Construction of an Artificially Randomized IgNAR Phage Display Library: Screening of Variable Regions that Bind to Hen Egg White Lysozyme

To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7?×?10⁷ PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I?IV): type I contains a single cysteine residue and type II?IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.     

Minichromosomal variable surface glycoprotein genes and molecular karyotypes of Trypanosoma (Nannomonas) congolense

Employing orthogonal-field-alternation gel electrophoresis (OFAGE), we have separated chromosome-sized DNA molecules from Trypanosoma (Nannomonas) congolense clones, the clones being derived from several distinct antigenic repertoires. Trypanosome clones that belong to a specific antigenic repertoire appear to have a chromosome pattern characteristic of that particular repertoire. Hybridization of the separated chromosomes with cloned DNA fragments encoding variable surface glycoproteins revealed the presence of two different T.(N.) congolense variable surface glycoprotein genes on mini-chromosomes (mc) and the modes by which these genes may be activated: one by duplicative and the other by non-duplicative activation.     

Trypanosoma congolense: host responses following tsetse transmitted infection of Kilifi isolates in goats

East African x Galla goats, when infected with Trypanosoma congolense isolates from the Kilifi area of Kenya by Glossina morsitans centralis, did not develop the characteristic chancre reaction at the bite sites, whereas bites of tsetse infected with the cloned T. congolense IL.1180 from Serengeti, Tanzania, resulted in chancres in the same goats. Histological changes could not be observed in skin biopsies collected 8 or 9 days after infection with Kilifi isolates. However, all goats became parasitemic about 10 days after challenge. It is concluded that the absence of chancre development is a characteristic feature of T. congolense parasites from Kilifi. The isoenzyme analysis of clones of two T. congolense Kilifi isolates and the T. congolense clone IL.1180 indicated that they belong to different zymodemes. Neutralizing antibodies to homologous metacyclic variable antigen types were detected in six out of seven (86%) of the sera from goats infected with a clone or stock of a T. congolense Kilifi isolate, 20 days after infection. Goats primed by tsetse transmitted infection with a stock or clone of T. congolense from Kilifi and treated with Berenil were, in three out of eight cases (37%), not immune to homologous challenge. It is suggested that the reduced immune response to metacyclic variable antigen types could be a result of the absence of cellular infiltration, i.e., chancre development in the skin at the tsetse bite site. It is concluded that the use of the chancre reaction as a marker for serodeme analysis of recently isolated stocks of T. congolense from Kilifi was not feasible.     

Attainment of 15-Fold Higher Affinity of a Fusarium-specific Single-Chain Antibody by Directed Molecular Evolution Coupled to Phage Display

Fusarium head blight (FHB) caused by Fusarium graminearum infection is a devastating disease of wheat, maize, and other cereals. A previously isolated chicken single-chain Fv antibody (scFv), CWP2, that conferred durable resistance in planta was subjected to directed evolution by error-prone PCR and DNA shuffling, generating a mutated library. Panning of the mutated library against cell wall-bound proteins (CWPs) from F. graminearum by phage display enriched phage clones that were used for a further round of DNA shuffling to construct a combinatorial library comprising 3?×?10(6) variants. Screening of this library by phage display for variants reactive against the CWPs led to the identification of a number of clones. Comparative enzyme-linked immunosorbent assay analyses revealed eight clones exhibiting a higher reactivity than the parent, CWP2, and containing four different single-chain antibody sequences. Surface plasmon resonance measurements confirmed that three mutated scFvs, CWPa, CWPb, and CWPd, displayed 15-fold, 11-fold, and 7-fold higher affinities, respectively, compared with CWP2. Three-dimension modeling of CWPa illustrates a conformational change bringing all six complementary domain regions on the antibody surface in one direction. These results provide promising unique resistance molecules for effective control of FHB and its associated mycotoxins in food/feed chains.     

Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

人源噬菌体抗体库的构建及抗VEGF抗体的初步筛选分析

应用噬菌体表面呈递技术构建人抗体组合文库 .筛选获得了结合血管内皮细胞生长因子( VEGF)的人噬菌体 Fab抗体 ,并对所获抗体的多样性进行了进一步分析 .从不同人群外周血淋巴细胞提取总 RNA,经反转录后采用家族特异性免疫球蛋白可变区基因引物与免疫球蛋白信肽序列引物 ,通过改变 PCR条件或半套式扩增分别获得全部亚型的轻、重链抗体 Fab段 ,并重组到噬粒载体 p Comb3H中 ,经电转化大肠杆菌 XL- 1 Blue,构建了 1 .5× 1 0 8完整组合抗体库 .利用 VEGF12 1对该库经过 4轮固相筛选后 ,获得 1 2个可与 VEGF特异结合的阳性克隆 .酶谱分析表明了所获抗体克隆的多样性 .为通过基因工程改造 ,进一步获得可用于临床的人源 VEGF抗体奠定了基础 .     

