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PubMed2003

The Cochrane Library can answer your nursing care effectiveness questions.

Systematic reviews help nurses to manage the overwhelming volume of available information by synthesizing valid data from primary studies and summarizing the results of interventions. One reliable source of systematic reviews of healthcare interventions is the Cochrane Library. This paper briefly describes the Cochrane Collaboration and the Cochrane Library. It also encourages nurse clinicians and nurse administrators to use the Cochrane Library and encourages nurse researchers to contribute to the Cochrane Library by conducting systematic reviews in the field of nursing care effectiveness.

Humans↗
PubMed2004

Generation of high affinity human single-chain antibody against PreS1 of hepatitis B virus from immune phage-display antibody library.

BACKGROUND: A single-chain antibody (ScFv) phage display library was created by cloning antigen-binding regions of V(H) (variable domain) and V(L) gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display human ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 x 10(8) clones. RESULTS: After 3 rounds panning, a high affinity (K=10(-7)-10(-8)mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the V(H) belonged to the V(H)4 family and V(lambda) to V(lambda)4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Antibodies, Monoclonal↗
PubMed2004

[Construction of a reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

OBJECTIVE: To inquire into the mechanism of prothrombotic state (PTS) and the roles of liver therein by constructing a reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS. METHODS: The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization. The rat model of PTS was induced by a high-carbohydrate diet. Poly A+ mRNAs were isolated from PTS and control rats, and cDNAs were synthesized from the mRNAs. After digestion by means of Ras I, cDNAs 400-600 bp in size were obtained. For suppression subtractive hybridization, cDNAs from PTS rat were used as Driver and the cDNAs from control rat as Tester. The Tester was divided into two parts and ligated to adaptor 1 and adaptor 2R respectively. After two times of subtractive hybridization and two times of nested PCR, the products of the last PCR amplification were inserted into T/A plasmid vectors to transform the Escherichia coli JM109 cells. The transformed cells were incubated at 37 degrees C overnight on a LB agar plate containing ampicillin (50 micrograms/ml), IPTG and X-gal. The colonies were counted. RESULTS: 78% of the colonies were white and the reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state was successfully constructed. CONCLUSION: Prothrombotic state caused by malfunction of homeostasis and fibrinolysis is an important risk factor of cardiovascular disease. Liver plays important roles in the development of PTS, for the majority of the factors in the coagulation as well as fibrinolytic cascades are generated by the liver and secreted into the bloodstream. The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS, successfully constructed in the present study, provides an efficient way to further investigate the mechanism of PTS and the relevant liver functions.

Animals↗
PubMed2003

[Construction of phage anti-idiotypic antibody library using sensitized in-vitro B-lymphocytes of nasopharyngeal cancer patients].

AIM: To construct phage human anti-idiotypic antibody library. METHODS: Peripheral blood mononuclear cells(PBMCs) of patients with nasopharyngeal cancer(NPC) were sensitized in-vitro and transformed by Epstein-Barr virus(EBV). V(H) and V(L) genes were re-amplified by PCR and combined to single-chain fragment of variable region(ScFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into MC1061 by electroporation to construct the ScFv-displaying phage library. RESULTS: Detection of Sandwich ELISA showed that of 10 NPC patients,8 patients' B cells transformed by EBV could produce anti-idiotypic antibodies to NPC. 5 types of V(H) genes and 7 types of V(L) genes were obtained by PCR re-amplification and then connected with (Gly(4)Ser)(3) linker to form 14 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into MC1061 via electroporation. Phage anti-idiotypic antibody library with sink size being 1.1x10(7) was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 70%. CONCLUSION: A strategy for preparing human single chain anti-idiotypic antibody by means of phage antibody library technique in combination with EBV transformation technique is feasible.

B-Lymphocytes↗
PubMed2003

[Construction and screening of hepatitis E virus-specific phage antibody combinatorial library].

