Objective To construct a novel recombinant human IFN-ε,and to analyze its physi-cal,chemical properties and biological characteristics.Methods Human genomic DNA was used as the template to synthesize IFN-ε gene by PCR.The sequence was cloned into plasmid vector pET-32a(+),and the recombinant plasmid pET-32a(+)/IFN-ε was transformed into E.coli BL2l(DE3).The form of ex-pression product was inclusion bodies.After purification and renaturation.high purity active protein IFN-εwas achieved.The final product was tested for its physical.chemical properties and biological characteristics including anti-viral and anti-proliferative acfivities.Results IFN-ε was expressed in inclusion body in E.coli.After the protein renaturation and purification,the purity was more than 95%.The rhIFN-ε protein had a specific anti-viral activity of about 1.2×103 IU/mg.Its anti-poliferative activity is obvious and can in-duce cells to produce anti-viral protein MxA.Conclusion Human IFN-ε protein was expressed successful-ly.and this protein has anti-virus and anti-prolireration activity.

Objective To investigate the histogenesis and clinicopathologic features of carcinosarcoma of the urinary bladder. Methods Two cases of carcinosarcoma (one man and one woman)were included.They were admitted to hospital with discontinuous,painless,macroscopic hematuria.Clinicopathalogic features of all findings of the 2 cases were reviewed. Results Cystoscopy of both cases showed that the tumors displayed polypous or cauliflower-like shape,and grew invassively.CT examination of the bladder wall confirmed the presence of parenchymatous tumor.Both of the 2 cases underwent partial cystectomy.Intraoperative examination showed the tumors were similar to carcinoma of bladder.Histopathology showed biphasic neoplasms with distinct high grade epithelial and mesenchymal components.Morphologic characteristic of the tumor was abnormally proliferative and the mitotic figures could be seen.One of the patients died within 11 months and the other,16 months following the operation. Conclusions Carcinosarcoma of the urinary bladder is a kind of rare malignant tumor that is most often in an advanced stage at presentation and has an unfavorable prognosis. It should be identified promptly and treated appropriately.Those who have discontinuous,painless,macroscopic hematuria should be warned of the risk of the disease.

Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influertzae(NTHi) was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a (+) to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q Sepharose~(TM) XL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein be-fore challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was com-pared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a rel-atively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group (P < 0.05). Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation.

The glycoproteins in the biological sample are low abundance and are susceptible to be inhibited and interfered by other non-glycoproteins.An enrichment step is usually required before the glycoprotein analysis, but the operation steps of conventional solid-phase-based glycoprotein enrichment methods are difficult to be compatible with the most classical enzyme-linked immune sorbent assay (ELISA) technique.In this study, a novel water-soluble dendrimer based boronic acid capture (DBC) material was developed using PAMAM 4.0 as the carrier and boronic acid as the affinity group.The method was applied to the detection of glycoproteins in human liver microsomes using ELISA.In this study, the DBC enrichment conditions were optimized by model glycoprotein, and then its sensitivity and anti-interference ability were investigated.This method was applied to the enrichment of glycoproteinsin human liver microsomal.The results showed that the enrichment selectivity of DBC for glycoprotein could be up to 100000 folds, and the enrichment signal of glycoprotein could be increased by 100 times.Therefore, the ELISA method using DBC as a novel enrichment material for glycoprotein had high sensitivity and selectivity in detection of biological samples with only one simple incubation step, which was useful for glycoproteins researches.

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).

The HIV-1 Rev protein facilitates nuclear export of unspliced and singly spliced viral transcripts containing RRE RNA through the CRM1 export pathway. Inhibition of Rev-mediated RNA nuclear export can arrest HIV-1 transcriptional process, which clearly, reveals a target for anti-HIV drug development. In this work, a cell-based assay has been established for screening anti-HIV compounds targeting the Rev-mediated RNA nuclear export. This assay utilized a codon-optimized green fluorescent protein (GFP) as reporter gene, which expression is in a Rev-dependent manner. Any compound that inhibits the Rev-mediated RNA nuclear export is identified by reducing emission of GFP. The Z' score of this model is 0.8220. Three thousands compounds were screened and the positive rate was 9.3% with a cutoff at 50% inhibition. IMB7C7, one of the positive compounds, efficiently inhibits viral production from HIV-1 infected cells.

ObjectiveTo detect the difference of cytokines and antibodies productions by immunologic system from mice and rabbits vaccinated with the M.RCAg-1 chimeric protein,expressed in E.coli,formulation with different adjuvants,including Freund's adjuvant and three clinically acceptable adjuvants,namely,Al(OH)3,Montanide ISA720 and Montanide ISA51.MethodsSix weeks female BALB/c mice were vaccinated with recombinant protein formulated with different adjuvants through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual Epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; Affinity assay between protein and immune IgG of rabbits with the biosensor,and the growth of Plasmodiumfalciparum in vitro to evaluate by growth inhibition assay(GIA).ResultsDifferent formulation can induce different levels of antibody titers,the effection of ISA51 adjuvant was most closely with Freund's adjuvant,and can induce a higher specific antibody of 11 epitopes within proteins,can effectively stimulate cellular immune response based on the IFN-γ,to avidity Montanide ISA51 adjuvant immune antibodies and M.RCAg-1 protein affinity than the other two adjuvants;and Montanide ISA720 adjuvants and Al (OH)3 adjuvant group in mice can't induce a significant IFN-γresponse(P＞0.05).On avidity assay,the Montanide ISA51 formulation group was better than the other two adjuvants; and Montanide ISA720 and Al (OH)3 adjuvant formulation group can't induce a significant IFN-γresponse in mice(P＞0.05) ; the inhibition rates were 60% and 100% in 3D7 and Dd2 Plasmodium falciparum at a concentration of 2 mg/ml IgG by Montanide ISA51 formulated protein,and IgG of Al( OH)3 formulation could not effectively inhibit the in vitro growth of Plasmodium falciparum( 10% ),while IgG of Montanide ISA720 formulation could not inhibit growth of parasite in vitro.ConclusionBy comparing three clinically acceptable adjuvants and Freund's adjuvant in BALB/c mice and New Zealand rabbit,Montanide ISA51 adjuvants can be acceptable formulated M.RCAg-1 protein induced humoral and cellular immune responses,can be used as one of the candidate adjuvants.

