The spike (S) and nucleocapsid (N) proteins, which are responsible for viral binding to cell surface receptors and the formation of ribonucleoprotein complexes during virion assembling, are major structure proteins of severe acute respiratory syndrome coronavirus (SARS-CoV). The expression of recombinant protein may give more accurate result for detecting SARS-CoV infection. A novel fusion protein,comprising of two fragments of N and S proteins from SARS-CoV, was prepared. Our computer-assisted analysis suggested that the immunodominant domains were located in the amino acid residues 1-227 of N protein and 450-650 of S protein, further the fusion of the two fragments did not change the immunochemical characteristics. The complementary DNA(cDNA) encoding N1-227 fused with S450-650 was obtained by sequence overlapping extension (SOE), and named NLS. It was cloned into pET-28a ( + ), an expression vector for His-tag fusion protein. This new constructed fusion protein was prokaryotic expressed in E. coli,and purified by metal chelate affinity chromatography with the purity over 95 %. The purified fusion protein was identified by anti-His monoclonal antibody and convalescence SARS patients serum. The NLS protein based ELISA showed that NLS maintained appropriate antigenicity and specificity to react with the sera of convalescent SARS patients. The functional NLS protein were successfully expressed and purified. And the fusion protein based ELISA can be used for detection of antibodies (Abs) against the S and N proteins of SARS-CoV. It may provided a novel diagnostic tool and have the potential application in developing of anti-SARS vaccine.

To study effects of arachidonic acid (AA) on the growth and differentiation of rat adipocytes, a cells culture system of rat primary preadipocytes was established. The cells treated by different concentration of AA supplemented based on DMEM medium containing 10% fetal bovine serum. Cell proliferation was measured by trypan blue exclusion and methyl thiazolyl tetrazolium (MTT) assay method. Hoechst33342 fluorescence staining observed AA induced morphological changes. Oil Red O staining extraction assay assess the degree of adipogenesis and differentiation, and cyclooxygenases-2(COX-2) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that rat preadipocytes treated with 120 μmol/L AA for 24-72 hours remarkably promoted the cells proliferation compared with control, 160 μmol/L AA treated for 48 hours could induce apoptosis of preadipocytes. 40, 80 μmol/L AA decreased the fat content in cells at 72 hours, and 40 μmol/L AA significantly up-regulated the expression of COX-2 mRNA at 24 hours. This results indicate that AA regulate adipocytes proliferation and differentiation depended on treatment time and concentration. 40-80 μmol/L AA maybe useful to control body fat, which may be associated with the increase of COX-2 mRNA.

Many overwintering organisms produce antifreeze proteins (AFPs) that can be adsorbed onto the surface of ice crystals and modify their growth. These proteins show great diversity in structures, and they have been found in a variety of organisms. AFPs from insects have higher thermal hysteresis activity than other organisms. Recent studies revealed the structures of AFPs and put forward different ice-binding models. No mechanism, however, can apply to all antifreeze proteins and the molecular interaction between AFPs and ice are not accurately resolved. AFPs can be applied extensively to agriculture, aquaculture and low temperature storage of organs, tissues, as well as cells. To confer transgenic plant cold resistance application of AFPs is essential, while the expression and regulation of antifreeze gene need to be elucidated.

A new fusion expression vector, pED-P8-Hi-lys was designed and constructed. It includes four parts, a 20 peptide sequence of hirudin that can maintain anticoagulant activity, the C-terminus of asparaginase as a fusion partner, basic octopeptide (KRKRKKSR) that makes the fusion partner easy to remove, and the unique acid-labile aspartyl-prolyl bond. It was transformed into E. coli BL-21 and the fusion protein (AnsB-C-P8-Hi-lys) was expressed effectively as inclusion bodies after inducing by lactose. The objective peptide Hi-lys was purified by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and DEAE-cellulose 52 column chromatography. The antithrombin activity of the purified Hi-lys peptide was about 50 ATU/mg by thrombin activity assays.

In order to investigate whether defense genes PAL, LOX, PBZ1, PR 1 a and Cht 1 participate in APR(adult plant resistance)to rice bacterial blight, their expression were analyzed using RT-PCR. Enzymatic activities of PAL and LOX were also measured. Results indicated that PAL was induced by pathogen and wounding in adult plants while it only induced by pathogen at the seedlings, and the expression of PAL was stronger in adult plants than that in seedlings. Expression of LOX was induced by pathogen both in seedlings and adult plants and it was stronger in adult plants than that in seedlings. Expression PBZ1 was induced by both pathogen and wounding in both seedlings and adult plants and it is earlier and stronger in adult plants than that in seedlings. No expected fragments were obtained for PR 1 a and Cht 1. Enzymatic activities of PAL and LOX were consistent with their mRNA accumulations, respectively. It is probable that activation of PAL,LOX, and PBZ1 play crucial roles in APR to rice bacterial blight.

