Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Predicting equations of main factors affecting regional climate in the"Three-North" Protective Forest Area

The relationship between the change of forest resources and climatic factor in the, “Three-North” region of China were studied in this paper. The predicting equations of climatic factor (dependent variable) with regional independent variable (longitude, latitude and altitude) and stand independent variable (forest coverage rate), were developed by extensively using the linear and nonlinear regression methods. With these models, we can calculate the ecological benefit of Shelter-belt forest.

Regional Variability of the Effects of Land Use Systems on Soil Properties

In order to explore the regional variability of the effects of land use systems on soil properties, Shouyang County in Shanxi Province and Danling County in Sichuan Province of China were selected as the study areas. Field soil samples of the four land use systems （natural forest, forest plantation, shrubland, and cropland） were collected, respectively, from the two areas. The general statistical tools were used to analyze soil data. The results showed that the influence of land use systems on soil properties was significant. In general, soils in slightly human-disturbed land use systems presented a higher fertility level than those in strongly human-disturbed land use systems in both areas. Furthermore, the impacts of the same land use systems on soil properties showed a distinct regional variability, and even in the same land use system, different farming systems and site management measures （such as irrigation, fertilization, and pesticides） could also lead to the regional heterogeneity in soil properties. The regional variability of land use effects on soil properties reveals the regional variability of the effects of human activities on environmental changes, and could explain the complex relationship between humans and the natural environment in certain ways.

CLONING AND SEQUENCING OF IMMUNOGLOBULINVARIABLE－REGION GENE OF A MONOCLONALANTIBODY SPECIFIC FOR HUMANHEPATOCARCINOMA

A murine monoclonal antibody HAb27 specific for human hepatocarcinoma has been developed for radioimmunolocalization in animal models. The isotype of this antibody was IgGl, k. In the present study, we used a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable region genes by the polymerase chain reaction. Sequence analysis of the heavy variable region indicated that the VH region was highly homologous to the plasmacytoma cell line MOPC21 gene, and closely related to germline genes of the VHⅢ family. The JH region was encoded by the JH3 gene. For the light chain, the VK segment of the antibody showed the highest homology to the germline VKOXl gene,and the JK region was JK5.

Study of Variable Step Size LMS Adaptive Algorithm Based on Variable Region

This paper puts forward a new variable step size LMS adaptive algorithm based on variable region. The step size p（k） in the algorithm varies with the variation of the region of deviation e （k） to ensure the optimization of the three performance objectives including initial convergent speed, trace ability of the time-varying system and steady disregulation. The paper demonstrates the convergence of the algorithm accompanied by random noise,

Genotyping of White Spot Syndrome Virus in Chinese Cultured Shrimp during 1998-1999

Recent studies showed that white spot syndrome virus(WSSV) isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15,and variable number of tandem repeats(VNTR) within ORF94.In this study,genotyping was performed according to these three variable regions among WSSV isolates collected during 1998/1999 from Southern China.These WSSV isolates contain a deletion of 1168,5657,5898,9316 and 11093 bp,respectively in the variable region ORF23/24 compared with WSSV-TW,and a deletion of 4749 or 5622 bp in the variable region ORF14/15 relative to TH-96-II.Four types of repeat units(RUs)(6,8,9 and 13 RUs) in ORF94 were detected in these isolates,with the shortest 6 RUs as the most prevalent type.Our results provide important information for a better understanding of the spatio-temporal transmission mode and the WSSV genetic evolution lineage.

A Study of Spatial Variability on Aquifer Hydraulic Conductivity K

A structural analysis of K of an aquifer system in the study area is presented, and the main direction and degree of the variability of K are found by using the unstationary regionalized variable theory of geostatistics. Optimal estimation of K has been made by universal kriging method (U K M ). Both spatial variability distribution map and division map of K are given.