ObjectiveTo explore the effects of vector fusion peptides on the conformation and immune reactivity of recombinant polyepitope antigens,M.RCAg-1.MethodsWe subcloned polyepitope artificial antigen gene,M.RCAg-1,into prokaryotic expression vectors,pDS-ex,that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site.Three recombinant proteins expressed by these vectors,named P312-1,P312-2,and P312-3,were purified and purity is greater than 95％.Then BALB/c mice were vaccinated with the three proteins formulated with Freund's adjuvant through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; and the growth of Plasmodium falciparum in vitro to evaluate by growth inhibition assay(GIA),and analyze secondary and tertiary structures of recombinant proteins from different expression vectors by bioinformatics and circular dichroism technique.ResultsThe P312-1,P312-2 have almost the same amino acid sequence,and the three proteins have the same immunogenicity in animal models(P＞0.05),however,the different proteins elicited various T-cell responses,the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assaysin vitro ( respectively,93.9％,14.7％,54.3％ ).The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors,analyzed by bioinformatics and circular dichroism technique,which demonstrated the change of protein molecule spaces conformation,and the obviously change of some epitope locations.ConclusionThese results suggest that the expressed polyepitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and,subsequently,the epitope exposure.Thus,these proteins are able to induce both distinct humoral and cellular immune responses in animal models,and they affect the efficacy of immune inhibition against the parasite,the enhancement or suppresses.

Objective To evaluate the indication and surgical technique for treating tetralogy of F allot with pulmonary atresia (TOF-PA).Methods From June 1984 to June 2009,66 patients with TOF-PA underwent 69 operations.Among them,34 were males and 32 females.Their age ranged from 6 months to 29 years.The anatomic characteristics of TOF-PA included 31 cases of Type Ⅰ,14 Type Ⅱ,12 Type Ⅲ and 9 Type Ⅳ.The operations included palliative aorto-pulmonary shunts in 11 cases,one-stage unifocalization with unpatched VSD in 2 cases,one stage complete repair in 40 cases,one-stage unifocalization with VSD repair in 13 cases,and delayed intracardiac repair after shunt procedures in 3 cases.Results There were 6 early deaths,including 1 death happened after aorta-pulmonary shunt and 5 after complete repair.The causes of death were severe low cardiac output in 3 cases,respiratory failure in 1,multiorgan function failure in 1 and severe wound infection with endocarditis in 1 after aorta-pulmonary shunt.The postoperative oxygen saturation of the patients undergone shunt and one stage unifocalization with unpatched VSD increased to 82％ ～ 91％.The postoperative ratio of right ventricular pressure/left ventricular pressure after complete repair was ＜ 0.5 in 31 cases,18 cases were between 0.5 and 7 cases ＞ 0.75.47 patients were followed up from 3 months to 15.5 years.The heart function(NYHA) of 44 patients were in class Ⅰ or Ⅱ and 3 in class Ⅲ or Ⅳafter operation.Conclusion The surgical strategy for TOF-PA mainly depends on the anatomic characteristics of the pulmonary and aortopulmonary collateral arteries.An individualized approach based on the anatomy of the pulmonary circuits permits a better result in the patients with TOF-PA.Patients with well developed pulmonary arteries should undergo one stage complete repair as early as possible.

Objective To study the expression of PINCH and vascular endothelial growth factor C (VEGF-C) in cervical squamous cell carcinoma.Methods We detected the expression of PINCH and VEGF-C by immunohistochemistry SP in 58 cervical squamous cell carcinoma cases and 30 normal cervical epithelial tissue and analyzed their relationship to the clinical pathological features.Results The expression of PINCH and VEGF-C in 58 cervical squamous cell carcinoma(62.1％,36/58 ;67.2％,39/58 ) were higher than that in normal cervical epithelial tissue(0,0/30).The difference was significant( x2 =31.512,12.534,P ＜ 0.001 ).The expression of PINCH protein was not significantly associated with the age,tumor size and tumor differentiation grade ( P ＞ 0.05 ),but was associated with the lymph node metastasis and clinical stage ( x2 =9.090,8.236,P ＜ 0.001 ).The expression of VEGF-C had no significant correlationship with the age and tumor size( P ＞ 0.05 ) but had a correlationship with the lymph node metastasis,tumor differentiation grade and clinical stage( x2 =10.775,13.496,5.001,P ＜ 0.05 ).The expression of VEGF-C was correlated with the expression of PINCH protein( C =0.341,P ＜ 0.01 ).Conclusion It is possible that VEGF-C and PINCH take part in the development and progress of cervical squamous cell carcinoma and play an important role in the invasion and metastasis mechanism altogether.