抗禽流感病毒H5N1 分泌型IgA 在中国仓鼠卵巢细胞中的表达

分泌型IgA (SIgA) 在机体的粘膜免疫中具有重要作用,在外分泌道中比单体IgA和IgG抗体具有更好的抗感染活性。为了表达抗禽流感病毒H5N1人-鼠嵌合分泌型IgA抗体,首先以本室先前构建的稳定表达IgA的中国仓鼠卵巢细胞 (CHO) 细胞系为基础,共转染分泌片和J链表达质粒,然后用抗生素Zeocin选择阳性克隆细胞,利用倍比稀释的方法筛选分泌SIgA的单克隆细胞,通过Western blotting分析培养上清中SIgA的表达情况。结果表明,在CHO细胞中成功表达了SIgA抗体,上述研究为研制分泌型     

人源中和性抗高致病性禽流感病毒H5N1基因工程全抗体的研制

运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5NI禽流感病毒基因工程抗体文库.用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/Viet Nam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达.通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定.结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFlulgG01、AVFlulgG03).微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade 2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于Clade Ⅰ的越南H5N1分离株A/Viet Nam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/Viet Nam/1203/2004,但是对属于Clade 2的所有中国H5N1分离株均具有中和作用.人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路.     

抗HEV嵌合抗体的构建及在CHO细胞中的表达

通过RT-PCR方法从分泌戊型肝炎(戊肝)病毒中和性鼠源单克隆抗体(单抗)8C11的杂交瘤细胞中克隆出抗体基因的重链可变区(VH)、轻链可变区(VK)序列,并分别克隆到含有人gamma 1重链和kappa轻链恒定区序列的pcDNA3.1/Hygro和pcDNA3.1( )质粒中,共转染中华仓鼠卵巢癌细胞(CHO)细胞.RT-PCR结果表明,转染的CHO细胞转录了嵌合重链及轻链基因,间接ELISA及Western blot结果表明:翻译出的两种多肽在细胞内正确组装成嵌合抗体分子,并可分泌至细胞外,表达的嵌合抗体保留了原鼠单抗的抗原结合特异性及对8H3结合抗原的增强作用.8C11嵌合抗体的成功表达可降低鼠源性,为探讨戊肝抗体治疗的可能性奠定了基础.     

抗人P185~(erbB2)嵌合抗体在CHO细胞中的高效表达及其活性分析

为降低人抗鼠抗体 (HAMA)反应并在CHO细胞中高效表达抗人P185 erbB2 人 /鼠嵌合抗体 ,将抗人P185 erbB2 单抗C2 5的轻、重链可变区基因分别克隆入具有人抗体恒定区基因组序列和弱化启动子驱动的选择标志基因的真核表达载体中 ,共转染CHO dhfr-细胞 ,经G418及氨甲喋呤 (MTX)梯度加压筛选进行了嵌合抗体的高效表达。采用RT PCR、ELISA、细胞ELISA、免疫荧光细胞化学等实验证实了所表达的抗人P185 erbB2 嵌合抗体的人源性及抗原特异性。培养上清中的抗体产量可达 10 0mg/L ,所表达的嵌合抗体具有抑制P185 erbB2 高表达肿瘤细胞增殖的作用     

抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化

目的：FGF2是肿瘤血管新生过程中最重要的因子之一,因此通过制备抗FGF2人鼠嵌合抗体中和其发挥作用,以达到抑制肿瘤生长的目的。方法：利用分泌抗FGF2抗体的杂交瘤细胞株IgG9B9和人B淋巴细胞,分别克隆抗体轻链可变区V_L、重链可变区V_H和人重链恒定区C_H基因,从pComb3λ载体中扩增出人λ 链恒定区C_L基因,通过重叠PCR,将V_L,V_H和C_L,C_H片段分别连接形成嵌合抗体的轻链L和重链H,将L/H链以单独构建或串联于同一载体的方式,构建抗FGF2嵌合抗体表达载体,并通过调控元件WPRE优化载体、共转染促生长因子aFGF以及调整表达温度等方式提高嵌合抗体在真核细胞中的表达。结果：成功构建了优化表达载体PLexm-WPRE、PLexm-aFGF;L、H链基因也成功构建,并以L、H或L-F2A-H（2A连接肽将L和H连接起来）的方式分别成功连接到PLexm,PLexm-WPRE载体中。转染细胞上清的ELISA鉴定结果表明,L/H链单独构建要比串联构建的方式具有更高的表达水平,WPRE能有效促进抗体的表达而aFGF并不能促进其表达,与31、37℃相比,33℃时抗体的表达量最高,同时嵌合抗体表现出了很好的结合活性及中和活性,竞争IC50=6.25μg/ml。通过亲和层析获得了高纯度的抗FGF2嵌合抗体。结论：在33℃下,人鼠嵌合抗体基因在WPRE存在下表达量最高,且与抗原FGF2有很好的结合活性及中和活性,为临床应用奠定了基础。     

抗人P185^erbB2嵌合抗体的CHO细胞中的高效表达及其活性分析

为降低人抗鼠抗体（HAMA）反应并在CHO细胞中高效表达抗人P185^erbB2人／鼠嵌合抗体,将抗人P185^erbB2单抗C25的轻、重链可变区基因分别克隆入具有人抗体恒定区基因组序列和弱化启动子驱地劝的选择标志基因的真核表达载体中,共转染CHO－dhfr^-细胞,经G418及氨甲喋呤（MTX）梯度加压筛选进行了嵌合抗体的高效表达,采用RT－PCR、ELISA、细胞ELISA、免疫荧光细胞化等实验证实了所表达的抗人P185^erbB2嵌合抗体的人源性及抗原特异性。培养上清中的抗体产量可达100mg/L,所表达的嵌合抗体具有抑制P185^erbB2高表达肿瘤细胞增殖的作用。     

抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体(IgG)的完整的恒定区连接起来,构建全抗型抗CD3嵌合抗体,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植,减少受体产生免疫排斥,提高移植器官的存活率。利用PCR方法从抗CD3 ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD₃抗体的轻链和重链可变区,将轻链和重链可变区组装到含有人抗体（IgG）恒定区的表达载体中,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN100和PG1D105,并用脂质体法共转染CHO细胞。结果证明,抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符,ELISA和Western blot检测结果证实转染细胞的培养上清中含有抗CD₃嵌合抗体IgG的表达,表达产物能与Jurkat细胞结合,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性,³H-TdR掺入实验表明, 抗CD3嵌合抗体与亲代抗体HIT3a一样,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达,表达产物有较好生物活性,具有潜在的临床应用价值。     

Pichia pastoris酵母中表达人源中和性抗甲型肝炎病毒scFv-Fc融合抗体的研究

为了探讨人源抗甲型肝炎（甲肝）病毒scFv—Fc融合抗体在酵母中的表达特性,将获得的人源抗甲肝病毒中和性单链可变区抗体（scFv抗体）基因克隆入含信号肽及人IgG1Fc抗体基因的酵母细胞表达载体中,获得了一株中和性人源抗甲肝病毒pPiscFv—FcHA16融合抗体的分泌表达,并对表达产物进行了纯化。同时对表达产物的生物学特性进行了一系列鉴定。表达的pPiscFv—FcHA16融合抗体为具有不同糖基化形式的同源二聚体,与相应的CHO细胞表达的IgG抗体相比,pPiscFv—FcHA16融合抗体仍保持很好的抗原结合活性,以及与中和性鼠抗甲肝病毒单克隆抗体的竞争抑制能力。同时也保持了对甲肝病毒的体外中和活性。这些结果表明,在酵母中表达的单链可变区（scFv）与IgG1Fc区的融合抗体具有很好的生物学活性,有希望用做体外诊断,用纯化相应的抗原,或者可能用于体内预防与治疗。     

