从免疫及天然噬菌体抗体库中筛选抗rP27Kip1单链抗体的研究

用重组p27Kip1蛋白(rP27Kip1) 免疫小鼠,从免疫和未经免疫的小鼠脾脏抽提mRNA并扩增小鼠H链(H链)及L链(L链)基因,分别组装成单链可变区片段(ScFv)基因,构建噬菌体免疫抗体库及天然抗体库. 文库仅经一轮抗原-抗体亲和筛选后,用TaqⅠ/HinfⅠ酶切分析转化子. 获自免疫抗体库的64个克隆中,有11个克隆的酶切片段相同,而天然抗体库的64个克隆的片段则都彼此不同,但有1个克隆的酶切片段与免疫抗体库的11个克隆酶切片段相同. 将这些酶切图谱相同的重组片段分别克隆入原核表达载体pET28b(+),并在大肠杆菌(E. coli)中表达,表达产物经ELISA分析,证实可特异结合rP27Kip1抗原,一方面说明在抗体筛选过程中辅以酶切图谱分析,可以有效提高筛选效率,另一方面,也说明从噬菌体抗体库筛选特异性抗体是制备单克隆抗体(McAb)的理想途径之一.     

抗P-选择蛋白人源性单克隆抗体的制备

目的:获得人源性抗P-选择蛋白(selectin)特异性抗体,为相关疾病治疗奠定基础。方法:在HEK293细胞中真核分泌表达人P-选择蛋白功能性片段,以此蛋白片段作为抗原,利用本室构建的大容量全合成人源性噬菌体抗体库进行筛选,经过3轮固相筛选后,阳性克隆得到富集;将其中富集效果最好的一株单链抗体A1改造成全抗体(IgG1),重组质粒转染H293细胞后,抗体得到表达;表达后在全抗体水平上用ELISA和Western印迹分别验证了A1抗体的特异性,并通过非竞争ELISA方法初步测定这株抗体的亲和力。结果:3轮筛选得到3株特异性噬菌体抗体,其中富集效果最好的单链抗体A1改造成全抗体形式后特异性良好,抗体亲和力Kd=2×10-8 mol/L。结论:筛选得到一株特异性较好的抗P-选择蛋白人源性单克隆抗体A1,其特异性和亲和力较好,有继续开发的价值。     

TNF-α纳米抗体的筛选、表达及特异性检测

目的:构建肿瘤坏死因子(tumor necrosis factor,TNF)纳米抗体的噬菌体文库,筛选并表达与TNF-α具有亲和特异性的纳米抗体。方法:(1)利用TNF-α免疫羊驼,提取外周血淋巴细胞总RNA,构建噬菌体文库;多次淘洗筛选到与TNF-α有亲和力的克隆。(2)通过ExPASy分析其分子量和亲疏水性等理化性质,并将筛选得到的VHH基因在大肠杆菌E.coli DH5α中表达。(3)表达的Nb_(TNF-α)蛋白质经过Ni金属螯合亲和层析纯化,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测蛋白质的抗原特异性和亲和力。结果:(1)通过噬菌体文库的构建和淘洗,筛选到8个与TNF-α有亲和力的VHH基因片段。(2)通过软件预测,这8个Nb_(TNF-α)蛋白均为亲水性蛋白,其分子量为19.6～20.1kDa;在大肠杆菌中重组表达这8个抗体蛋白。(3)ELISA检测结果表明,有5株纳米抗体Nb_(TNF-α)-1、Nb_(TNF-α)-2、Nb_(TNF-α)-3、Nb_(TNF-α)-4和Nb_(TNF-α)-5能与TNF-α特异性结合。结论:成功筛选并表达了5株具有TNF-α特异性的纳米抗体,可能成为抗TNF-α的候选药物。     

