Objective To clone and bioinformatically analyze the full-length sequence of F protein binding protein 1 (FBP1). Methods Yeast two-hybrid system was employed to obtain an unknown gene and named it as FBP1.The coding sequence of FBP1 was cloned using molecular biological techniques. Results The coding sequence of FBP1 was cloned successfully. Conclusion FBP1, a cellular protein binding to F protein of HCV, plays an important role in the interaction of virus protein and host cell protein.

Objective To construct prokaryotic expression vector of NS5A transactivating protein 7 of hepatitis C virus (NS5ATP7), induce the expression of NS5ATP7 in E. coli, and to predict its structure and function by bioinformatics analysis. Methods NS5ATP7 gene was amplified by reverse transcription PCR (RT-PCR) using HepG2 cells mRNA as template, and was ligated into pGEM-T cloning vector. After verifying the sequence of the inserted fragment by restriction enzyme digestion identification and sequencing, NS5ATP7 was cloned into inducible prokaryotic expression vector pET-32a(+) and transfected into competent E. coli BL21. The protein expression was induced with IPTG and the product was analyzed by SDS-PAGE and Western blotting hybridization. The structure and function of NS5ATP7 were predicted using bioinformatics techniques. Results NS5ATP7 gene with about 891bp was amplified by RT-PCR, which was coincident with that we expected, and it was then inserted into expression vector pET-32a(+). Under the induction of IPTG, the recombinant NS5ATP7 was expressed successfully. SDS-PAGE and Western blotting assay showed that this recombinant protein possessed good immunogenicity. Bioinformatics method such as ExPASy, TMHMM and SignalP analysis indicated that NS5ATP7 was full of helices, had neither transmembranous structure nor signal peptide. Conclusions The recombinant NS5ATP7 gene could be successfully expressed in prokaryotic expression system of E. coli, which might lay the foundation of studying the immunogenicity and biological charactersitics of the NS5ATP7. Bioinformatics analysis may provide a new method to analyze the structure and function of a new protein.

Objective To investigate the activating effect of HBV X protein on transcription of calgizzarin S100A11 gene promoter. Methods The sequence of calgizzarin S100A11 gene promoter was identified in GenBank by bioinformatics and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was cloned into pCAT3 reporter vector. The HepG2 cells were transfected by pCAT3-S100-p, and then co-tranfected by pCAT3-S100-p and pcDNA3.1(-)-X. The choloraphenical acetyltransferase(CAT) activity was detected by enzyme-linked immunosorbent assay(ELISA) kit. Results It was shown that pCAT3-S100-p could regulate the expression of CAT in HepG2 cells. The expression of CAT in co-transfection of pCAT3-S100-p and pcDNA3.1(-)-X was 2.1 fold higher than pCAT3-S100-p plasmid. Conclusion HBV-X protein can trans-activate the expression of S100A11 protein, and it further proved our previous results by SSH and biochips.

Objective To construct prokaryotic expression vector of hepatitis C virus NS5ATP1 gene, and to induce its expression in E. coli. To purify the fusion protein and obtain its polyclonal antibody from immunized New Zealand rabbits. Methods The NS5ATP1 gene, which was cut from the vector pGBKT7-NS5ATP1, which was self-constructed by the authors, was cloned into plasmid pET32a(+) to construct the pET32a(+)-NS5ATP1 prokaryotic expression vector. It was proved that the recombinant plasmid was constructed correctly by sequencing. And then the expression vector was transformed into the competent E. coli DH5? and BL21. After being induced with IPTG, the NS5ATP1 fusion protein was expressed and analyzed with SDS-PAGE and Western blot. The transformed bacteria were fragmented by ultrasonic and then separated by SDS-PAGE. The fusion protein formed inclusion body. They were then purified and re-natured through Ni+ affinity column chromatography. The purified pET32a(+)-NS5ATP1 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and immunizing potence of polyclonal antibody were evaluated by Western blot and ELISA. Results After transferring pET32a(+)-NS5ATP1 plasmid into DH5? and BL21 and induced with IPTG, the NS5ATP1 fusion protein of about 56kD was highly expressed. SDS-PAGE analysis showed that the fusion protein products were mainly in inclusion body and expressed in the highest level at 4.5h of induction. The purified protein and polyclonal antibody were obtained successfully. ELISA manifested the titer of polyclonal antibody was over 1∶512 000. The high specificity was testified by Western blot. Conclusions The successful expression and purification of NS5ATP1 fusion protein and the preparation of NS5ATP1 specific polyclonal antibody will be valuable for the study on the biological function of NS5ATP1.