The Geostatistical Framework for Spatial Prediction

Geostatistics provides a coherent framework for spatial prediction and uncertainty assessment, whereby spatial dependence, as quantified by variograms, is utilized for best linear unbiased estimation of a regionalized variable at unsampied locations. Geostatistics for prediction of continuous regionalized variables is reviewed, with key methods underlying the derivation of major variants of uni-vafiate Kriging described in an easy-to-follow manner. This paper will contribute to demysti- fication and, hence, popularization of geostatistics in geoinformatics communities.

The Geostatistical Information Method of Exploratory Engineering

The geostatistical information method consists in the use of the principle of quantitative evaluation in exploratory engineering research. The principle of uantitative evaluation is based on the mathematical model of the ore body. The selection of optimal exploratory scheme and optimal engineering positions has been studied in this paper.

Effect of treatment failure on the Cag A EPIYA motif in Helicobacter pylori strains from Colombian subjects

AIM To evaluate effect of treatment failure on cag A and vac A genotypes in Helicobacter pylori(H. pylori) isolates from Colombia.METHODS One hundred and seventy-six participants infected with H. pylori from Colombia were treated during 14 d with the triple-standard therapy. Six weeks later, eradication was evaluated by 13C-Urea breath test. Patients with treatment failure were subjected to endoscopy control; biopsies obtained were used for histopathology and culture. DNA from H. pylori isolates was amplified using primers specific for cag A and vac A genes. The phylogenetic relationships among isolates obtained before and after treatment were established by conglomerate analysis based on random amplified polymorphic DNA(RAPD) fingerprinting.RESULTS Treatment effectiveness was at 74.6%. Of the par-ticipants with treatment failure, 25 accepted subjected to a second endoscopy. Prevalence of posttreatment infection was 64%(16/25) and 40%(10/25) by histology and culture, respectively. Upon comparing the cag A and vac A genotypes found before and after therapy, multiple cag A genotypes(cag A-positive and cag A-negative) were found before treatment; in contrast, cag A-negative genotypes decreased after treatment. vac A s1m1 genotype was highly prevalent in patients before and after therapy. The 3'cag A region was successfully amplified in 95.5%(21/22) of the isolates obtained before and in 81.8%(18/22) of the isolates obtained after treatment. In the isolates obtained from patients with treatment failure, it was found that 72.7%(16/22) presented alterations in the number of EPIYA motifs, compared to isolates found before treatment.CONCLUSION Unsuccessful treatment limits colonization by lowvirulence strains resulting in partial and selective eradication in mixed infections, and acts on the cag A-positive strains inducing genetic rearrangements in cag A variable region that produces a loss or gain of EPIYA repetitions.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

Mutation Analysis of IgVH Gene in B-Cell Chronic Lymphocytic Leukemia

Summary: The variable heavy chain region (VH) genes of 3 untreated patients with B cell chronic lymphocytic leukemia (B CLL) were cloned and analyzed. The VH family used was VH3 11, VH3 72 and VH3 33. More than 2 % difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post GC cells.

Gene Cloning,Expression and Immunoreactivity of a Single Chain-Fv-Fragment Antibody PS-9

The gene encoding the heavy- and light-chain Fv regions of monoclonal antibody PS-9, which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma, was cloned by PCR techniques. The light and heavy chains were connected by a flexible linker to form a single chain variable fragment (ScFv) gene with 720 bp, which was in turn fused to pCANTAB 5 phage. The single chain Fv was expressed as fusion protein displayed on the phage surface. The phagemid is used to transform competent E. Coli TG1 cells, then infected with M13K07 helper phage to rescue the phagemid and antibody ScFv gene. All randomized 12 clones were shown reacting with colon cancer cell line Ls174t, which expresses S-Tn antigen. The recombinant phage has been infected E. Coli HB2151 cells to produce soluble antibody, which can be used for immunodetection and immunotherapy for cancer.

