Identification of an epitope of SARS-coronavirus nucleocapsid protein

The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SAR,S-CoV. Furthermore, it was confirmed that Nl peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

AIM: To construct ItB-ureBfusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacter pylori (H pylon) strain Y06 and the ItB gene from Escherichia coli (E. coli) strain 44851 were linked into ItB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of Hpylori and a self-prepared rabbit anti-rUreB serum, respectively,and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB.Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA.RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E, coli BL21DE3 to express the rLTB-UreB.The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins, rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB.CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of Hpylorigenetically engineered vaccine.

LRH-1/hBlF and HNFl synergistically up-regulate hepatitis B virus gene transcription and DNA replication

Enhancer Ⅱ (ENⅡ) is one of the critical crs-elements in the Hepatitis B Virus (HBV) genome for the hepatic viral gene transcription and DNA replication. The liver-specific activity of ENII is regulated by multiple liver-enriched transcription factors, including LRH-1/hBlF, HNF1, HNF3β, HNF4 and C/EBP. Knowledge on the interplay of these important factors is still limited. In this study, we demonstrate a functional synergism between the orphan nuclear receptor LRH-1/hBlF and the homeoprotein HNF1 in up-regulating the liver-specific activity of ENII. This synergism is sufficient for initiating the viral gene transcription and DNA replication in non-hepatic cells. We have defined the activation domains in hB1F and HNF1 that contribute to the synergism. We further show that hB1F and HNF1 can interact directly in vitro and have mapped the domains required for this interaction.

Loss of heterozygosity on chromosome 10q22-10q23 and 22q 11.2-22q 12.1 and p53gene in primary hepatocellular carcinoma

AIM: To analyze loss of heterozygosity (LOH) and homozygous deletion on p53 gene (exon2-3, 4 and 11), chromosome 10q22-10q23 and 22q11.2 -22q12.1 in human hepatocellular carcinoma (HCC).METHODS: PCR and PCR-based microsatellite polymorphism analysis techniques were used.RESULTS: LOH was observed at D10S579 (10q22-10q23) in 4 of 20 tumors (20%), at D22S421 (22q11.2-22q12.1) in 3 of 20(15%), at TP53.A (p53gene exon 2-3) in 4 of 20 (20%), at TP53.B (p53gene exon 4) in 6 of 20(30%), and at TP53.G (p53gene exon 11)in 0 of 20(0%). Homozygous deletion was detected at 10q22-10q23(8/20; 40%), 22q11.2-22q12.1(8/20; 40%), p53 gene exon 2-3(0/20;0%), p53gene exon 4(6/20; 30%), and p53gene exon 11(2/20; 10%).CONCLUSION: There might be unidentified tumor suppressor genes on chromosome 10q22-10q23 and 22q11.2-22q12.1 that contribute to the pathogenesis and development of HCC.

Distribution and expression of non-muscle myosin light chain kinase in rabbit livers

AIM: To study the distribution and expression of non-muscle myosin light chain kinase (nmMLCK) in rabbit livers.METHODS: Human nmMLCK N-terminal cDNA was amplified by polymerase chain reaction (PCR) and was inserted into pBKcmv to construct expression vectors. The recombinant plasmid was transformed into XL1-blue. Expression protein was induced by IPTG and then purified by SDS-PAGE and electroelution, which was used to prepare the polycolonal antibody to detect the distribution and expression of nmMLCK in rabbit livers with immunofluorescene techniques.RESULTS: The polyclonal antibody was prepared, by which nmMLCK expression was detected and distributed mainly in peripheral hepatocytes.CONCLUSION: nmMLCK can express in hepatocytes peripherally, and may play certain roles in the regulation of hepatic functions.

A note on overshoot estimation in pole placements

In this note we show that for a given controllable pair (A, B) and any λ > 0, a gain matrix K can be chosen so that the transition matrix e {(A+BK)t} of the system x = (A + BK) x decays at the exponential rate e ?λt and the overshoot of the transition matrix can be bounded by Mλ L for some constants M and L that are independent of λ. As a consequence, for any h > 0, a gain matrix K can be chosen so that the magnitude of the transition matrix e (A+BK)t can be reduced by 1/2 (or by any given portion) over [0, h]. An interesting application of the result is in the stabilization of switched linear systems with any given switching rate.

Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene,CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

AIM:To construct a prokaryotic expression system of a Helicobacter py/ori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody,so as to understand the manner in which the infection of CagA-expressing Hpylori (CagA^+ Hpylori) isolates cause diseases. METHODS:Hpyloristrains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated.PCR was used to detect the frequency of cagA gene in the 109 Hpyloriisolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06.A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE.Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and ant igenicity of rCagA1,respectively. Two ELISAs were established to detect CagA expression in 109 Hpyloriisolates and the presence of CagA antibody in the corresponding patients'sera,and the correlations between infection with CagA+ Hpyloriand gastritis as well as peptic ulcer were analyzed. RESULTS:Of all the clinical specimens obtained,80.8% (126/156) were found to have Hpyloriisolates and 97.2% of the isolates (106/109) were positive for cagA gene.In comparison with the reported data,the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively.The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein,rCagA1 was able to bind to the commercial antibody against the whole-cells of Hpyloriand to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4.A proportion as high as 92.6% of the Hpyloriisolates (101/109) expressed CagA and 88.1% of the patients'serum samples (96/109) were CagA antibody-positive.The percentage of CagA^+ H pylori strains(97.9%)isolated from the biopsy specimens of peptic ulcer appeared to be higher than that from gastritis(88.5%),but the difference was not statistically significant(x^2=3.48,P>0.05). CONCLUSION:rCagAl produced by the prokaryotic expression system constructed in this study possesses good immunoreactivity and antigenicity,and the established ELISAs can be used to detect CagA of Hpyloriand its antibody. H pylori isolates show high frequencies of cagA gene and CagA expression,but the infections by CagA^+ H pylori strains are not the most decisive factors to cause gastric diseases.

