BACKGROUNDTo determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris).

METHODSBy using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P. pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E. The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested.

RESULTSThe minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120. No cross-reaction was found with sera from patients with any other types of hepatitis. The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C. The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P. pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli.

CONCLUSIONThere may be more extensive conformational epitopes in the rHEV ORF2 from P. pastoris. The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

Gene Expression ; Hepatitis Antibodies ; blood ; Hepatitis Antigens ; genetics ; immunology ; Hepatitis E ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

Objective：This study was designed to establish and optimize the research methods for proteome,and to analyze the proteome components of human lung squamous carcinoma cell line NCI H520. Methods： A series of methods, including immobilized pH gradient two dimensional polyacrylamide gel electrophoresis(2DE), silver staining, PDQuest 2DE analysis software, peptide mass fingerprint based on matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI TOF MS) and SWISS PROT database searching, were used to separate and indentify the proteome of human lung squarmous carcinoma cell line NCI H520. Results： The good 2DE pattern including resolution and reproducibility was obtained. After silver staining, the 2DE image analysis by PDQuest 2DE software had detected average of 1146± 116 spots, and 851± 95 spots were matched. The average matching rate was 73.3％ . There had a good reproducibility of spot position in 2DE map, with average deviation in IEF direction of 1.52± 0.22 mm， while in SDS PAGE direction it was 1.97± 0.13 mm. Sixty spots were incised from silver staining gel randomly and digested in gel by TPCKtrypsin. Fifty four peptide mass fingerprints (PMF) maps were obtained by MALDI TOF MS. The typic peptide masses were searched in the SWISS PROT database by PeptIdent software. Forty four proteins were preliminarily identified. Some of them were cell cycle related proteins such as Cyclin H, some were signal transduction related proteins such as mitogen activated protein kinase, protein kinase C and receptor protein tyrosine kinase ERBB 2, some were oncogene related proteins such as Ras related protein RAB 36, etc. Conclusions： The main proteome research system including IPG 2DE, image analysis, MALDI TOF MS derived PMFs and database searching has been established. The data of NCI H520 obtained by above methods will be useful for the establishment of human lung squamous cell proteome database.

OBJECTIVETo identify the type of the intermediate filament (IF) protein of Angiostrongylus cantonensis and analyze its tissue localization.

METHODSRecombinant pET-IF of antigen IF was expressed in E.coli with IPTG induction, and the expression products were purified by His.Bind column and identified for determining the type of the IF protein by Western blotting. Anti-IF antibody was prepared by multi-spot subcutaneous injection into mouse and used to detect the tissue slices of A. cantonensis by immunohistochemical analysis.

RESULTSThe antigen IF were correctly expressed and purified, and identified as a keratin located in the intestine wall and cytoplusma.

CONCLUSIONThe antigen IF is distributed in the intestine wall of A. cantonensis.

Angiostrongylus cantonensis ; cytology ; metabolism ; Animals ; Cell Nucleus ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Intermediate Filament Proteins ; classification ; genetics ; isolation & purification ; metabolism ; Protein Transport

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology

OBJECTIVETo establish a simple and rapid system for carrier detection and prenatal diagnosis in hemophilia A (CHA) family.

METHODSLong distance-polymerase chain reaction (LD-PCR) was selected for detection factor VIII intron 22 inversion. Polymorphism of factor VIII intragenic restriction fragment length polymorphism (RFLP) of Xba I and Hin d III, short tandem repeat (STR) within intron 13 and 22, as well as extragenic DXS52 (ST 14) variable number of tandem repeat (VNTR) were assayed by PCR and linkage analysis.

RESULTSSeventy-one females were diagnosed as carriers within 52 HA families. Twenty-one families were diagnosed to be factor VIII intron 22 inversion and 28 families were diagnosed by linkage analysis, whereas 3 families could not been diagnosed. Seventeen of 18 fetuses at risk were male. Ten of 17 male fetuses were shown to be affected and were subsequently aborted. Seven male fetuses were diagnosed to be not affected. One female fetus was identified to be HA carrier. One-year follow-up study demonstrated that these babies were normal and living well.

CONCLUSIONLD-PCR combined with multiple locus linkage analysis enables the direct and indirect detection of HA for carrier testing and prenatal diagnosis.

Factor VIII ; genetics ; Family Health ; Female ; Hemophilia A ; diagnosis ; genetics ; Humans ; Male ; Minisatellite Repeats ; genetics ; Pedigree ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Pregnancy ; Prenatal Diagnosis ; methods

OBJECTIVETo detect serve acute respiratory syndrome-associated coronavirus (SARS-CoV) and SARS-like-CoV in fruit bats captured in Guangzhou and its vicinity.

METHODSTotally 927 bats of 9 species (Cynopterus sphinx, Rousettus leschenaulti, Miniopterus schreibersi, Hipposideros pratti, Rhinolophusasinicus, Scotophilusakuhlii, Hipposideros Pomona, Rhinolophus affinis, and Rhinolophus pusillus) captured in Guangzhou and its vicinity from September 2004 to November 2005 were available for this investigation, from which 3,043 samples (813 throat swasb, 524 sera, 853 lung tissues and 853 colorectal tissue specimens) were obtained. SARS-Cov and SARS-like-CoV were detected in these specimens using diagnostic kit for novel coronavirus N protein (ELISA), SARS-CoV Virus RNA detection kit, fluorescence PCR, Genchip, RT-PCR and cell isolation culture methods.