AIM: To construct HEV-specific phage combinatorial anti-body library and screen anti-HEV antibodies with neutralizing activity from the library. METHODS: The total RNA was extracted from B-lymphocytes of 6 HE patients. Kappa chain and Fd segment of IgG gene were amplified respectively by RT-PCR using a set of Fab-specific primers. The amplified gene were inserted successively into vector pComb3 and electrotransformed E. coli XLI-Blue cells. Furthermore, the recombinant phage was rescued by being concultured with helper phage VCSM13 to construct HEV-specific phage anti-body library. RESULTS: Fab displayed on the surface a as fusion protein with the N terminal of coat protein III, and 1. 8 x 10(7) clone library was established. Specific antibodies to HEV ORF2 recombinant antigen were acquired after five rounds of panning with HEV ORF2 recombinant antigen including neutralizing epitope. CONCLUSION: Four clones exhibited specific binding to HEV ORF2 recombinant antigen including neutralizing epitope is identified by ELISA. The results show that we have got the recombinant phage antibodies.

Hepatitis Antibodies↗
PubMed2003

[Construction and characterization of osteosarcoma 9901 cell cDNA library].

AIM: To construct human osteosarcoma 9901 cell cDNA expression library for screening osteosarcoma-specific antigens. METHODS: Total RNA was extracted from human osteosarcoma cell line 9901 and mRNA was purified. cDNA was synthesized by reverse transcription, and cDNA fragments larger than 400 bp were ligated with dephosphorylated arms of lambdagt11. The recombinants were packaged in-vitro, and a small portion of packaged phage was used to infect E.coli Y1090 for titration. The size of cDNA inserts and the diversity of library were detected by PCR. RESULTS: The osteosarcoma 9901 cell line cDNA library consisting of 1.5x10(6) recombinant bacteriophages was constructed. The average length of exogenous inserts in the recombinants was about 1.4 kb. CONCLUSION: The cDNA library reported herein is suitable for screening osteosarcoma-specific antigens.

Bone Neoplasms↗
PubMed2004

[Construction and characterization of cDNA library for lung cancer cell line YTMLC-90 isolated from Gejiu, Yunnan province].

AIM: To construct the cDNA library for Yunnan Gejiu human lung cancer cell line YTMLC-90. METHODS: The total RNA was extracted from YTMLC-90 cells and the first-strand cDNA was synthesized by reverse transcription with a modified oligo(dT) primer (containing Sfi I B digestion site). Simultaneously, the SMART oligonucleotide (contained Sfi I A digestion site) was utilized as a template that the first-strand of cDNA could be extended out the 5' terminal of mRNA. The ds cDNA was amplified by LD-PCR (long-distance PCR) and then digested with Sfi I(IA & IB). After fractionation of cDNA through CHROMA SPIN column, the ds cDNA was cloned into lambdaTripIEx2 vector which was then packaged. RESULTS: The unamplified cDNA library for human lung cancer cells consisted of 1.01 x 10(9) pfu/L independent clones in which the recombinant rate was about 93.2%. The clone number in the amplified cDNA library reached 5.24 x 10(12) pfu/L and the length of inserted exogenous cDNA sequence was 750-3 000 bp. CONCLUSION: The constructed cDNA library for YTMLC-90 cells has an excellent quality, which lays a solid foundation for further screening and cloning novel tissue-specific genes of human lung cancer.

Carcinoma, Squamous Cell↗
PubMed1992

The CEPH YAC library.

Because of their large size, YACs provide is a powerful tool for physical mapping studies of complex genomes. As it will be advantageous to have genomic libraries of clones with large inserts for analyzing megabase sized regions of the human genome, we have investigated a number of parameters in order to increase the insert size of the YACs. We constructed a genomic library currently containing more than 85,000 YAC clones. Mean sizes of YACs produced at several stages of construction of the library range from 430 kb to 1,200 kb, representing 13 haploid equivalents of the human genome. This library was organized in order to allow rapid screen of YACs for large scale physical mapping of the human genome.

Chromosome Walking↗
PubMed2004

Protective effect of vaccination with DNA of the H. Pylori genomic library in experimentally infected mice.

Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.

Animals↗
PubMed2004

[Construction and screening of the human phage antibody library from a patient with melanoma].