6C6鼠-人嵌合抗体的纯化及鉴定

采用基因工程技术 ,将小鼠 6C6单克隆抗体可变区基因与人抗体恒定区基因连接 ,构建了鼠 人 6C6嵌合抗体基因 ,并在CHO细胞中高效表达 .利用ProteinA亲和层析柱从细胞培养上清中分离纯化 6C6嵌合抗体 ,得到电泳纯度大于 98%的 6C6嵌合抗体 ,其重链 (5 5kD)和轻链 (2 4kD)符合IgG相对分子质量的理论值 .Western印迹、细胞免疫荧光和免疫组织化学实验结果均呈阳性 .表明6C6嵌合抗体可识别人乳腺癌细胞表面上的肿瘤相关抗原 ,保持了 6C6单克隆抗体的特性 ,为后续的研究工作奠定了基础     

重组抗A型肉毒毒素人鼠嵌合单克隆抗体的瞬时表达及中和活性研究

为了制备重组抗A型肉毒毒素(botulinum neurotoxin A,BoNT/A)人鼠嵌合单克隆抗体并分析中和活性,以BoNT/A作为抗原筛选鼠源抗BoNT/A噬菌体单链抗体库,将筛选得到的鼠源抗BoNT/A特异性单链抗体的重链和轻链可变区分别克隆到pGP5KCγ和pGP5KCκ载体构建表达质粒。表达质粒共转染HEK-293EBNA1细胞,瞬时表达抗BoNT/A人鼠嵌合单克隆抗体(monoclonal antibody,mAb)。蛋白A亲和纯化mAb,分子排阻高效液相色谱(size exclusion high performance liquid chromatography,SEC-HPLC)分析mAb纯度,小鼠中和试验评价mAb中和活性。结果显示,经过筛选得到4个序列正确且各不相同的抗BoNT/A单链抗体。4个抗BoNT/A人鼠嵌合mAb表达质粒均构建成功并转染HEK-293EBNA1细胞,根据转染效率指示绿色荧光蛋白的表达推测,50%～62. 5%的细胞表达抗BoNT/A抗体。表达上清经过蛋白A亲和纯化,浓度均200μg/mL,单体纯度均90%。小鼠中和试验显示mA1、mA2、mA3和mA4单株抗体均不能完全中和4 LD_(50)的A型肉毒毒素,但从延迟小鼠生存时间可以得出4个抗体的中和活性为mA3mA2mA1mA4。mA1、mA2、mA4中任何一个抗体与mA3连用中和活性为80 LD_(50)/mg。本研究制备了4个具有不同程度中和活性的抗BoNT/A人鼠嵌合mAb,为A型肉毒神经毒素鸡尾酒疗法奠定了基础。     

甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析

目的：采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法：设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果：巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论：建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了基础,并为研究甲型H1N1流感病毒血凝素与抗体的结合位点提供了实验数据。     

Oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza.

Mice immunized with two intragastrically administered doses of a replication-deficient recombinant vaccinia virus containing the hemagglutinin and nucleoprotein genes from H1N1 influenza virus developed serum anti-H1 immunoglobulin G (IgG) antibody that completely protected the lungs from challenge with H1N1. Almost all of the mice given two intragastric doses also developed mucosal anti-H1 IgA antibody, and those with high anti-H1 IgA titers had completely protected noses. Intramuscular injection of the vaccine protected the lungs but not the noses from challenge. We also found that the vaccine enhanced recovery from infection caused by a shifted (H3N2) influenza virus, probably through the induction of nucleoprotein-specific cytotoxic T-lymphocyte activity. A replication-deficient, orally administered, enteric-coated, vaccinia virus-vectored vaccine might safely protect humans against influenza.     

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.     

Completion of the analysis of the primary structure of the variable domain of a homogeneous rabbit antibody to type III pneumococcal polysaccharide