SHN3多克隆抗体纯化与鉴定

目的:利用SHN3免疫特异肽段亲和层析纯化抗SHN3多克隆抗体。方法:将制备多克隆抗体SHN3免疫原片段(99bp)划分为4段互相重叠等长的基因片段(48bp),构建原核表达重组质粒并转化E.coil的BL21(DE3)菌株。IPGT诱导重组蛋白表达,Western blotting筛选并人工合成与抗体免疫结合特异性片段,亲和层析纯化抗体,Western blotting检测纯化结果。结果:成功地筛选到SHN3免疫原特异肽段part-3,以亲和层析的方式纯化多克隆抗体,纯化后免疫特异性提高。结论:亲和层析的方式纯化得到的SHN3多克隆抗体抗体,有效地提高免疫特异性,为后续SHN3蛋白功能研究和应用奠定了基础。     

甲型副伤寒结合疫苗人免疫血清破伤风类毒素抗体亲和力的检测与分析

目的 建立甲型副伤寒结合疫苗人免疫血清中破伤风类毒素(tetanus toxoid, TT)抗体亲和力的检测方法,探究甲型副伤寒结合疫苗免疫人体后,免疫系统对载体蛋白质TT的亲和力成熟情况,并分析与免疫后抗体含量增长情况的关系。方法 采用间接ELISA检测甲型副伤寒结合疫苗免疫前后血清中TT抗体含量,利用硫氰酸钾(potassium thiocyanate, KSCN)洗脱测定抗体亲和力。检测免疫后抗体含量高增长倍数组(免疫后相比免疫前抗体含量增长>10倍)和低增长倍数组(免疫后相比免疫前抗体含量增长<2倍)血清,计算相对亲和力指数。结果 KSCN洗脱对TT与酶标板之间的物理性吸附没有影响(P>0.05)。KSCN的最适洗脱浓度为3.1 mol/L。对于高抗体含量增长倍数组,免疫后相比免疫前抗体亲和力明显成熟,免疫前与免疫后抗体亲和力差异有统计学意义(P=0.005 0,P<0.05),对于低抗体含量增长倍数组免疫后抗体亲和力成熟不明显,免疫前与免疫后抗体亲和力差异无统计学意义(P=0.852 2,P>0.05)。结论 KSCN可用于甲型副伤寒结合疫苗人免...     

抗GM-CSF纳米抗体的筛选与鉴定

目的:制备抗粒细胞-巨噬细胞集落刺激因子(GM-CSF)纳米抗体,并测定其亲和力。方法:分离提取GM-CSF免疫后羊驼外周血淋巴细胞总RNA,PCR扩增得到纳米抗体基因片段,与载体pHEN1重组后克隆至大肠杆菌TG1以构建初始文库,经拯救构建得到噬菌体展示纳米抗体文库,并对其进行生物淘选;通过大肠杆菌BL21(DE3)原核表达阳性纳米抗体克隆,并测定其亲和力。结果:构建了多样性良好且库容量为1.37×10~9cfu的纳米抗体初始文库,3轮淘选后共筛选得到5株氨基酸序列差异性较大的纳米抗体,并对其中一株纳米抗体G1进行表达纯化,SDS-PAGE分析表明纳米抗体G1纯度较高,且有较高的亲和力(K_D=2.95×10~(-8)mol/L)。结论:制备了高亲和力的抗GM-CSF纳米抗体,可应用于相关炎症抗体药物研制和疾病监测等方面。     

鼠源抗B型肉毒毒素单链抗体噬菌体文库的构建筛选及抗体免疫学活性的初步研究

B型肉毒毒素重链C-端片段(BoNTB/Hc)经金属螯和层析法纯化后免疫Balb/c小鼠,从其脾淋巴细胞中提取总RNA,反转录成cDNA,用抗体可变区混合引物进行全套抗体重、轻链可变区基因的扩增,体外随机装配成单链抗体(scFv)。将其克隆至pCANTAB5E中,构建单链抗体噬菌体抗体库。结果表明经过4轮"吸附-洗脱-扩增"的富集过程,筛选获得高亲和力的克隆。序列测定符合抗体可变区结构特点。     