AIM: To construct and screen the human phage antibody library from a patient with melanoma. METHODS: Total RNA was extracted from the peripheral blood mononuclear cells (PBMCs) of a patient with melanoma who has survived for twenty years. The heavy chain Fd fragments and kappa light chains were amplified by RT-PCR. The amplified products were cloned into phagemid vector to construct Fab antibody library. The library was panned with two kinds of melanoma cells for 4 rounds. RESULTS: Antibody library contained 1.2 x 10(8) different clones. Specific phage antibodies against one of the two melanoma cells were obtained after 4 rounds of panning. CONCLUSION: Specific human phage antibodies against melanoma cells were obtained.

Antibodies, Neoplasm↗
PubMed2004

Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection.

BACKGROUND: A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj). METHODS: A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients. RESULTS: Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%. CONCLUSIONS: The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

Animals↗
PubMed2004

[The construction and screening of genomic library based on transformable artificial chromosome (TAC) vector in Lotus japonicus].

Many vectors, especially artificial chromosome vectors, have been developed for genome-scale mapping and sequencing. As the first artificial chromosome, YAC has been extensively used in the genomic library construction and mapping. Shizuya et al. (1992) devised the bacterial artificial chromosome (BAC) originated from F factor of E.coli, which is much easy to handle and isolated with large harboring capacity. Liu et al. (1999) designed the transformation-competent artificial chromosome (TAC) vector, derived from PAC vector. TAC could shuttle a large-scale DNA fragment between bacteria and Agrobaceria. Further, it could integrate a large targetted DNA fragment into plant genome, as being documented in Arabidopsis and rice genome research (Liu et al. 2000, 2002). The time-saving virtue of TAC should be significant in genomics research in Lotus japonicus, a model plant of legume. Using a nuclei-based method of Liu and Whittier, high molecular weight DNA was isolated from Lotus japonicus (Gifu ecotype). The DNA was digested partially with Hind III and size-fractionated in the 10- to 20-kb size range as described (Liu and Whittier 1994). The partially digested and size selected DNA fragments were ligated with Hind III-digested pYLTAC7 and then used for transformation of E. coli DH10B by electroporation. Transformants carrying inserts were selected on LB agar plates containing 25 mg/L kanamycin and 5% sucrose. The library, 6 haploid genome equivalents, was pooled in 12 96-well microtiter plates at about 150 transformants per well. The TAC library was then arrayed in nylon membranes and subjected to screening. The probe, a homolog fragment of CEN gene controlling the structure of inflorescence Antirrhinum, was used for screening. 0.5 muL solution from the positive pool was titered and the secondary screening was conducted in the same way. Finally, these positive clonies were confirmed by Southern blot. These data showed that this genomic library was reliable for further molecular research in Lotus japonicus.

Chromosomes, Artificial, Bacterial↗
PubMed2005

[Construction and screening of human phage antibody library against SARS virus].

AIM: To construct human phage antibody library against SARS virus. METHODS: Human Fab genes were amplified by RT-PCR from lymphocytes of a convalescent SARS patient and cloned into vector pComb3 to construct phage antibody library. Antibodies against SARS virus were screened by biopanning with immobilized virus antigen. The binding specificity of phage antibody to SARS virus was detected by ELISA. RESULTS: 13 Ig genes were obtained by PCR. A human phage antibody library consisting of 1.3 x 10(6) clones was constructed by electrotransformation and 60 percent of the clones contained Fab genes. After three rounds of panning with SARS virus antigen, phage antibodies against SARS virus were specifically enriched. ELISA detection verified 10 positive phage antibodies that were highly specific for SARS virus. CONCLUSION: Human phage antibody library against SARS virus has been constructed successfully, from which 10 anti-SARS virus antibodies were obtained.

Animals↗
PubMedDate not supplied

[Immunization with Leishmania amazonensis subgenomic libraries protects BALB/c mice against the challenge].

