Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica最新文献

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.

Proteomics has its origins in two-dimensional gel electrophoresis (2-DE), a technique developed more than twenty years ago. 2-DE has a high-resolution capacity, and was initially used primarily for separating and characterizing proteins in complex mixtures. 2-DE remains an important tool for protein identification, but is now normally coupled with mass spectrometry (MS), a technique which has advanced considerably in recent years. The recent completion of human genome project has produced a large DNA database which can be utilized through bioinformatics, and the next challenge for scientists is to uncover the entire proteome of a particular organism. The integration of genomic and proteomic data will help to elucidate the functions of proteins in the pathogenesis of diseases and the ageing process, and could lead to the discovery of novel drug target proteins and biomarkers of diseases. This review describes recent advances in proteomic technology and discusses the potential applications of proteomics in biomedical research.

tyrR gene encodes a global regulatory protein (TyrR), which plays an important role in the transcriptional regulation of eight transcription units (including tyrR gene itself) whose protein products catalyze key steps in aromatic amino acid biosynthesis and/or transport. The aroP gene encodes an integral membrane protein (AroP) that transports aromatic amino acids through the cell membrane. The transcription of aroP was reported to be repressed by TyrR. In this work, aroP(p) (aroP gene carrying its own promoter), aroP (aroP gene without promoter) and tyrR genes were amplified by PCR from genomic DNA of E. coli K12 and introduced into E. coli WT5. The expression of aroP and tyrR were detected and the activities of AroP and TyrR were determined. The introduction of either aroP(p) or aroP elevated the strain's transport activity by 1.40 or 1.46-fold respectively. Transformant carrying tyrR gene showed an ATPase activity 1.69-fold compared with the control. When the genes were linked in tandem and co-expressed in a plasmid, the relative AroP transport activity of the strain harboring aroP(p) -tyrR (0.95) was significantly lower than that of aroP-tyrR (1.31). The results indicated that TyrR might be able to reduce the expression of aroP gene by binding with the aroP promoter region in E.coli.

To evaluate the type-specific prevalence of eight common types of human papillomavirus (HPV) in patients with cervical cancer living in Shanxi, China, with fluorescence polarization detection, crude DNA extracted from 137 samples of early-stage cervical cancer (within stage IIa) and chronic cervicitis was subjected to HPV L1 consensus GP5+/GP6+ system. Then, the HPV-positive products identified by GP5 + /GP6+ PCR were genotyped based on template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP): the PCR products were respectively hybridized with designed type-specific probes within the GP5+/GP6+ amplicons for eight common HPV types (HPV 6, 11, 18, 16, 31, 33, 35, and 58), and specific fluorescence-labeled ddNTPs (TAMRA-ddTTP or R110-ddGTP) were directly incorporated to the ends of the corresponding probes under directing of the corresponding template in PCR products, which was reflected and read by high FP values for TAMRA or R110. HPV DNA was detected in 38.89% (28/72) cases of chronic cervicitis, and 87.69% (57/65) cases of cervical cancer. There was a significant difference in HPV prevalence between these two groups. The four commonly identified types in patients with cervical cancer were HPV 16 (45.6%), HPV 18 (22.8%), HPV 58 (17.5%), and HPV 31 (7.02%), and in those with chronic cervicitis were HPV 16 (35.7%), HPV 11 (32.1%), HPV 6 (21.4%), and HPV 18 (10.7%). 57.14% of HPV types detected in patients with chronic cervicitis were high-risk types. HPV 16 was the most common viral type identified in both groups. Type-specific prevalence of HPV DNA has some characteristics in patients with chronic cervicitis and cervical cancer living in Shanxi, China and the worldwide uncommon type HPV 58 is relatively common in both kinds of cases. The high prevalence of HPV 58 in Chinese women should been considered in diagnosis and vaccine designs of HPV.

Myostatin is a recently discovered member of transforming growth factor beta (TGFbeta) superfamily and shares similar structure features with other members of TGFbeta superfamily. For a better understanding of molecular mechanism of myostatin function, the production of C-terminal truncated form of recombinant myostatin protein (rMSTN) in E. coli was previously reported. Herein, the functional role of the recombinant myostatin in regulating myogenesis in a chicken embryonic myoblasts (CEMs) system was determined. By using flow cytometric analysis, the myostatin was found to inhibit cell cycle transition from G1 to S phase and result in a cell cycle arrest at G1. In addition, myostatin blocked the multi-nucleus myotube formation and caused a decreased expression of the muscle cell differentiation markers (myogenin and MHC) in CEMs. In this study, a rabbit polyclonal antibody against myostatin was produced and high affinity and specificity of this anti-myostatin antibody to recombinant and endogenous myostatin were assayed by Western blot analysis. Further studies showed that the antibody could also recognize the tissue endogenous myostatin of human, mouse and rat. A specific 40 kD band was detected in chicken muscle, which suggested that chicken myostatin might have different splicing pattern. Immunofluorescence assay indicated that myostatin predominantly existed in the cytosol in C2C12 cells. Taken together, the results show that myostatin inhibits chicken muscle cells proliferation and differentiation and down-regulates expression of two differentiation marker gene in CEMs. Remarkably, production of functional recombinant myostatin protein and its specific antibody provides important reagents for unraveling molecular mechanisms underlying myostatin action during myogenesis.

