Cloning and expressing of tissue inhibitor of metalloproteinases I gene fragment and preparation of monoclonal antibodies against the recombinant protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.</p><p><b>METHODS</b>TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.</p><p><b>RESULTS</b>The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.</p><p><b>CONCLUSION</b>The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.</p>

Analysis of hemagglutination inhibition antibody level in patients with influenza A H1N1 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the hemagglutination inhibition antibody level in patients with influenza A H1N1.</p><p><b>METHODS</b>Sera from 28 patients with influenza A H1N1 at different time points after illness onset were collected and measured by hemagglutination inhibition assay.</p><p><b>RESULTS</b>The serum hemagglutination inhibition antibody titers at 1, 5, 15, 22, 37, 49 and 58 days after illness onset were 5.36, 9.39, 39.02, 57.99, 137.92, 55.19 and 57.99 respectively. The top geometric mean titer of hemagglutination inhibition antibody was 148.55. The antibody seroconversion rate and seroprotection rate were occurred in 96.4% (27/28) of patients.</p><p><b>CONCLUSION</b>The patients with influenza A H1N1 have effective immune response.</p>

Construction of recombinant plasmid expressing S1 gene of new type of reovirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the recombinant plasmid containing S1 gene of new type of reovirus, and to study the expression of protein sigma1 in Vero cells.</p><p><b>METHODS</b>The recombinant plasmid, named pC-S, was constructed by cloning S1 gene into vector pCAGGS/MCS. Then Vero cells were transfected with pC-S and collected at 24, 48, 72 h post transfection followed by SDS-PAGE and Western-Blot assay.</p><p><b>RESULTS</b>Results both SDS-PAGE and Western-Blot assay indicated that sigma1 protein could be expressed well and the highest expression level was 72 h post transfection.</p><p><b>CONCLUSIONS</b>Sigma1 protein could be expressed well in Vero cells by transfected with recombinant plasmid containing S1 gene, and could give some implications for subsequent research on virus-host interactions.</p>

Clinical study on hybrid bioartificial liver supporting system for acute on chronic liver failure patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct an hybrid bioartificial liver supporting system, and observe its effectiveness and safety on patients with acute on chronic liver failure.</p><p><b>METHODS</b>Hybrid bioartificial liver supporting system (HBALSS) was constructed using bioreactor with HepG2 cells transfected with human augmenter of liver regeneration (hALR) gene. 12 acute on chronic liver failure patients were divided into 2 groups randomly. The treatment group was treated with the hybrid bioartificial liver support system. The group underwent plasma exchange was used as control.</p><p><b>RESULTS</b>In the treatment group, four patients recovered, one patient died of hepatic encephalopathy, one patient died of hepatorenal syndrome, one patient recovered, but died of gastrointestnal bleeding after 1 year. In control group, two patients recovered, one patient underwent orthotropic liver transplantation, and three patients died of liver failure.</p><p><b>CONCLUSION</b>The hybrid bioartificial liver supporting system with HepG2 cell line was established successfully and have certain safety and effectiveness on acute on chronic liver failure patients.</p>

Construction and identification of attenuated Salmonella which harboring enterovirus 71 VP1 gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop attenuated Salmonella which harboring enterovirus 71 (EV71) VP1 gene.</p><p><b>METHODS</b>The plasmid which expressed VP1 protein of EV71 was constructed by gene recombination. Cellular expression was assessed by Western Blot analysis. The recombinant plasmid was then transformed into attenuated Salmonella SL7207.</p><p><b>RESULTS</b>EV71 VP1 gene sequence was inserted into a eukaryotic expression plasmid VR1012. VP1 protein was detected by Western Blot analysis in the culture supernatant. And the attenuated Salmonella harbored the plasmid stable.</p><p><b>CONCLUSION</b>The plasmid was constructed successfully and it can express effectively in vitro. The bacteria which harboring the plasmid were constructed successfully. This has provided a basis for further research of an oral EV71 vaccine.</p>

Construction and identification of a vector inserted with gene of T7 RNA polymerase / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a system to rescue virus by intracellular expression of T7 RNA Polymerase.</p><p><b>METHODS</b>The gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-1a was then identified. VR-1a and EV71 infectious plasmid were co-transfected in Vero cell. CPE was observed and viral gene viral antigen were detected.</p><p><b>RESULTS</b>The gene of T7 RNA Polymerase was successfully cloned into vector VR1012. Vero cell developed to CPE after being transfected VR-1a and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA.</p><p><b>CONCLUSION</b>The method can be used to rescue virus. It could apply to immunologic research of EV71 DNA vaccine.</p>