Establishment of transgenic mice carrying the gene of human nuclear receptor NR5A2 (hB1F)

AIM: Human hepatitis B virus enhancer II B1 binding factor (hBIF) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hBIF transgenic mouse model to promote the functional study of hB1F.METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice.The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR analysis.RESULTS: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages.Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably.CONCLUSION: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

Screening for PreS specific binding ligands with a phage displayed peptides library

AIM:To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV). METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. Ecoli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay. RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreSl epitope on the phage surface with a high efficiency. A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thio-PreS with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined. CONCLUSION: A phage library has been constructed, with random peptides displaying as pVIII-fusion proteins. Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.

Expression of hepatitis C virus hypervariable region 1 and its clinical significance

AIM: To explore the properties of hypervariable region 1(HVR1) in the envelope 2 gene of hepatitis C virus by analyzing the reactivity of HVR1 fusion proteins from different Chinese HCV strains with sera of patients with chronic hepatitis C and by comparing their reactivity between interferon therapy responders and non-responders.METHODS: Gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were amplified from pGEMT-E2 plasmids and sub-cloned into pQE40vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli strain TG1. The purified DHFRHVR1 proteins were then used to detect the anti-HVR1antibodies in 70 serum samples of patients with chronic hepatitis C.RESULTS: Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320-800 ug fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b,SD1b and SD2a) reacted with 72.8 % (51/70), 60 % (42/70), 48.6 % (34/70), and 58.6 % (41/70) of the anti-HCV positive patients' sera respectively by ELISA. 57.1% (4/7) of non responders reacted with all four HVR1 fusion proteins, while only 15.3 % (2/13) of responders reacted with all of them. The O.D. values of sera from IFN therapy responders were significantly higher than those of non responders (P＜0.05).CONCLUSION: The selected HVR1 fusion proteins expressed in E. coli can broadly react with HCV-infected patients' sera. The intensity and/or quality of the immune response against HCV may be a critical factor determining the response to interferon treatment. With the evolution of virus strains, anti-HVR1 antibodies can not neutralize all the quasispecies. A polyvalent and high immunogenic vaccine comprising a mixture of several HVR1 sequences that cover the reactivity of most HCV isolates may be useful.

Gene expression profile in liver of hB1F transgenic mice

AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hBIF transgenic mice. METHODS: Transgene expression was analyzed with RT-PCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of bhe differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hBIF were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hBIF transgene may cause changes of gene expression profiles in the liver of transgenic mice.

Gene expression profiling of rat livers with Yin-deficiency-heat syndrome

OBJECTIVE： To explore the nature of ＂Yin internal heat caused by Yin-deficiency＂ in terms of the theo- ry of Traditional Chinese Medicine, by studying en- ergy metabolism in rats with Yin-deficiency-heat syndrome and analyzing the gene expression pro- file of their livers. METHODS： A Yin-deficiency-heat syndrome model was induced in rats using three Chinese medicinal herbs. Glycogen and triglycerides in blood plasma, and the enzyme activity of ATP in livers were mea- sured colorimetrically. Triiodothyronine （T3）, thyrox- ine （T4）, and thyroid stimulating hormone levels in blood plasma were also measured with enzyme linked immunosorbent assay. The gene expression profile of livers was detected with gene chip analy- sis. Differentially expressed genes were screened out and classified according to Gene Ontology. The accuracy of results were examined with reversetranscription-polymerase chain reaction RESULTS： Compared with the control group, body weight （P〈0.05） and hepatic glycogen （P〈0.05） were significantly lower in the Yin-deficiency-heat syndrome group. Moreover, toe temperature （P〈 0.01） and triglyceride （P〈0.05）, Na ＋-K＋-ATPase （P〈 0.01）, Mg2＋-ATPase （P〈0.01）, T3 （P〈0.05）, and 1-4 （P〈 0.01） levels were significantly higher. There were 99 differentially expressed genes in livers from the Y/n-deficiency-heat syndrome group. Genes were mainly related to sterol synthesis （Pc=0.0392）, de- fense response （Pc=0.0448）, and sterol metabolism （Pc=0.0533）. CONCLUSION： Abnormal expression genes in rats with Yin-deficiency-heat syndrome prompted the synthesis and metabolism of cholesterol, increased energy consumption, and reduced defense re- sponse. This gene expression might be the molecu- lar mechanism underlying ＂internal heat caused by Yin-deficiency＂ in the rats with Yin-deficiency-heat syndrome.