亲环素A抗原表位氨基酸序列及三维结构研究

目的：研究人抗亲环素A（CyPA)McAb D4识别的抗原表位的氨基酸组成及其在蛋白分子上的三维定位。方法：以抗人亲环素A McAb D4作捕获抗体,对噬菌体展示肽库进行淘筛,并对筛选出的噬菌体中插入片段进行DNA序列测定和氨基到序列分析,再用RasMol蛋白质三维结构分析软件在亲环素A分子的三维结构中查找相应的抗原表位。结果：经过三轮淘筛和扩增,选择7个克隆进行序列,提示该抗原表位是构象性的。用RasMol蛋白质三维结构分析软件在亲环素A分子的三维结构中,找到由W121、S99、L98、Q111、S110、F112、L122构成的疏水代状结构,可能就是McAb D4的抗原表位。结论：人亲环素A分子上W121、S99、L98、Q111、S110、F112、L122构成一个构象型抗原表位。     

丙型肝炎病毒非结构蛋白3人源单链抗体的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（ＨＣＶ）非结构蛋白（ＮＳ）３的人单链抗体（ＳｃＦｖ）,以解决鼠单抗蝗免疫原性问题,为ＨＣＶ的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术,以重组的ＨＣＶＮＳ３为包被抗原,从噬菌体单链楞变区抗体库中经过５轮“吸附－洗脱－扩增”筛选过程,获得抗原结合活性较强的ＨＣＶＮＳ３人单链抗体（ＳｃＦｖ片段）阳性克隆,并对其进行免疫体及序列测定。结果 筛选出来的ＳｃＦｖ片段     

东方田鼠抗日本血吸虫抗性相关靶基因筛选

目的 寻找东方田鼠抗日本血吸虫抗性相关靶基因。方法利用东方田鼠血清以亲和淘洗方法筛选日本血吸虫成虫噬菌体展示cDNA文库，将所得阳性克隆进行测序及生物信息学分析，确定目的基因。结果经3轮筛选，特异性噬菌体得到有效富集（857倍），随机挑取58个克隆进行序列测定，获得了10条表达序列标签（EST）序列，其中7条与GenBank中登陆的日本血吸虫基因序列具有99％～100％同源性，1条与类人猿锌指蛋白基因有82％同源性。功能预测结果表明，大部分EST编码蛋白或多肽的功能主要是参与血吸虫基因表达调控。结论发现了多个可能与东方田鼠抗血吸虫抗性相关的靶基因，为进一步阐述东方田鼠抗血吸虫病的分子机理奠定了基础。     

HCV非结构蛋白NS5A人源单链可变区抗体基因的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（HCV）非结构蛋白NS5A的人源单链可变区抗体（ScFv)的编码基因,为HCV NS5A蛋白质生物学功能的研究及抗HCV的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术,以重组纯化的HCV非结构蛋白NS5A为固相抗原,从噬菌体单链可变区抗体半合成库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性和特异性较强的HCVNS5A人源单链可变区抗体基因片段的阳性克隆,并对其进行免疫检测及序列测定,结果 筛选得到的ScFv片段基因核苷酸序列为789nt,编码由262个氨基酸残基组成的多肽,具有典型的免疫球蛋白轻链和重链可变区的结构特点,基因编码产物具有HCV NS5A蛋白反应的免疫学活性和特异性。结论 利用噬菌体抗体库技术,成功获得了HCV NS5A人单链可变区抗本ScFv编码基因,并获得了可溶性单链抗体的表达。     

Isolation of specific peptides that home to dorsal root ganglion neurons in mice

We isolated peptides that home to mouse dorsal root ganglion (DRG) from a phage library expressing random 7-mer peptides fused to a minor coat protein (pIII) of the M13 phage. An in vitro biopanning procedure yielded 113 phage plaques after five cycles of enrichment by incubation with isolated DRG neurons and two cycles of subtraction by exposure to irrelevant cell lines. Analyses of the sequences of this collection identified three peptide clones that occurred repeatedly during the biopanning procedure. Phage-antibody staining revealed that the three peptides bound to DRG neurons of different sizes. To determine if the peptides would recognize neuronal cells in vivo, we injected individual GST-peptide-fusion proteins into the subarachnoid space of mice and observed the appearance of immunoreactive GST in the cytosol of DRG neurons with a similar size distribution as that observed in vitro, indicating that the GST-peptide-fusion proteins were recognized and taken up by different DRG neurons in vivo. The identification of homing peptide sequences provides a powerful tool for future studies on DRG neuronal function in vitro and in vivo, and opens up the possibility of neuron-specific drug and gene delivery in the treatment of diseases affecting DRG neurons.     

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.     