Minor fibrillar collagens,variable regions alternative splicing,intrinsic disorder,and tyrosine sulfation

Minor fibrillar collagen types V and XI,are those less abundant than the fibrillar collagen types I,II and III.The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region.Genomic variation and,in some cases,extensive alternative splicing contribute to the unique sequence characteristics of the variable region.While unique expression patterns in tissues exist,the functions and biological relevance of the variable regions have not been elucidated.In this review,we summarize the existing knowledge about expression patterns and biological functions of the collagen types V and XI alpha chains.Analysis of biochemical similarities among the peptides encoded by each exon of the variable region suggests the potential for a shared function.The alternative splicing,conservation of biochemical characteristics in light of low sequence conservation,and evidence for intrinsic disorder,suggest modulation of binding events between the surface of collagen fibrils and surrounding extracellular molecules as a shared function.

Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro

OBJECTIVE: To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. METHODS: Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation. RESULTS: Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC. CONCLUSIONS: Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

A study of Hodgkin/Reed-Sternberg cells using single cell polymerase chain reaction

OBJECTIVE: To investigate the characteristics of Hodgkin/Reed-Stemberg (H/R-S) cells found in patients with various types of Hodgkin's disease (HD). METHODS: H/R-S cells were micropicked from frozen sections of tissues affected by HD. The DNA from these cells was amplified by polymerase chain reaction (PCR) using immunoglobulin heavy chain gene FR III a/JH primers and light chain gene family-specific primers. RESULTS: A total of 52/135 (35.8%) isolated cells showed the specific products in the reactions. IgH and V kappa 4 rearrangements were repeatedly found in many cells from a lymphocyte predominance type sample; repeated V kappa 4 and individual IgH/V kappa 2,4 rearrangements and individual IgH, V lambda 3/ V kappa 4 rearrangements were found in two different cases of the nodular sclerosis type; repeated IgH/ V lambda 3 and individual V lambda 2,4 rearrangements, repeated V kappa 2,4 rearrangements, repeated V kappa 4 and individual IgH/ V kappa 3 rearrangements, repeated IgH and individual V kappa 3/ V lambda 4 rearrangements were detected in 3 cases of the mixed cellularity type. Repeated and individual IgH rearrangements were found in other 2 cases. CONCLUSION: The H/R-S cells isolated from the lymphocyte predominance subtypes of HD have IgH and V lambda 4 gene rearrangements. This suggests that the lymphocyte predominance type is a proliferation of neoplastic B cells. The cells isolated from the mixed cellularity and nodular sclerosis types derive from B lineage cells at various stages of differentiation because of the presence of their IgH, kappa and/or lambda gene rearrangements. To our knowledge, this is the first time that the lambda gene rearrangement was detected in H/R-S cells.

Development of the expressed immunoglobulinμchain repertoire during maturation of mice B cells

In the bone marrow and spleen,the developing B cell populations undergo both negative and positive selections to shape their B cell receptor repertoire.To gain insight into the shift of the immunoglobulin heavy(IgH)chain repertoire during B cell development,we undertook large scale Igμchain repertoire analysis of pre-B,immature B and spleen B cell populations.We found that the majority of VH gene segments,VH families,JH and D gene segments,were observed to have significantly different usage frequencies when three B cell populations were compared,but the usage profile of the VH,D,and JH genes between different B cell populations showed high correlations.In both productive and nonproductive rearrangements,the length of CDRH3 shortened significantly on average when B cells entered the periphery.However,the CDRH3 length distribution of nonproductive rearrangements did not follow a Gaussian distribution,but decreased successively in the order 3n–2,3n–1 and 3n,suggesting a direct correlation between mRNA stability and CDRH3 length patterns of nonproductive rearrangements.Further analysis of the individual components comprising CDRH3 of productive rearrangements indicated that the decrease in CDRH3 length was largely due to the reduction of N addition at the 5′and 3′junctions.Moreover,with development,the amino acid content of CDRH3 progressed toward fewer positively charged and nonpolar residues but more polar residues.All these data indicated that the expressed Igμchain repertoire,especially the repertoire of CDRH3,was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.