Construction and identification of a vector inserted with complete genome of poliovirus strain Sabin I / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a vector inserted with complete genome of poliovirus strain Sabin I.</p><p><b>METHODS</b>The 3 fragments of the complete genome of Sabin I was amplified and cloned to pEASY-T3 by molecular biological technology. These cloned pEASY-T3 were then digested by Restriction enzymes and ligated to pWSK29 step by step and identified.</p><p><b>RESULTS</b>The complete genome of poliovirus strain Sabin I was successfully cloned into vector pWSK29 with 9 nucleotide mutations.</p><p><b>CONCLUSION</b>The complete genome plasmid was constructed and it provided a basis for further research of the function of Sabin I.</p>

Construction and experimental study on off-line hybrid bioartificial liver supporting system with human liver cell line / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct an off-line hybrid bioartificial liver supporting system with human liver cell line, and study it's effect on the plasma from patients with liver failure.</p><p><b>METHODS</b>We established the bioreactor using Psu-2s (Fresenius) cultured with Hep G2 cell transfected with human augmenter of liver regeneration (hALR) gene, then constructed a hybrid bioartificial liver supporting system, at last using the bioartificial liver support system to purify the plasma treated 2 hours with serum bilirubin absorbent, separated from acute on chronic liver failure patients infected by hepatitis B virus.</p><p><b>RESULTS</b>Bioreactor was successful constructed. The cell viability in perigastrum of bioreactor is 85.2% and cell propagated rapidly. Before and after treating with bilirubin absorbent, serum total bilirubin was (176.19 +/- 54.14) micromol/L and (50.1 +/- 16.85) micromol/L respectively (P = 0.0002). While there were no significance difference in the level of albumin, urea and glucose. At the begin and end of treatment with bioartificial liver, serum total bilirubin was (50.10 +/- 16.85) micromol/L and (30.27 +/- 15.02) micromol/L respectively (P = 0.000), the urea and albumin increased, urea has significantly difference, but the change of albumin hasn't.</p><p><b>CONCLUSION</b>The off-line hybrid bioartificial liver supporting system with human liver cell line were builded successfully and have synthesis and metabolism functions for acute on chronic liver failure patients.</p>

Construction and expression of hepatitis B virus envelope protein combined with core protein with two multiple cloning sites vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a coexpression plasmid which expressing envelope protein and nucleoprotein of hepatitis B virus and know of its expressing efficiency.</p><p><b>METHODS</b>The plasmid coexpressing envelope protein and nucleoprotein of hepatitis B virus under the CMV promoter respectively was constructed by gene recombination. Cellular expression was assessed by ELISA.</p><p><b>RESULTS</b>Multiple cloning site was inserted into expression vector contain hepatitis B virus PreS2-S gene. And expression unit containing hepatitis B virus PreC-C was cloned into it. HBsAg and HBeAg was detected both in the culture supernatant and in the cells.</p><p><b>CONCLUSION</b>The coexpressing plasmid was constructed successfully and it can express effectively in vitro. This has provided a basis for further research of the therapeutic HBV DNA vaccine.</p>

Genome analysis of a newly isolated enterovirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To demonstrate molecular characterization of a newly isolated enterovirus.</p><p><b>METHODS</b>Virus were isolated from patient with unknown-causing disease and tested by reverse transcription-polymerase chain reaction (RT-PCR) and 5'3'RACE (rapid amplification of cDNA ends, RACE), in an attempt to obtain the sequence of this newly isolated enterovirus.</p><p><b>RESULTS</b>Sequence analysis showed that this newly isolated enterovirus shared 83%-94% nucleotide identity and 91%-100% amino acid identity with enterovirus 89. Phylogenetic analysis indicated that it was probably a new subtype of enterovirus 89.</p><p><b>CONCLUSION</b>This newly isolated enterovirus in the stool specimen from patient has the same serotype with enterovirus 89, but it was probably a new subtype of enterovirus 89.</p>

Level and clinical significance of soluble CD40 in patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the level and clinical significance of soluble CD40 in patients with chronic hepatitis B.</p><p><b>METHODS</b>Detecting the concentration of sCD40 from 176 cases with chronic hepatitis B by ELISA and analyzing its relationship with different grades of inflammation and necrosis in liver tissue.</p><p><b>RESULTS</b>sCD40 from patients with chronic hepatitis B was significantly higher than those from healthy. And that the concentration of sCD40 was positive correlation with severe clinical disease and liver inflammation and necrosis. In patients whose ALT lower than 80 IU/L and sCD40 higher than 80 pg/ml, it showed that 65.85% cases have high grade of liver inflammation and necrosis, which was significantly higher than patients with sCD40 lower than 80 pg/ ml.</p><p><b>CONCLUSION</b>The concentration of sCD40 is positively related with the grade of liver inflammation and necrosis. This information could help us to evaluate the status of chronic hepatitis B as an immunological index.</p>

