A novel method for quickly cloning genes with multiple DNA fragments--one step cloning technique using isoschizomer-heterotail restriction endonuclease (IHRE) is described. Up to six DNA segments are ligated by using only one restriction endonuclease in this method. Comparing with routine method,it is simple, fast, economical and generates products with higher purity and achievement. Light chain of human enterokinase, DNA multi-epitope vaccine to HSV2 have been designed and successfully constructed via this method.

Objective To observe the effect of AS1411-mediated signal transduction and activator of transcription 3 (STAT3) antisense oligonucleotide (ASO) targeting tumor cells.Methods RNA was used as coupling molecules to link the targeting molecules AS1411 and effector molecules ASO.Prediction and analysis of the secondary structure of the pre-synthesis of chimeric molecules by RNA Structure software.Agarose gel electrophoresis was used to test the stability of chimeric molecules in serum and cell lysis solution.Using flow cytometry and confocal fluorescence microscope were used to estimate the internalization of AS1411-mediated STAT3 ASO.Inhibitory effect of ASO on the growth of tumor cells was detected by CCK-8 kit.RT-PCR and Western blot was used to measure the expression levels of tumor related genes.Results STAT3 ASOs mediated by AS1411 can enter tumor cells efficiently to inhibit the transcription and translation of C-myc,Cyclin D1,Bcl-xl and PD-L1 gene,and also can inhibit the growth of Du145 cells.Conclusions AS1411-mediated STAT3 ASO can enter tumor cells and act as anti-tumor medicine.

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo-and endocytic pathways. Though there are many reports on Rab proteins, the function of these small proteins still remain in speculation. And no report has ever clarified the character of human Rab26. Here it was reported that a novel Rab protein Rab26 is membranous organelle related and in volved inendocytosis of HeLa cells. By using RT-PCR method a novel Rab26 cDNA full-length cDNA of Rab26 that is 1656 bp was identified.The cDNA sequence that at 1197 is 'A' other than 'G', while 'C' at 956 substitutes for 'T', and has 'GCC' insertion at 48 to 50 compared with published sequences. The complete open reading frame (ORF) is 771 bp in length encoding 256-residue protein with a calculated molecular mass of 27.9 ku (GenBank accession No.AY646153), rather than a shorter one with 190-amino acid residue as reported previously. GFP labeled full-length Rab26 expression showed that Rab26 was mainly sublocated in membranous organelles and could enhance endocytosis which means could took PE labeled protein as an endocytic tracer. RT-PCR analysis showed Rab26 was detected to express in several kinds of adenocarcinoma cell lines such as Acc2, AccM, SPC-A1 and HeLa cell lines, which indicated that Rab26 expression might be associated with some carcinomas.

We searched for metastasis-related genes in adenoid cystic carcinoma by suppression subtractive hybridization analysis of high and low metastasis cell lines. Twelve genes (ten previously identified and two novel sequences) were identified as being expressed at lower levels in high metastasis cell line Acc-M when compared to low metastasis cell line Acc-2. The known sequences corresponded to the genes for cysteine-rich angiogenesis induction factor (cyr61), chromosome 7 RP11-52501 clone, G-protein, WAS familial ferritin I heavy chain, jumping translocation breakpoint, eukaryotic translation elongation, folate receptor and three ribosomal proteins. Among them, the G protein and ferritin I heavy chain genes contained mutations in the high metastasis cell line. The two novel gene sequences have been named ACC metastasis-associated RNH and ACC metastasis-associated suspected protein (GenBank # AF522024 and AF522025, respectively). Taken together, these results suggest that reduced expression and/or mutation of several genes in the tumor cell line Acc-M are associated with high tumor metastasis, providing important molecular biological materials for further study of metastasis control and possible targets for cancer gene therapy.
Blotting, Northern ; Carcinoma, Adenoid Cystic/*genetics/secondary ; *Gene Expression ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Human ; In Vitro ; Molecular Sequence Data ; Neoplasm Metastasis/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

Human hepatitis B virus surface antigen (HBsAg) binding protein(SBP) shows a specific binding ability to HBV surface antigen HBsAg. Previous work proved an ability of SBP to enhance the immune response of HBsAg vaccine. To investigate the function and mechanism of this protein, we constructed SBP-expression strains with Pichia pastoris expression system. We screened these strains and have got an expression strain with high protein expression quantity. Fermentation product was collected and purified to gain a large amount of purified protein. Identification of purified SBP with SDS-PAGE, High performance liquid chromatography, Western blotting and mass spectrometry suggested that the protein was highly purified and with a good integrity. ELISA test of purified SBP showed a significant binding ability to HBsAg, suggesting a good protein activity. This work offers a solid foundation to the research of SBP function and mechanism of immune enhancement.
Binding Sites ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Pichia ; genetics ; metabolism ; Receptors, Virus ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification

To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED₅₀ of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.