The antinociceptive effect of epidural administration of huwentoxin-I was elucidated in a tonic visceral pain rat model produced by acute colon inflammation. The nociceptive behaviors were induced by perendoscopically injecting dilute formalin (50 μl) into the depth of the colonic wall in rats. Both ω-conotoxinMVIIA and morphine hydrochloride were given epidurally as positive control while saline as negative control.Similar to ω-conotoxin-MVIIA and hydrochloride morphine, the epidural administration of HWTX-Ⅰ significantly reduced the nociceptive responses in a dose-dependent manner in tonic visceral pain rat model ( P ＜ 0.05). The suppression effects of both huwentoxin- Ⅰ and ω-conotoxin-MVIIA at 20 μg/kg were kept steady compared with the saline group and reached their maximum effects at the doses of 50 ～ 75 μg/kg within 1 hour when the nociception had been observed. It was also found that at the same doses, huwentoxin- Ⅰ was less effective in antinociception than ω-conotoxin-MVIIA. However, ω-conotoxin-MVIIA, but not huwentoxinⅠ , caused an obvious motor dysfunction at these doses. The action of morphine hydrochloride was initiated faster, but lasted for a shorter time than that of huwentoxin- Ⅰ and ω-conotoxin-MVIIA. Thus, huwentoxinⅠ , a potent blocker of neuronal N-type voltage-sensitive calcium channels, induced a remarkable dosedependent restrain effect similar to ω-conotoxin-MVIIA and morphine on the tonic visceral pain produced by colonic wall injection of formalin in conscious rats.

A full-length cDNA sequence encoding for the precursor of a venom peptide (named BmKCT) with homology to chlorotoxin has been isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The sequence of BmKCT is similar (68 % identities) to that of chlorotoxin isolated from Leiurus quinquestriatus quinquestriatus. To understand the biological function of BmKCT, this peptide was expressed using pGEX expression system and purified using GST affinity column and gel filtration.Whole cell patch-clamping recording showed that BmKCT could significantly inhibit chloride currents of gliomas cells, and the inhibitory effect was reversible. These results suggested that BmKCT might belong to the class of short chain toxins blocking the chloride ion channels.

Mimecan belongs to a family of leucine-rich proteoglycans that are secreted into the extracellular matrix. In order to investigate the function of mimecan, the coding region of mimecan was amplifed from a human pituitary cDNA by PCR and the recombinant prokaryotic expression vector pGEX-M was constructed. The vactor was transformed into E.coli BL21(DE3)and the GST-M fusion protein of 38 Kd was ecpressed in the bacteria under induction of IPTG. After purification, the fusion protein was infucted into New Zealand rabbits to prepare polyclonal antibody. The antibody was tested by Western blotting for their specificity and sensitivity. Using the antibody it was found the mimecan was expressed highly in certain types of human pituitary tumor tissues. These results make it possible for studying the biological function of mimecan.

In order to separate chiral D-phenylalanine from L-phenylalanine,the enzyme catalytic method by papain and immobilized papain were used in this investigation.It indicated that the yield rate of N-acetyl-DL-phenylalanine synthesized from DL-phenylalanine was 88.7%.The ionic strength was the strongest influencing factor in the synthesis of N-acetyl-L-phenylalanylaniline by both papain and immobilized papain on sodium alginate-ehitosan(IPSAC).But the reaction temperature was the strongest influencing factor in the synthesis by immobilized papain on nylon cloth(IPN).The ionic strength and the pH were the important influencing factors in the synthesis.The yield rates of the best synthesis system of N-acetyl-L-phenylalanylaniline by papain,IPSAC,IPN were 61.2%,54.7%,36.3%,respectively.The yield rate of L-phenylalanine from N-acetyl-L-phenylalanylaniline hydrolyzation was 59.2%.The optical purity of the product was 96.6%.The vield rate of D-phenylalanine from N-acetyl-D-phenylalanine hydrolyzation was 61.7%.The optical purity was 95.7%.

Three pSIREN-siRNA plasmids were constructed using a pSIREN-RetroQ vector to silence the expression of muhidrug resistance-associated protein (MRP1) gene, and subsequently characterized by restriction endonuclease digestion and DNA sequencing. A truncated MRP1 and a full-length MRP1 were cloned into pEGFP-N2 and PeDNA3.1 respectively as pEGFP-MRP1T and pcDNA-MRP1. The plasmid pEGFP-MRP1T was co-transferred with each of the three pSIREN-siRNAs into HEK293 cells for MRP1T-GFP targeted silencing, and pSIREN-siRNA1 was used as the negative control, pSIREN-siRNA2 and pSIREN-siRNA3 appeared to be more effective to silence MRP1T-GFP compared to pSIREN-siRNA1 as shown by fluorescence microscopy. For the silencing of full-length MRP1 expression, HEK293 ceils were co-transferred with pcDNA-MRP1 and either of the three pSIREN-siRNAs, then subjected for Western blot analysis and MTT assays, pSIREN-siRNA2 and pSIREN-siRNA3 were able to inhibit the expression of 190 kD MRP1, but not pSIREN-siRNA1. The MDR of MRP1-transfected HEK293 ceils was abolished with pSIREN-siRNA2 or pSIREN-siRNA3 transfections. RNA secondary structure predictions demonstrated that the mRNA local free energy (△G) of the siRNA1 targeted sequence was lower, as the GC content and Tm value of siRNA1 were higher than those of siRNA2 and siRNA3. These data suggest that the local structure siRNAs and target mRNA may influence the silencing efficiency of MRP1 expression.