To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.

In order to obtain new gene and to develop the new vaccine candidate of immunoprotection against Schistosoma japonicum (Sj), Sj adult worm cDNA library was screened with anti-sera to soluble male adult worm antigen and resulted in the discovery of a novel gene designated as Sj-MA. Sequence analysis showed that Sj-MA as a complete cDNA contains one open reading frame. It was deduced to contain 249 amino acid residues and encode a 28.8 kD soluble protein with plenty of phosphorylation sites, supposing its action in signal transduction. Furthermore, Sj-MA cDNA was cloned into a prokaryotic expression vector pGEX-5X to construct the recombinant plasmid which was transformed and highly expressed in E. coli as a 54.8 kD glutathione-S-transferase (GST) fusion protein. The fusion protein rSj-MA/GST could be recognized with both anti-male adult worm sera and anti-GST sera in Western blotting. Mice vaccinated with the fusion protein revealed significant worm reduction rate of 34.29% (P<0.001), compared with the control groups. Taken together, the novel gene Sj-MA can be expressed in E. coli as a fusion protein that can elicit immunity against Schistosoma japonicum, suggesting its potential as a new vaccine candidate.

To check the feasibility of expression of the immunogenic gene of avian coronavirus infectious bronchitis virus (IBV) in plants, the transformation of S1 gene of IBV into potato and the immunogenicity of its expression product was studied. The S1 gene of IBV-ZJ971 strain was inserted into plasmid pBI121 under the control of 35 S promoter. Agrobacterium fumefaciens EHA105 with the recombinant vector pBI121 was obtained by tri-parental mating method. So, an efficient potato transformation system mediated by Agrobacterium fumefaciens was established. The rates of calli and shoots differentiation were 100%, and more than 95% respectively, for transgenic potato with S1 gene of IBV. PCR and Southern blot analyses showed that IBV S1 gene was integrated into genomic DNA of the potato plant and most transgenic plants had two copies of S1 gene of IBV. In our experiments, 47 transgenic plantlets have been obtained. Northern blot and ELISA analyses indicated that most transgenic plants could normally transcribe and translate S1 gene of IBV, though the levels of transcription and translation were different in various transgenic plants. Immunity assay with BALB/C mice showed that expression products of transgenic potato with S1 gene of IBV were immunogenic, and ELISA antibody titer reached 1:20 to 1:40 and 1:80 to 1:160 with doses of 0.5 g and 1 g, respectively. Virus neutralization (VN) antibodies were detected by tracheal organ cultures, and the results showed that VN titers reached respectively 1:160 to 1:320 and 1:320 to 1:2048 with doses of 0.5 g and 1 g.

A novel neurotoxic peptide, named huwentoxin-III, and a natural mutant have been isolated from the venom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang). The average molecular weights of the two peptides were determined as 3853.35 and 3667.40 by mass spectrometry, respectively. Huwentoxin-III has 33 amino acid residues containing 6 cysteine residues. Its natural mutant is only truncated a tryptophan residue from C-terminals of huwentoxin-III. The sequences of the two peptides show 70.5% sequence similarity with that of lectin-like peptide SHL-I previously isolated from the venom of the same spider, while they cannot agglutinate human erythrocytes. Huwentoxin-III can reversibly paralyze cockroaches for several hours with an ED(50) of (192.95 +/- 120.84) microg/g (P=0.95) (mean +/- SD) and can enhance the muscular contractions elicited by stimulating the nerve of the isolated rat vas deferens, however, the mutant of huwentoxin-III has no such effect, which suggests that Trp33 was an important residue related to the biological function of huwentoxin-III.

The orphan nuclear receptor hB1F (also known as NR5A2, LRH-1, FTF or CPF) plays important roles in regulating the expression of several cellular and viral genes actively involved in a wide range of biological processes such as the bile acid biosynthesis, liver specific gene regulatory network and hepatitis B virus replication. The activity of nuclear receptors is regulated by multiple mechanisms, including coactivation and corepression. In this study, it was found that the silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) specifically represses the transcriptional activity of hB1F, on either GAL4 dependent reporter system or the hB1F-responsive HBV enhancer II/core promoter. The repression imposed by SMRT is observed in different cell lines. Interestingly, hB1F couldn t interact with SMRT directly, as demonstrated by mammalian two-hybrid analysis or GST pull-down assay. Taken together, it can be concluded for the first time that the transcriptional activity of hB1F is regulated specifically by the corepressor SMRT via an indirect mechanism.