RESULTS AND CONCLUSIONNo SARS-CoV and SARS-like-CoV were detected in the 3043 samples, indicating the current absence of SARS-CoV and SARS-like-CoV in the bats captured in Guangzhou and its vicinity.

Animals ; China ; epidemiology ; Chiroptera ; virology ; Disease Vectors ; Enzyme-Linked Immunosorbent Assay ; Humans ; Nucleocapsid Proteins ; metabolism ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; isolation & purification ; metabolism ; Severe Acute Respiratory Syndrome ; epidemiology ; transmission ; virology

OBJECTIVETo study the influence of the mutants of hepatitis B surface antigen on the cell immunity.

METHODSThe recombinant plasmids of NS2Swt, NS2S126, NS2S133, NS2S141 and NS2S145 were transfected into Chinese hamster ovary (CHO) cells and the expressed proteins were detected by means of ELISA. Following PHA-activated lymphoblasts proliferation assay and IFN-gamma, IL-2, IL-10 induction assay were done with these proteins.

RESULTSIt was identified that these proteins of HBsAg could stimulate human lymphoblasts proliferation. Besides, there were no significant difference between the mutants and the wild. It was deserved to point out that the HBsAg with T126S mutation could increase the expression of IFN-gamma in the culture medium while the HBsAg with M133T mutation induced more expression of IL-10.

CONCLUSIONThe results suggested that the cellular immune response to mutants of HBV might not be strengthened or weakened. But it should not be ignored that HBV T126S or M133T mutation may assert a potential impact on the cell immunity.

Animals ; CHO Cells ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cricetinae ; Cricetulus ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B Surface Antigens ; genetics ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Lymphocytes ; cytology ; drug effects ; metabolism ; Mutant Proteins ; genetics ; Mutation ; Plasmids ; genetics ; Recombinant Proteins ; pharmacology ; Transfection

OBJECTIVETo establish an improved method for stereotactic location of the supraoptic nucleus in rats.

METHODSTwenty-four SD rats were randomly divided into experimental group (12 rats) and control group (12 rats) for oblique (20° to the left) stereotactic puncture (OSP group) and vertical stereotactic puncture (VSP group), respectively, both targeting the supraoptic nucleus (SON). The surgical data and postoperative (within 24) mortality of the rats were compared between the two groups.

RESULTSThe nucleus locating time was longer in OSP group than in VSP group (59.55∓3.64s vs 27.44∓2.18 s, P=0.000), and the postoperative mortality rate of the rats did not differ significantly between the groups (0 vs 44.4%, P=0.082). In OSP group, compared with VSP group, the procedure was associated with a lowered rupture rate of the superior sagittal sinus (11.1% vs 88.9%, P=0.003), a shortened hemostatic time after craniotomy (52.89∓24.05 s vs 157.445 ime a s, P=0.000) and after puncture (24.33 reas 45 s vs 133.89∓28.81 s, P=0.000), and also a shortened operation time (178.89 on tims vs 362.44 timees, P=0.000).

CONCLUSIONThe improved method for locating supraoptic nucleus in rats is convenient, stable and reproducible, and helps to avoid important blood vessels and specific nuclei according to the needs of different experiments and allows the operators to choose different surgical paths.

OBJECTIVETo obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis.

METHODSBicistronic expression vector P35-IRES-P40 was constructed for the simultaneous translation of IL-12 p35 and p40 cDNA subunit through internal ribosomal entry sites (IRES). pCI-dhfr-P35-IRES-P40 vector was constructed for expression in CHO-DHFR- cells. Positively cloned cells were screened by means of ELISA. Pools of clones with increased expression of IL-12 could be generated by selection in methotrexate. To determine the biological activities of rhIL-12, PHA-activated lymphoblasts proliferation assay and IFN-gamma induction assay were used in this study.

RESULTSGenetically engineered cells expressing hIl-12 were obtained and all the cell lines showed the stabile expression of rhIL-12 in high efficiency and good growth properties.

CONCLUSIONrhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.

Animals ; Blotting, Western ; CHO Cells ; Cell Proliferation ; drug effects ; Cricetinae ; Cricetulus ; Dose-Response Relationship, Drug ; Genetic Vectors ; genetics ; Humans ; Interleukin-12 ; biosynthesis ; genetics ; pharmacology ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; metabolism ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; Transfection

OBJECTIVETo observe the clinical efficacy of integrative medical therapy in treating post-craniocerebral traumatic mental disorder (PCT-MD).

METHODSSixty patients with confirmed diagnosis of PCT-MD were randomly assigned to the treated group and the control group equally. All were treated by conventional comprehensive Western medicine, but to patients in the treated group, modified Xufu Zhuyu Decoction (XFZY) was additionally given and the therapeutic course for both groups was 20 days. Changes in mental symptoms were observed and recorded on the 10th and 20th day and clinical efficacy as well as cranial CT image was estimated after termination of the treatment.

RESULTSThe clinical effective rate in the treated group and the control group was 96.67% and 83.30% respectively. Comparison between them showed significant difference (P<0.05). Significant differences were also shown in the comparisons between the two groups in improving mental symptoms, either on the 10th or on the 20th day (P<0.05 and P<0.01 respectively), and in post-treatment cranial CT image (P<0.05).

CONCLUSIONBetter efficacy could be obtained by integrative medical therapy in treating PCT-MD.

Adult ; Aged ; Brain Injuries ; drug therapy ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; adverse effects ; therapeutic use ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; adverse effects ; Mental Disorders ; drug therapy ; Middle Aged ; Tomography, X-Ray Computed ; Treatment Outcome