抗人胎盘酸性铁蛋白单链抗体的构建、表达与鉴定

目的:制备人胎盘酸性铁蛋白(PAF),构建、表达并鉴定抗人PAF单链可变区抗体片段(scFv)。方法:设计以SfiⅠ、NotⅠ为酶切位点、以(Gly4Ser)3为linker的2对引物,从抗人PAF单克隆抗体可变区基因的克隆载体中扩增VH和VL基因,用重叠延伸PCR在VH和VL基因间引入连接短肽,构建VH-linker-VL的scFv基因。经SfiⅠ、NotⅠ酶切后克隆到原核分泌型表达载体pUC19/119上,转化大肠杆菌TG1和筛选后测序验证,IPTG诱导表达,SDS-PAGE鉴定其相对分子质量(Mr)。以人PAF为抗原,scFv为一抗,抗His单克隆抗体为二抗,间接ELISA鉴定其抗体活性。结果:重叠延伸PCR扩增产物经凝胶电泳可见约700bp的条带,DNA序列分析证明2株抗体具有完整的scFv序列,并含有c-myc和His。IPTG诱导阳性菌表达产物经SDS-PAGE鉴定有Mr约27000的显示条带,符合scFv与表达标签融合蛋白的理论值;间接ELISA证明表达产物可与PAF结合。结论:成功构建并表达了抗人PAF单链抗体,为进一步建立检测PAF的间接ELISA奠定了基础。     

幽门螺旋杆菌单链抗体的筛选及鉴定

为了获得幽门螺旋杆菌特异性单链抗体scFv,通过噬菌体展示技术,首次直接用幽门螺旋杆菌细胞Hp对噬菌体单链抗体文库Tomlinson进行单链抗体的筛选,经5轮筛选后,通过ELISA方法检测,从随机挑选的96个克隆中获得了8株阳性克隆。再分别将阳性克隆与10种常见菌进行ELISA的交叉反应,最终得到1株特异性表达抗Hp的scFv的噬菌体JH1。随后又进一步对JH1所表达的scFv基因进行PCR扩增,分别得到scFv的VH片段、VL片段,全长基因分别为527bp、368bp和935bp,这些包含着部分载体序列的DNA片段与理论值相符。通过对scFv全长基因进行测序,在NCBI中进行基因序列比对,与已报道的一种植物RNA病毒的复制酶单链抗体基因序列有96％同源性。     

抗乙型肝炎病毒表面抗原T7噬菌体抗体库的构建

构建T7噬菌体单链抗体(scFv)库筛选抗乙型肝炎病毒表面抗原抗体.从抗-HBs阳性患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第1条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接.体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库.以乙型肝炎病毒表面抗原进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性.所建抗体库库容为1.53×107,扩增后初级库滴度为2.42×1010pfu/mL.以乙型肝炎病毒表面抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与HBsAg抗原特异结合的噬菌体抗体,成功构建了抗HBsAg蛋白T7噬菌体抗体库.     

人源抗汉坦病毒核衣壳蛋白T7噬菌体抗体库的构建

构建人源T7噬菌体单链抗体(scFv)库筛选抗汉坦病毒核衣壳蛋白(NP)抗体。从肾综合征出血热恢复期患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第一条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接。体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库。以基因工程表达NP进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性。所建抗体库库容为1.35×107,扩增后初级库滴度为2.12×1010pfu/mL。以NP抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与NP抗原特异结合的噬菌体抗体。结果表明,研究成功构建了人源抗NP蛋白T7噬菌体抗体库。     

抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.     

