Proteomics data analysis using multiple statistical approaches identified proteins and metabolic networks associated with sucrose accumulation in sugarcane.

BACKGROUND: Sugarcane is the most important sugar crop, contributing > 80% of global sugar production. High sucrose content is a key target of sugarcane breeding, yet sucrose improvement in sugarcane remains extremely slow for decades. Molecular breeding has the potential to break through the genetic bottleneck of sucrose improvement. Dissecting the molecular mechanism(s) and identifying the key genetic elements controlling sucrose accumulation will accelerate sucrose improvement by molecular breeding. In our previous work, a proteomics dataset based on 12 independent samples from high- and low-sugar genotypes treated with ethephon or water was established. However, in that study, employing conventional analysis, only 25 proteins involved in sugar metabolism were identified . RESULTS: In this work, the proteomics dataset used in our previous study was reanalyzed by three different statistical approaches, which include a logistic marginal regression, a penalized multiple logistic regression named Elastic net, as well as a Bayesian multiple logistic regression method named Stochastic search variable selection (SSVS) to identify more sugar metabolism-associated proteins. A total of 507 differentially abundant proteins (DAPs) were identified from this dataset, with 5 of them were validated by western blot. Among the DAPs, 49 proteins were found to participate in sugar metabolism-related processes including photosynthesis, carbon fixation as well as carbon, amino sugar, nucleotide sugar, starch and sucrose metabolism. Based on our studies, a putative network of key proteins regulating sucrose accumulation in sugarcane is proposed, with glucose-6-phosphate isomerase, 2-phospho-D-glycerate hydrolyase, malate dehydrogenase and phospho-glycerate kinase, as hub proteins. CONCLUSIONS: The sugar metabolism-related proteins identified in this work are potential candidates for sucrose improvement by molecular breeding. Further, this work provides an alternative solution for omics data processing.

[Preparation and identification of a single-chain antibody fragment against high pathogenic H5N1 avian influenza virus].

Previously, an mAb 10F7 was developed against H5N1 hemagglutinin, which was highly specific to 34 different H5N1 strains and showed good neutralizing activity. In the present study, the single-chain fragment of the antibody was cloned into a prokaryotic vector and then expressed in E. coli. The activity of the scFv was tested in hemagglution inhibition and neutralization experiment. Two H5N1 virus strains were inhibited to bind erythrocyte cells by the scFv while the H9 virus was not. Also, five H5N1 virus strains were neutralized during infecting MDCK cells. These results showed an approachable method for developing therapeutic antibody to H5N1 virus.

Isolation of human antibodies against hepatitis E virus from phage display library by immobilized metal affinity chromatography.

OBJECTIVE: To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC). METHODS: Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. RESULTS: His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. CONCLUSION: Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

[Construction and preliminary application of a human natural phage antibody library with diversity].

AIM: To construct a human natural phage single-chain antibody (scFv) library with diversity. METHODS: V(H) and V(L) genes were amplified by RT-PCR and hemi-PCR from peripheral blood lymphocytes of healthy persons. The V genes were assembled to form scFv by overlap PCR and cloned into phagemid pCANTAB-5E, and then transformed into E. coli TG1 by electroporation to construct a human natural phage scFv library. The diversity and gene family of antibody gene were analysed by sequencing and the specific antibodies against various antigens were screened through bio-panning. RESULTS: A human natural phage scFv library with diversity and 2x10(8) sink size was constructed successfully. The specific human scFvs against 5 antigens were obtained by bio-panning. CONCLUSION: A human natural phage scFv library with diversity is constructed successfully and can be applied to human antibody preparation.

[Preliminary study on the conditions of solid-phase screening phage antibody library].

AIM: To explore the conditions of solid-phase screening phage antibody library and to provide the experimental basis for the design of screening project. METHODS: Diverse antibodies including HEV NE2-specific and non-specific humanized phage antibodies were used to study the screening conditions, such as the binding time of phage antibodies to antigen, the concentration of coating antigen, the washing times and elution method. RESULTS: The best binding time of positive phage antibody to antigen was 1 min. The highest positive rate of screening was obtained under the conditions of washing for 20 to 30 times and pH value of the washing solution being 5. The concentration of the coating antigen had no obvious influence on the positive rate of screening. Higher positive rate was obtained by using 10 mg/L antigen to competitively elute for 1 h. CONCLUSION: Solid-phase screening of the phage antibody library is a very complex process, in which there are close relationship between the conditions, therefore, appropriate readjustment should be made for screening conditions according to concrete conditions.