Cloning of genomic sequences of human prointerleukin 1 beta

The human brain library carried in the EMBL3 vector was employed for isolating prointerleukin 1 beta genomic sequences using three synthetic 20-member oligonucleotides. Oligonucleotides were homologous to the following mRNA regions: 3'-nontranslated region/C1/, 3'-translated region of mRNA/C2/ and the sequence coding N-terminal of mature protein/N1/. The oligonucleotide labeling utilized the terminal nucleotidyltransferase and [alpha-32P] dATP and specific activity of labeled oligonucleotides reached 1.6.10(10) cpm. The sizes of the synthesized labeled sequences (tails) were about 10 b.p. Hybridization probe C1 was used for the first screening and 24 hybridization positive clones were detected. For the next screening probe C2 was used and only 2 hybridization positive clones with different level of hybridization were detected from 24 clones. Probe N1 was used for the third screening and allowed to identify the only positive clone. The characterization by restriction mapping and Southern blot analyses have shown that recombinant phage DNA contains all three exons, coding the mature interleukin 1 beta. Some fragments were recloned to tg130 vector phage and nucleotide sequences of exon 5 (completely) and exon 7, intron 4 and 5 (partially) were determined.     

Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells.

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.     

Identification of epitopes of trichosanthin by phage peptide library

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.     

从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体

目的:从天然的大容量噬菌体抗体库中筛选特异的抗结核分枝杆菌晶体蛋白( alpha-crystallin Acr)的人源抗体.方法:以结核分枝杆菌Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮“吸附-洗脱-扩增”的过程从大容量抗体库中筛选特异性抗结核分枝杆菌Acr蛋白的抗体,并对可变区序列进行了测序分析.将特异性的噬菌体抗体感染HB2151菌,经IPTG诱导表达,制备了抗结核分枝杆菌Acr蛋白的可溶性单链抗体;对其序列和抗原结合活性进行分析鉴定.结果:经过4轮筛选,获得了43个与结核分枝杆菌Acr蛋白结合的阳性克隆,其中29个特异结合的克隆;测序分析有26不同的可变区片段;通过可溶性单链抗体(scFv)表达筛选到14株特异性结合Acr蛋白的可溶性单链抗体克隆;经过基因测序,分析了可变区基因的亚群.成功制备了可溶性单链抗体.Westren blotting分析证实筛选的人源单链抗体能与天然蛋白结合.结论:利用单链大容量抗体库获得抗结核分枝杆菌Acr蛋白的噬菌体抗体并且成功制备抗结核分枝杆菌Acr天然蛋白的可溶性单链抗体,为今后的研究和应用奠定基础.     

Two bovine genes for cytochrome c oxidase subunit IV: a processed pseudogene and an expressed gene

We have isolated and analyzed 17 clones from a bovine genomic library in phage lambda Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.     

囊素与杂交瘤细胞的结合及结合肽的鉴定

【目的】囊素(BS)是禽类和哺乳动物中具有重要免疫调节功能的多肽,能有效促进杂交瘤细胞抗体的分泌,为探讨杂交瘤细胞是否有BS受体分子的表达。【方法和结果】本研究采用荧光显微镜、激光扫描共聚焦显微镜和流式细胞仪分析的方法,检测BS与杂交瘤细胞的结合特性。实验结果证实BS与杂交瘤细胞的结合具有特异性、趋饱和性和可逆性。为进一步分析BS与杂交瘤细胞的结合位点,实验中以BS分子作为靶标,对噬菌体随机12肽库进行了4轮亲和筛选,ELISA和竞争抑制试验显示2个噬菌体克隆能特异性与BS结合。对阳性噬菌体克隆进行序列测定分析表明,其插入的12肽分别为:ACTKHLCLLQPL、MSCNDTLCLLPN,保守序列为LCLL。体外实验表明,2个人工合成的12均都能在一定程度上抑制BS与杂交瘤细胞的特异性结合。【结论】本研究表明杂交瘤细胞具有BS结合的受体,这为进一步研究BS促杂交瘤细胞抗体分泌的信号传导通路奠定了基础。     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

Composite human VK genes and a model of their evolution.

A phage library and two cosmid libraries were screened for human VK genes. Two recombinant phage and four cosmid clones were analysed in detail by restriction mapping and sequencing. Each one contained a single VKI sequence. Two of these six sequences are potentially functional VK genes and four are pseudogenes. Two pseudogenes derived from different genomic DNAs are highly homologous and are therefore either allelic variants or the products of a recent duplication event. Comparisons of our sequences with all fully determined human VKI amino acid and DNA sequences reveal identical segments which at first sight appear like minigenes. But these segments do not coincide with the subregions and some of the segments include both, framework and complementarity determining regions (FR, CDR, ref. 2). The findings may be explained by an evolutionary model generating composite genes by gene conversion and selection.