A genomic library of Leishmania amazonensis in expression vector of eukaryote cells (pEF1HisA, pEF1HisB, pEF1HisC) was prepared. Also two subgenomic libraries having each 500 clones approximately were created and BALB/c mice were immunized with 50 mg/0,1 mL of DNA from each. Two immunizations were administered intramuscularly at 15-day interval. Groups of control mice were immunized with DNA from empty plasmid pEF1His, with soluble parasite antigen (100 mg/0,1 mL) and saline solution. The size of lesions was measured for 12 weeks and at the end of the experiment, the parasite load at lesion sites was determined by plaque microtitration method. In mice immunized with subgenomic library DNAI and with soluble antigens,the size of lesions was controlled, which reached an statistical difference (p< 0,05) in relation to the rest of groups whose lesions increased. The parasite load found in lesion sites confirmed the previous results; the number of promastigots was significantly lower in those mice already protected. It was concluded that in subgenomic library DNA1 there should be genes or gene fragments whose in vivo expression induces protective immune response against the challenge in the murine model used.

Animals↗
PubMedDate not supplied

[Evaluation of the humoral immune response by Western Blot test in mice immunized with expression genomic library of Trypanosoma cruzi].

An expression genomic library of Trypanosoma cruzi was built by using plasmid pc DNA3 as a vector. This library served to immunize by intramuscular administration BALB/c inbred mice. A positive control group to which T. cruzi soluble antigens were administered and other group which was given the same plasmid used for the building of the genomic library were included in this study; another group was not immunized. Blood was extracted from the retrorbital plexus of all the mice two weeks after the third vaccination so as to study the specific antibody response to soluble parasite anigens through the Western Blot technique. The antibody response was shown in animals immunized with the expression genomic library and with soluble parasite antigens.

Animals↗
PubMed2005

Current directions in the evolution of compound libraries.

Synthetic compound libraries have been available for over a century, but in the last 15 years they have become increasing important in early-stage drug discovery projects. The qualities that these libraries possess impart a dramatic effect on hit discovery, on the subsequent steps of hit-to-lead progression, and on lead optimization by medicinal chemists. More recently, synthetic libraries have found an important application in chemical genetics. Concurrently, ideas have evolved regarding the application of the different types of synthetic approaches that are variously described as combinatorial chemistry, high-throughput synthesis, or diversity-oriented synthesis. This review will discuss current directions of synthetic chemistry, natural product chemistry and chemoinformatics, which relate to the various types of compound libraries that are in use in drug discovery and chemical genetics.

Animals↗
PubMed2004

[Construction of phage display antibody library to MCF-7 cells and screening of single-chain antibodies against breast cancer cells].

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.

Animals↗
PubMed2005

[Construction and primary analysis of subtractive library induced by soybean mosaic virus (SMV)].

SMV is one of main diseases of soybean, which could affect yields and quality of soybean seriously. It was effective to soybean breeding by studying the expression of resistant gene to SMV with molecular technology. In this study, a soybean resistance line, DongNong 8143, was used to construct a subtractive cDNA library by SSH from soybean leaves inoculated by SMV No.1 at primary stage. cDNA dominantly or specifically expressed in infected leaves was purified using PCR Purification Kit and cloned into pGEM-T easy vector. Colonies were grown on LB-agar plates containing ampicillin, X-gal and IPTG. A subtractive plasmid library was constructed by SSH. Then the library was transformed to host bacteria E. coli DH5alpha, and the titer of the library was measured as 2 x 10(3) . 64 clones were picked up randomly and sequenced. Of them there is 50 clones which result of sequenced were good. The length of EST fragment varied from 136bp to 691bp, and the average length is 456bp. Among them, 41 sequences has poly(A). Through ESTs were compared with sequences in unigene database of GeneBank with BLASTn and BLASTx algorithm, 38 ESTs of them had comparatively clear results and the percent of them in acquired ESTs is 74%. The EST expression profile showed that the resistance-related genes include cell protection, signal transduction, restrict pathogen growth, system acquired resistance, and house-keeping gene. There are 12 ESTs, which have not comparatively clear results, that maybe new genes.

Expressed Sequence Tags↗