Child, Preschool ; Fever ; etiology ; Histoplasmosis ; complications ; diagnosis ; pathology ; Humans ; Jaundice ; etiology ; Male

Objective To construct recombinant full length genotype B and C hepatitis B virus (HBV)and to examine HBV DNA replication and hepatitis B surface antigen(HBsAg),hepatitis B e antigen(HBeAg)expressions in Huh7 cells. Methods The full length genotype B and C HBV DNA were extracted and amplified from two HBV infected patients. The recombinant plasmids were constructed by inserting the amplified HBV fragments into the eukaryotic expression vector,pHY106,which were then transfected into Huh7 cells. The cells transfected with blank pHY106 vector were used as control. HBV DNA replication at 72 hours of transfection was detected by Southern blot. The HBV DNA levels in Huh7 cells at 24,48,72,96 and 120 hours of transfection were determined by real-time polymerase chain reaction(PCR).Meanwhile, the HBsAg and HBeAg expression levels in the supernatants at 24,48,72,96 and 120 hours were determined by enzyme linked immunosorbent assay(ELISA).Results The recombinant plasmids expressing genotype B or C HBV DNA were successfully constructed.The HBV replicative intermediates in HBV core particles,including rcDNA dsDNA and ssDNA,were detected by Southern blot.HBV DNA level could reach 8 lg copy/mL which was by real-time PCR. HBsAg and HBeAg levels determined by ELISA peaked at 72 hours after transfection and then declined gradually. Conclusions The recombinant plasmids inserted with genotype B or C HBV DNA are constructed successfully, which can express high levels of HBsAg and HBeAg in Huh7 cells. This system provides a platform for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs against HBV.

A competency dictionary of advanced health informatization application persons was compiled by inter-viewing 20 health information persons with behavior events interview , their competency characteristics were coded By Nvivo, the coding frequency and characteristics score in good performance group and ordinary group were ana-lyzed by variance analysis, which showed 8 characteristics of key competency and 12 characteristics of basic compe-tency in advanced health information application persons.

Objective To investigate the expression of p53 in breast cancer and its clinical significance .Methods The clinical pathological data of 214 cases of breast cancer patients were collected ,and retrospective analyzed of p53 protein expression and its correlation with clinical pathological features .Results The positive rate of p53 was 52 .3% .The expression p53 was no correlation with age ,the size of the lesion(P> 0 .05) ,it was correlation with lymph node metastasis ,histological classification and molecular subtype(r=0 .396 ,0 .309 ,0 .167 ,P=0 .000 ,0 .000 ,0 .014) ,and also have negative correlation with the expression rate of ER ,PR (r= -0 .561 ,-0 .315 ,P=0 .000 ,0 .000) ,positive correlation with HER2 and Ki67(r=0 .374 ,0 .153 ,P=0 .000 ,0 .026) .In the group of triple negative breast cancer (TNBC) and the group of non-TNBC ,the difference was statistically significant of expression of p53 (P<0 .05) .Conclusion p53 protein has closely related with the development ,relapse and metastases of breast cancer ,the detection of p53 can be used to guide the individualized therapy and as a reference index to evaluate the prognosis of breast cancer .

AIM:To perform a new method for orbital implant and contracted socket through one time and its results. ·METHODS:Totally 114 patients 114 eyes, from January 2008 to June 2014, with contracted socket participated in this study. We incised the bulbar conjunctiva horizontally and excised scar tissue, then implanted the hydroxyapatite in the four extraocular muscles and tightly sutured the Tenon ' capsule. After that, we put the superior and inferior conjunctival petals backwards and sutured them to the Tenon ' s capsule. All the patients were divided into four groups according to the vertical diameter length of the conjunctival defect area:GroupⅠ:≤5mm; Group Ⅱ: 6-10mm; Group Ⅲ: 11-15mm; and Group Ⅳ: ≥16mm. These patients were followed up for 6mo to 3y to observe the conjunctival sac shaping and growth of conjunctiva. ·RESULTS:There were 64 cases in GroupⅠ, 31 cases in Group Ⅱ, 16 cases in Group Ⅲ and 3 cases in Group Ⅳ. All patients ' conjunctival defect was covered by new conjunctiva and scar tissue 4 to 6wk after surgeries. Ten cases had contracted socket; 2 cases had orbital implant exposure, requiring reoperation. Of the 114 cases, 8 had contracted socket and could use a smaller conformer, 106 could use a normal size conformer. ·CONCLUSION: When the conjunctival defect was ≤15mm, this new method can address the orbital implant and contracted socket at the same time. While it was ≥16mm, flap transplantation is necessary.

To establish a convenient and rapid method for identification of Pseudostellariae Radix by molecular identification, the rDNA-ITS sequences of Pseudostella riaheterophylla and its adulterants had been aligned to find out specific fragment. The specific primers against the fragment were designed and the PCR amplification conditions were optimized. The fluorescence reaction of the PCR products colored by 100 x SYBR Green I was observed under UV. The concentration of reaction buffer included 5.5 μL DNA Taq polymerase premix, 10 pmol Tzs-2F and 10 pmol Tzs-2R, 20-80 ng template DNA, and plus double sterile distilled water to 25 μL. The PCR thermal profile was as follows: predenaturation at 95 degrees C for 1 min, followed by 30 cycles of denaturation at 95 degrees C for 5 seconds, primer annealing and extension at 56 degrees C for 15 seconds, then it was extension at 72 degrees C for 30 seconds. The fluorescence reaction of Pseudostellariae Radix showed green fluorescence, while adulterants had not. Extraction, amplification DNA and all steps of molecular identification could be completed successfully in 40 minutes. The approach could amplify DNA template of Pseudostellariae Radix specificity, and its product with 1 μL 100 x SYBR Green I could engender green fluorescence under UV. The method was simple and accurate, so it could be used for identification of Chinese traditional medicine.
Caryophyllaceae ; classification ; genetics ; DNA Primers ; genetics ; Drug Contamination ; prevention & control ; Plant Roots ; classification ; genetics ; Polymerase Chain Reaction ; methods