。结论：成功构建了尘螨变应原Der f1真核表达载体,并转染CHO细胞表达蛋白质。     

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.