半抗原特异性抗体的筛选及亲和力成熟

噬菌体抗体库技术的出现开创了一条便捷的基因工程抗体生产路线,为小分子半抗原抗体的制备提供了一条新途径,具有极大的应用潜力,但得到的抗体片段的亲和力普遍较全分子差,需要优化筛选策略及抗体的体外亲和力成熟来解决这一问题。     

抗CD5单链抗体基因克隆和表达

以分泌抗CD5单克隆抗体的杂交瘤细胞poly(A) mRNA为模板,通过RTPCR扩增出抗CD5单克隆抗体的重链可变区(VH)和轻链可变区(VL) cDNA片段组装出抗CD5单链抗体(ScFv) cDNA片段。该ScFv片段被克隆到pCANTAB 5E载体上,以E.coli TG1为宿主,进行噬菌体表面呈现。通过Molt4细胞表面分子CD抗原,对噬菌体表面呈现的ScFv进行免疫亲和富集筛选。经细胞ELISA鉴定,得到4株高亲和力克隆。DNA序列分析得知,单链抗体全长732碱基,其中VH为339碱基,VL为300碱基。抗CD5 ScFv在E.coli HB2151中以可溶形式分泌表达,产物主要分布于周质之中,占周质中总蛋白的20%。     

人间隙连接蛋白 31 相互作用蛋白Annexin Ⅱ的筛选与证实

间隙连接蛋白 31 (connexin31 , Cx31) 是间隙连接蛋白 (connexin) 家族的一员,目前对于 Cx31 的功能及其调节方式知之甚少 . 采用固相多肽合成的方法合成 Cx31 羧基端一个多肽片段 (250~266) ,经 HPLC 纯化后偶联到匙孔血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化、经蛋白质印迹、细胞免疫荧光染色、免疫沉淀证实得到的抗体为特异性抗 Cx31 的抗体 . 运用制备的抗 Cx31 多克隆抗体免疫沉淀, SDS- 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分离,蛋白质条带回收,蛋白质胶块酶解, Q-TOF 质谱分析,数据库扫描筛选可能相互作用蛋白,运用抗体 pull-down 实验,筛选到可能相互作用蛋白 annexin Ⅱ,经过免疫共沉淀、细胞免疫共定位等实验证实 annexin Ⅱ与 Cx31 相互作用 .     

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (K_D < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.     

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10⁸ clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR‐L3 and CDR‐H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three‐dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire. Copyright © 2010 John Wiley & Sons, Ltd.     

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V_H and V_L genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Production of Humanized Fab Fragment against Human High Affinity IgE Receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (K_D < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.     

Glycosaminoglycan—lipoprotein interactions: 2. Structure of heparin-releted variants with high affinity for low density lipoprotein

A particular heparan sulphate fraction which possessed the largest proportion of high affinity variants for human low density lipoprotein contained almost equal proportions of the repeating units l-iduronosyl(O-sulphate)N-sulphamidoglucosamine and d-glucoronosyl-N-acetylglucosamine. The heparan sulphate was fractionated on lipoprotein-agarose into three populations. Results of periodate oxidation—alkaline elimination indicated that the size of the completely N-sulphated block regions increased with increasing affinity. In contrast, the number of consecutive l-iduronosyl(O-sulphate)-containing repeats decreased with increasing affinity towards lipoprotein. After selective periodate oxidation—alkaline scission of d-glucoronic acid residues only a portion of the heparan sulphate fragments retained high affinity for lipoprotein. This portion consisted of fragments larger than dodecasaccharide which contained both l-iduronic acid-O-sulphate and non-sulphated uronic acid residues (−) 2:1). No affinity or little affinity was displayed by fragments (of comparable size) that contained only sulphated l-iduronic acid residues.     

Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V_H region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG₁ antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.     

Single chain Fab (scFab) fragment

Background  

The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.     

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.