The amino acid sequence between residues 70 and 116 of the V (variable) region of the H (heavy) chain derived from rabbit antibody BS-5, specific for type III pneumococcal polysaccharide, was determined. The sequence of this section of the H chain which includes the hypervariable residues 94 to about 112 was unique, although minor variant sequences present in the H chain preparation would not have been detected by the techniques used in this work. Taken together with the known sequences of the N-terminal 69 residues of H chain BS-5 (Jaton & Braun, 1972) and of the V region of the light chain (Jaton, 1974b), the data establish the complete sequence of the V domain of a rabbit immunoglobulin G. The V region of H chain BS-5 is compared with the basic sequences of the three human V region subgroups known to date, with one mouse H chain, and with guinea-pig pooled H chains. Even though chains from guinea pig and mouse clearly belong to the subgroup III of variability (V(HIII)), rabbit H chain BS-5 (allotypic variant a(1)) appears more closely related to the subgroup V(HII) than to the subgroups V(HIII) or V(HI). The homology between V(L) and V(H) regions of antibody BS-5 (28%) is not greater than that observed between the V(H) region of antibody BS-5 and the V(L) regions of different rabbit antibodies.     

Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection

Attenuated recombinant H5N1 influenza virus was constructed to develop a safe H5N1 influenza vaccine. The immunogenicity and protective effect of the vaccine prepared from haemagglutinin-modified recombinant H5N1 influenza virus was evaluated in mice intranasally co-administered with cholera toxin B subunit containing a trace amount of holotoxin (CTB*), synthetic double-stranded RNA, poly (I:C) or chitin microparticles (CMP) as adjuvants. Intranasal administration of recombinant H5 HA split vaccine with CTB* or poly(I:C) and/or CMP elicited an immunological response with both anti-H5 HA IgA in the nasal wash and anti-H5 HA IgG antibody in the serum, and showed a protective against lethal H5N1 A/Hong Kong/483/97 (HK483) infection. We also demonstrated that intranasal co-administration of antigen with both poly (I:C) and CMP enhanced the expression of Toll-like receptor (TLR) 3, TLR7 in the spleen. These results indicate that poly (I:C) and CMP are highly effective as mucosal adjuvants for use with the nasal H5N1 vaccine.     

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Background

The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).

Methodology/Principal Findings

The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.

Conclusions/Significance

Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

我们采用RT-PCR,从小鼠杂交瘤细胞中扩增并克隆了抗破伤风类毒素(TT)抗体轻、重链可变区,重链Fd区基因,测定了其VH、Vk序列。并在大肠杆菌中表达了Fd片段,ELISA分析的结果表明Fd片段具有抗原结合的能力,但特异性很差。进一步采用SOE,和PCR技术,将VH、VK基因与ScFv连接片段组装成单链抗体(ScFv)基因片段,以及将人重链CH1和Fab基因连接片段组装成Fab基因片段。将它们分别插入含噬菌体fd外壳蛋白3基因的phagem-id pHEN 1中,在辅助噬菌体M 13-VCS作用下,噬菌体表面表达了抗TT的噬菌体单链抗体(phage-ScFv)与噬菌体Fab(phage-Fab),经ELISA检测,表明它们都能与TT特异结合。     

Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model

Three defense functions of immunoglobulin A (IgA), immune exclusion, intracellular neutralization, and virus excretion, were assessed in a measles virus model using polarized epithelial cells expressing the polymeric immunoglobulin receptor and monoclonal antibodies against the viral H and F envelope proteins and the internal N protein. Anti-H IgA was the most effective antibody at preventing infection via the apical surface, i.e., immune exclusion. This IgA was also the most effective at intraepithelial cell neutralization after infection at the apical surface and endocytosis of IgA at the basolateral surface, although an antibody against the internal N protein was also effective. In the intracellular neutralization experiments, confocal immunofluorescence microscopy showed prominent colocalization of anti-H IgA and H protein inside virus-infected cells, whereas colocalization of anti-F and F protein and of anti-N and N protein was much less, in agreement with the neutralization results. Combinations of IgA anti-H, anti-F, and anti-N showed no synergistic effects in intracellular neutralization. In the immune excretion experiments, virus immune complexes with either anti-H or anti-F IgA placed beneath polarized epithelial cells could be transported to the apical supernatant. Anti-F IgA, which was relatively poor at immune exclusion and intracellular neutralization, was the most robust at virus excretion. Thus, the studies collectively demonstrated three different antiviral functions of IgA in relation to epithelium and also suggested that the particular viral component with which a given IgA antibody reacts is an important determinant of the magnitude of the antiviral effect.