A novel small heat shock protein 12.6 (HSP12.6) from Brugia malayi functions as a human IL-10 receptor binding protein

Phage display cDNA expression library of the third stage larvae (L3) of Brugia malayi was screened for identifying target(s) that bound to the human interleukin-10 receptor (huIL10R). This iterative screening identified an insert that showed significant homology to Caenorhabditis elegans HSP12.6. The gene was designated B. malayi HSP12.6 (BmHSP12.6) and has orthologues in several gastrointestinal nematode genome (Ancylostoma caninum, Ascaris lumbricoides and Ascaris suum) but the gene or gene product has not been studied further in these parasites. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs). BmHSP12.6 has a short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain suggesting that this protein belongs to a novel class of small HSPs. BmHSP12.6 appears to be differentially transcribed with highest expression in the vertebrate stages of the parasite (L4, adult and mf) compared to its mosquito vector stage (L3). More importantly recombinant BmHSP12.6 bound to huIL10R in a dose dependent fashion and inhibited the binding of human IL-10 (huIL10) to huIL10R in vitro. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10. This study thus describes a novel small HSP from B. malayi that has the capacity to bind to huIL10R, block binding of huIL10 to huIL10R and function similar to huIL10.     

Cloning and characterization of a novel immunogenic protein 3 (NIP3) from Brugia malayi by immuno screening of a phage-display cDNA expression library

Iterative screening of a phage display cDNA expression library of the third-stage larvae (L3) of Brugia malayi with sera from putatively immune individuals (endemic normal, EN) identified a novel clone with insert showing significant homology to Onchocerca volvulus novel immunogenic protein-3 (Ov-NIP3) gene and Caenorhabditis elegans NIP3-like protein and hence the gene was designated Brugia malayi NIP3-like protein (BmNIP3). EST database analysis showed that ESTs from several gastrointestinal nematodes such as Ancylostoma caninum, Teladorsagia circumcincta, Haemonchus contortus and Strongyloides stercoralis has BmNIP3 homologues, but the gene has not been described from these parasites. Sequence analyses showed that BmNIP3 has three potential mucin-type O-glycosylation sites and several serine/threonine phosphorylation sites. As expected, BmNIP3 protein isolated from the parasite was serine/threonine phosphorylated. Further analyses showed that BmNIP3 is differentially transcribed, with highest level of expression present in the larval (L3 and L4) stages. Mice immunized with rBmNIP3 developed strong antibody responses predominantly of the IgG1 and IgG2a subtype. A similar analyses of the sera samples from EN individuals showed that they also carry high levels of IgG1 and IgG2 antibodies against BmNIP3, whereas, chronically infected patients carry largely IgG3 antibodies and MF individuals carry high levels of IgG1 antibodies against BmNIP3. This study thus describes a novel protein from B. malayi that appears to be highly immunogenic in both humans and mice.Nucleotide sequence data reported in this paper is available in Genbank under the accession number: BmNIP3: AY464549     

Bacteriophages and their applications in the diagnosis and treatment of hepatitis B virus infection

Hepatitis B virus (HBV) infection is a major global health challenge leading to serious disorders such as cirrhosis and hepatocellular carcinoma. Currently, there exist various diagnostic and therapeutic approaches for HBV infection. However, prevalence and hazardous effects of chronic viral infection heighten the need to develop novel methodologies for the detection and treatment of this infection. Bacteriophages, viruses that specifically infect bacterial cells, with a long-established tradition in molecular biology and biotechnology have recently been introduced as novel tools for the prevention, diagnosis and treatment of HBV infection. Bacteriophages, due to tremendous genetic flexibility, represent potential to undergo a huge variety of surface modifications. This property has been the rationale behind introduction of phage display concept. This powerful approach, together with combinatorial chemistry, has shaped the concept of phage display libraries with diverse applications for the detection and therapy of HBV infection. This review aims to offer an insightful overview of the potential of bacteriophages in the development of helpful prophylactic (vaccine design), diagnostic and therapeutic strategies for HBV infection thereby providing new perspectives to the growing field of bacteriophage researches directing towards HBV infection.     

Selection and characterization of single domain antibodies against human CD20

CD20 is a membrane protein with four integral membrane regions and a large extracellular loop between residues 142 and 187, which serves as a target binding region for rituximab (RTX) and most other anti-CD20 monoclonal antibodies. It is highly expressed in B-lymphoma cells and B lymphocytes and often serves as a target for immunotherapy. In this study, we developed single domain antibodies (sdAbs) for the sensitive detection of CD20. To achieve this, an immune sdAb library was prepared from llamas immunized with a fusion between the large loop from CD20 and Hoc, a highly antigenic protein from the T4 bacteriophage, (CD20-Hoc). By subtracting binders to recombinant Hoc during the biopanning, potential anti-CD20 sdAbs were selected, sequenced, and characterized for their binding affinity to CD20-Hoc fusion versus Hoc. Twenty five clones grouped into three different families based on CDR3 sequence were identified as potential CD20 binders. The binding kinetics of representative sdAbs from each class and RTX were evaluated by surface plasmon resonance (SPR). Most of the sdAbs that were evaluated show binding affinities to CD20-Hoc in the nM range, and class A sdAbs, exhibited ≥40-fold increase in affinity for CD20-Hoc versus Hoc. When the binding of the sdAbs to CD20 on SU-DHL-4 cells was evaluated by flow cytometry, only class A sdAbs displayed strong binding to CD20 and recognized DHL cells in a concentration dependent manner.