SOE-PCR法合成狂犬病毒单链抗体基因Fv57

目的:通过一系列短引物拼接合成狂犬病毒糖蛋白单链抗体基因Fv57.方法:采用SOE-PCR的方法,利用多条短的寡核苷酸引物,经过6轮PCR,拼接合成800bp左右的狂犬病毒糖蛋白单链抗体基因Fv57,并利用此方法修正了上述PCR过程中产生的多处突变,从而获得正确的单链抗体基因序列.结果:采用SOE-PCR法合成的基因序列经测序及酶切鉴定,与预期结果一致.结论:成功地利用SOE-PCR法合成了狂犬病毒糖蛋白单链抗体基因Fv57,为其下一步构建原核表达载体,在大肠杆菌中进行表达奠定基础.     

抗肠炎沙门氏菌单链抗体制备及其特异性分析

目的:利用基因工程技术制备抗肠炎沙门氏菌的单链抗体.方法:从抗肠炎沙门氏菌单克隆抗体的杂交瘤细胞中纯化RNA,反转录后扩增出抗体的重链可变区(VH)和轻链可变区(VL)基因片段,采用重叠延伸的方法,用柔性多肽Linker接头(Gly4 Ser)3按VL-Linker-VH方式将VH基因和VL基因拼接成单链抗体基因片段后,连接到pGEX-4T-1载体上,进行重组转化.挑取阳性克隆,经IPTG诱导后,通过GST柱进行亲和层析,最后利用ELISA检测抗体的活性.结果:成功构建了表达抗肠炎沙门氏菌单链抗体的基因工程菌株,经SDS-PAGE和ELISA检测结果表明,诱导表达的单链抗体scFv分子量约为60 kDa,其能特异与肠炎沙门氏菌结合,但与副甲伤寒沙门氏菌、鸭沙门氏菌、鼠伤寒沙门氏菌有轻度交叉反应.结论:成功构建了抗肠炎沙门氏菌单链抗体的表达菌株,表达的单链抗体scFv可作为沙门氏菌的检测的候选抗体分子.     

在DH5α中拼接构建ADAM10全长真核表达载体的基因突变

目的:构建ADAMI0真核表达载体,为进一步研究其生物学功能打基础.方法:将人ADAM10的上下两部分基因片段(分别为全长基因的1 ～910bp和911 ～2 247bp片段),依次与真核表达载体pcDNA3.1相连,以大肠杆菌DH5α或BL21(DB)作为感受态宿主菌用于转化连接产物,拼接成全长的阳性克隆通过PCR、酶切和测序鉴定.结果:ADAM10下段基因与已正确连入上段的pcDNA3.1重组质粒拼接时,若用DH5α为感受态菌,则下半段出现碱基插入增加512bp,测序结果显示为ADAM10基因第1 531 bp～2 042 bp间的序列有紧邻的双份;若用BL21(DE3)为感受态,则无突变.结论:将ADAM10基因与pcDNA3.1真核表达载体依次拼接构建重组质粒时,以DH5α为宿主菌可出现基因序列增加的罕见突变,而以BL21(DE3)为宿主则无突变,由此成功构建ADAM10全长基因与pcDNA3.1的重组质粒.     

抗四环素单链抗体基因的构建与序列测定

目的：构建抗盐酸四环素的单链抗体（scFv）基因。方法：以抗盐酸四环素单克隆抗体杂交瘤细胞株的总RNA为模板,用RT-PCR法扩增全套抗体轻、重链基因;经重叠延伸反应,以编码柔性多肽（GIy4Ser）3的基因为接头,将轻、重链基因组装为完整的seFv基因,并克隆到pGEMT-Easy载体中进行测序分析。结果：所克隆的四环素scFV基因全长为735 bp,为VH-Linker-VL结构,VH基因为354bp,Linker为（Gly4Ser）3多肽的核酸序列,VL基因为336 bp。结论：构建了抗四环素的单链抗体基因,为进一步用于四环素的残留枪测奠审某础．     

