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Objective To investigate the influence of long sea voyage working environment on the symbiotic microorganisms and their relationship with their hosts .Methods The periumbilical microbial sam-ples from the operating workers of long sea voyage before and after operation were collected .Then 16S rRNA V4 section amplification ,sequencing and whole metagenome shotgun high-throughput sequencing were per-formed .Moreover the bacterial community structure ,kinds and microorganism metabolic function change were analyzed .The peripheral blood was collected from the workers of long sea voyage operation and shore-based operation for conducting the blood routine analysis .Results After 105 d ocean sailing ,the diversity of perium-bilical microbial community in the workers with long sea voyage operation decreased and the relative abun-dance of Firmicutes increased ,w hile w hich of Proteobacteria decreased ;w hich of Staphylococcus increased , while which of Corynebacteria decreased ,the differences were statistically significant (P<0 .05) ,the relative a-bundance of pathogenic bacteria or conditional pathogenic bacteria ,especially Staphylococcus epidermidis and Staphylococcusaureus aureus ,increased significantly .T he functional gene analysis indicated that the expres-sion of periumbilical microbial infection related genes increased after the long sea voyage operation .Compared with shore-based operation workers ,the proportion of workers with peripheral blood lymphocytes abnormal elevation in the long sea voyage group increased significantly ,the difference was statistically significant (P<0 .05) .Conclusion The periumbilical skin symbiotic microorganisms may reflect the health conditions in the workers with long sea voyage operation .
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Circulating endothelial cells(CEC)are mature endothelial cells,which have been shed from the vascular cell lining and enter into blood circulation.Rare in healthy individuals,increased CEC in peripheral blood reflects significant vascular damage and dysfunction.Increased CEC have been documented in many human diseases characterized as vascular damage,including different types of tumors.Clinical data suggest that CEC count is a promising tool for monitoring disease activity with potential to assess tumor prognosis and response to treatment.
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ObjectiveTo explore the relationship between circulating tumor cells(CTCs) in the peripheral blood of patients with esophageal squamous cell carcinoma(ESCC) and the physiopathological characteristics of esophageal neoplasms.MethodsUsing negative selection system,we depleted red blood cells(RBCs) in red blood cell lysis buffer,depleted white blood cells (WBCs) with Miltenyi magnetic beads and enriched the rare cells from ESCC patients'peripheral blood.Immunofluorence staining (IF) was adopted to identify CTCs.ResultsCirculating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma was closely related to cell differentiation grade,the invasion of primary cancer,lymph node status,P-TNM stages,and was rarely related to the sex,age or the location of tumor.ConclusionThe results suggest that circulating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma may express the development of esophageal cancer and may be served as a tumor marker to evaluate the biological behavior of esophageal cancer.
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Objective:To clone human anti-ricin antibodies from large phage antibody library.Methods:Panning for a large phage library against ricin toxin was conducted to select specific antibodies against ricin. The binding activities and specificities were tested by ELISA method. Soluble ScFvs were prepared through infecting E coli. HB2151 with the selected phage antibodies and induction with IPTG. Results:Forty positive clones were obtained after 5 rounds of panning, and 12 clones had specific binding ability to ricin toxin. DNA fingerprinting showed 7 different band patterns indicating 7 different positive clones. DNA sequencing showed that variable regions of these ScFvs belonged to different subgroups.Conclusion:Human anti-ricin antibodies were successfully obtained from large phage antibody library.
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Objective:To improve the affinity of an anti TNF? scFv.Methods:Starting from an anti TNF? scFv gene a mutant phage antibody library was generated by error prone PCR.Affinity improved clones were selected and subjected to staggered extension process to shuffle the mutated sites.Mutants with further improved affinity were selected by bio panning.Affinity was judged by dot blot ELISA and thiocyanate elusion ELISA.Results:Seven affinity improved mutants were obtained from library constructed by error prone PCR.By StEP mediated shuffling of these 7 clones and via bio panning,mutants with further improved affinity were obtained.Conclusion:Combination of error prone PCR and StEP could be used to improve the affinity of antibodies. [
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Objective:To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E.coli.Methods:Fd and ? genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results:Fd and ? genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells(NIH3T3/erbB-2 cells). Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of ? chain N terminal sequences was shown no expected result.Conclusion:Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.
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Objective:To construct bispecific minibody by using anti HBsAg and anti RBC single chain Fv(ScFv).Methods:A “knob” variant T366W was first obtained by replacement of a small amino acid with a larger one in the human IgG1 CH 3 domain.The knob was designed to insert into a “hole” in another CH 3 domain which was created by replacement of three large residues with three smaller residues:T366S:L368A:Y407V.The “knob into hole” mutation:S354C:T366W/Y349C:T366S:L368A:Y407V.Then a disulfide bond was engineered in combination with previously designed “knob” or “hole” CH 3 was connected to anti HBsAg or anti RBC ScFv genes respectively.Then the two genes were combined together to form a bispecific minibody expression vector.The bispecific minibodies were expressed in E.coli.Results:Three different form of anti HBsAg and anti RBC minibody expression vectors were constructed.They contained wild type CH 3,“knob into hole”CH 3 or “knob into hole” plus disulfide bond CH 3 respectively.The results indicated that these three different types of bacterially expressed minibodies had similar HBsAg and RBC binding activities.The second and third type of minibody could cause agglutination of human RBC when HBsAg was present,which demonstrated bispecific function.Conclusion:Engineered interface of CH 3 can promote formation of heterodimers of different antibodies and faciliates the formation of bispecific antibodies(bispecific minibody) in E.coli expression system.
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Objective:To clone soluble human anti-digoxin antibodies from large phage antibody library and construct a vector that expresses diabody.Methods:Soluble ScFvs were prepared through infecting E coli.HB2151 with the selected phage antibodies and induced with IPTG.The diabody vector was modified by enzyme digestion and checked by SDS-electrophoretogram.Results:It was showed by ELISA that soluble ScFv had specific binding ability to digoxin.The vector to express a soluble diabody was obtained by genetic modification,which was shown by Western blot.Conclusion:Soluble human anti-digoxin antibodies were successfully obtained from phage antibodies.The vector is efficient in creating diabody.
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Objective:To create a large human phage antibody library from which easy to get diabody and select human antibody clones.Methods:VL and VH genes were amplified by RT-PCR from RNA which came from normal adult peripheral blood and new-born cord blood lymphocytes. VL and VH genes were reamplified to add a region of overlap in the ScFv linker to form ScFv genes in which VH genes were flanked by two non-homologous loxp sites. The ScFv genes were cloned into PDF to obtain a primary library. This primary library was used to infect bacteria Bs1365(expressing cre recombinase) with high multiplicity of infection(MOI) 200∶1. This procedure resulted in a very large phage antibody library through VL and VH recombined by the cre recombinase. Following recombination, phagemide were derived from these bacteria and used to infect bacteria not expressing cre(XL1-Blue) at MOI