Liposome and QS-21 Combined Adjuvant Induces theHumoral and Cellular Responses of Acellular Pertussis Vaccine in a Mice Model.

The resurgence of pertussis in vaccinated communities may be related to the reduced long-term immunity induced by acellular pertussis vaccines. Therefore, developing improved pertussis vaccine candidates that could induce strong Th1 or Th17 cellular immunity is an urgent need. The use of new adjuvants may well meet this requirement. In this research, we developed a novel adjuvant candidate by combining liposome and QS-21 adjuvant. Adjuvant activity, protective efficacy, the level of neutralizing antibody against PT, and the resident memory T (TRM) cells in lung tissue after vaccination were studied. We then performed B. pertussis respiratory challenge in mice after they received vaccination with traditional aluminum hydroxide and the novel adjuvant combination. Results showed that the liposome + QS-21 adjuvant group had a rapid antibody and higher antibody (PT, FHA, Fim) level, induced anti-PT neutralizing antibody and recruited more IL-17A-secreting CD4+ TRM cells along with IL-17A-secreting CD8+ TRM cells in mice, which provided robust protection against B. pertussis infection. These results provide a key basis for liposome + QS-21 adjuvant as a promising adjuvant candidate for developing an acellular pertussis vaccine that elicits protective immunity against pertussis.

Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli.

Protein secretion in Escherichia coli is usually led by a signal peptide that targets the protein to specific secretory pathways. In this study, we demonstrated that the superfolder green fluorescent protein (sfGFP) could be served as a non-signal peptide to guide protein auto-secretion in E. coli. This auto-secretion was characterized as a three-step process through the sub-cellular localization analysis: inner membrane trans-location followed by anchoring at outer membrane, and then being released into culture media. We further determined that the beta-barrel structure and net negative charges of sfGFP played important roles in its auto-extracellular secretion property. Using sfGFP as a carrier, heterologous proteins ranging from peptide to complex protein, including antibacterial peptide PG4, endo-beta-N-acethylglucosamindase H (Endo H), human arginase-1 (ARG1), and glutamate decarboxylase (GAD) were all successfully expressed and secreted extracellularly when fused to the carboxyl end of sfGFP. Besides facilitating the extracellular secretion, sfGFP fusion proteins can also be correctly folded and formed the active complex protein structure, including the trimetric human ARG1 and homo-hexametric GAD. This is the first report that sfGFP can guide the secretion of recombinant proteins out of the cells from cytoplasm in E. coli without affecting their conformation and function.

Toward an understanding of the protein interaction network of the human liver.

Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.

An advanced blue-white screening method for construction of shRNA expression vectors.

Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue-white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue-white screening system is a powerful tool for the high-throughput assay of RNAi libraries.