柑桔溃疡病菌重组单链抗体的高效表达及活性检测

运用PCR方法扩增利用核糖体展示技术筛选的抗柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,XAC)的单链抗体(ScFv95)基因片段,将单链抗体基因重组到原核表达载体pET30a( )中,构建单链抗体高效表达载体pET30a( )-XAC-ScFv。再将pET30a( )-XAC-ScFv质粒转化进大肠杆菌BL21(DE3)后诱导表达,并对表达产物进行纯化、复性及活性检测。获得了抗XAC单链抗体的高效表达蛋白,以包涵体形式存在的表达蛋白大小约32kDa。包涵体蛋白经过变性、纯化和复性后,初步获得有功能的单链抗体。同时用Biacore分析XAC-ScFv-95与XAC LPS作用,结果表明复性后的XAC-ScFv-95具有较高的亲和力,从而为柑桔溃疡病菌XAC的免疫诊断和防治研究提供了新的工具和途径。     

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r2 values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.     

Rapid gene splicing and multi-sited mutagenesis by one-step overlap extension polymerase chain reaction

Gene splicing and site-directed mutagenesis (SDM) are important to introduce desired sequences in target DNA. However, introducing mutations at multiple sites requires multiple steps of DNA manipulation, which is time-consuming and labor-intensive. Here, we present a rapid efficient gene splicing and multi-sited mutagenesis method that introduces mutations at two distant sites via sequential connection of DNA fragments by one-step overlap extension polymerase chain reaction (OE-PCR). This bottom-up approach for DNA engineering can be broadly used to study protein structure-function, to optimize codon use for protein expression, and to assemble genes of interest.     

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene.

In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.     

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.     

Construction of a fully synthetic human scFv antibody library with CDR3 regions randomized by a split-mix-split method and its application

The randomization scheme of hypervariable region takes crucial role in construction of a synthetic antibody library. The codon bias and inevitable 'stop' codon of conventional 'NNK' and 'NNS' codons limit their applications. Here we report a split-mix-split DNA synthesis method that can control over the amino acid composition and distribution of randomized sequences effectually. A fully synthetic human antibody library with a diversity of 1.56 x 10(9) was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that >60% of colonies had completely correct scFv genes and the amino acid composition and distribution were designed well in accordance. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3 x anti-ovarian carcinoma bispecific antibody). These results proved the feasibility of the split-mix-split DNA randomization strategy in library construction and site-directed mutagenesis.     

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.     

重叠延伸PCR法构建小鼠Sumf1基因的定点突变真核表达载体

前期通过染色质免疫沉淀技术（chromatin immunoprecipitation assay,CHIP）筛选出了转录因子activator protein．2 alpha（AP-2α）的靶基因Sumf1．采用重叠延伸PCR定点诱变技术,对AP．2α在Sumf1内含子片段上的两个结合位点的碱基进行定点突变,并构建定点突变表达载体．DNA测序表明,Sumf1内含子片段103—111bp,411～419bp两处的碱基已分别由GCCGTCAGG突变为GAAGTCCTG,由GCCTCTAGG突变为GGATCTCTG,成功实现定点诱变,为进一步研究AP-2α对基因Sumf1的表达调控奠定了基础．     

高效构建卵清白蛋白scFv噬菌体文库及其筛选

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scFv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重链和小鼠轻链基因。通过无缝连接酶一步将小鼠重链基因、轻链基因和linker DNA连接起来,插入噬菌体表达载体中,构建OVA scFv噬菌体展示文库。测定文库容量,对文库进行富集筛选,ELISA鉴定阳性克隆,测序后构建真核表达载体,转入Expi-CHO悬浮细胞进行真核表达,利用Western blot进行鉴定。结果:成功获得库容量为1. 2×10~7cfu的OVA scFv噬菌体展示文库,并从中筛选出8个阳性克隆,选取效价最高的2号克隆,在Expi-CHO悬浮细胞中表达获得可溶性抗体。结论:建立了一种高效构建scFv噬菌体文库的方法,筛选获得高结合活性的OVA单链抗体,并成功进行了真核表达,为OVA ELISA检测试剂盒的研制奠定了基础。     

In vitro evolution of single-chain antibodies using mRNA display

Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved ~30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.