Primry research of separation and culture of adult hepatocytes / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the separation and culture method of adult hepatocytes.</p><p><b>METHODS</b>The isolated adult hepatocytes were cultivated by RPMI 1640 medium at 37 degrees C in vitro. The characteristics of the growing hepatocytes were observed. Their synthesis of urea was detected. The transformation efficiency and density's change of lidocaine were analyzed.</p><p><b>RESULTS</b>Hepatocytes were successful separated from adult liver. And they were cultivated in common condition and hollow fiber reactor. The functional capacity of hepatocytes was for lidocaine metabolism and urea excretion.</p><p><b>CONCLUSION</b>The adult hepatocytes have been successful separated from liver. And they can be cultivated in common condition and hollow fiber reactor. And it could provide a great quantity and high activity of hepatocytes for bioartificial liver.</p>

Immune effects of mutated hepatitis B virus precore-core DNA vaccines in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the immune effect of DNA vaccines encoding mutated HBV pre-c/c gene (VE2,VE4) in mice.</p><p><b>METHODS</b>Three kinds of plasmid VEC(DNA vaccines encoding HBV pre-c/c gene), VE2 and VE4 were injected into the thigh muscles of different group of BALB/c mice.Blood and splenocytes from mice were isolated at 4 weeks after immunization. We also have mouse groups immunized with three of these plasmid combined with IFN-gamma gene plasmids. The anti-HBc and anti-HBe antibody in peripheral blood in mice were detected by enzyme linked immunosorbent assay (ELISA), antigen-specific cell immune responses were detected by CTL test and enzyme linked immunospot assay(ELISpot).</p><p><b>RESULTS</b>We found that anti-HBe titers of VE2 and VE4 immunizing groups are higher than VEC group (P < 0.05). We also observed that VE2 and VE4 could induce stronger antigen-specific immune responses than VEC and when combined with IFN-gamma plasmid,the antigen-specific immune responses are stronger than those without combination immunization in mice (P < 0.05).</p><p><b>CONCLUSIONS</b>The DNA vaccine VE2 and VE4 could induces stronger antigen-specific immune responses than VEC, and when combined with IFN-gamma plasmid,the antigen-specific immune responses are improved in mice.</p>

A five-year analysis of HBV mutations in a multidrug-resistant patient with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate HBV mutations in reverse transcriptase (RT) gene and precore/basal core promoter (PC/BCP) regions in a chronic hepatitis B patient and to analyze the link between the mutations and drug resistance or HBeAg sero-conversion.</p><p><b>METHODS</b>Eighteen serum samples were collected from a chronic hepatitis B patient during his 14 hospitalizations from June 2002 to September 2007. HBV DNA was extracted and nested PCR was employed for amplification of target gene fragments. Direct sequencing of PCR products was performed followed by analysis with NTI software. The significance of the results was analyzed in combination with the clinical data of the patient.</p><p><b>RESULTS</b>Several mutations were identified in succession, including LAM-resistant mutations M204I/V and L180M+M204V, ETV-resistant mutation S202G, and HBeAg nonsense mutation G1896A. The results were in accordance with the disease progression of the patient.</p><p><b>CONCLUSION</b>Sequencing of HBV RT and PC/BCP regions is valuable for comprehensively checking the viral mutations and thus it is helpful in the surveillance of patients in clinics as a way for adopting reasonable antiviral therapy.</p>

Construction and antigenic evaluation of a recombinant MVA virus-like particle expressing HBV C gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the virus-like parcel expressing hepatitis B virus (HBV) C gene and identify its immunogenicity.</p><p><b>METHODS</b>HBV C gene was cloned into the shuttle vector pSC11, and the resulted plasmid pSC11-C was transfected into modified vaccinia virus Ankara (MVA).</p><p><b>RESULTS</b>pSC11-C was correctly constructed as verified by sequence analysis and PCR, and the recombinant virus-like parcel possessed good immunogenicity.</p><p><b>CONCLUSION</b>The MVA-C expressing HBV C gene has been successfully constructed to provide important basis for gene therapy research of chronic HBV infection.</p>

Establishment and evaluation of the diagnostic kit for anti-HIV1/2 antibody and P24 antigen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish and evaluate an Enzyme Immunoassay diagnostic kit combined with anti-HIV1/2 antibody and P24 antigen for shortening the examination window period of HIV infection in HIV laboratory diagnosis.</p><p><b>METHODS</b>The enzyme-linked reaction plates was coated by anti-HIV P24 monoclonal antibody and HIV 1/2 antigen. Labeling HIV1/2 antigen and anti-HIV P24 polyclonal antibody with horseradish peroxidase, setup an integrated ELISA kit for detecting anti-HIV-1/2 antibody and HIV P24 antigen, and evaluate the specificity and sensitivity of this kit.</p><p><b>RESULTS</b>The sensitivity of testing P24 antigen was up to 0.2 ng/ml. 78 serum samples of patients with AIDS, 85 serum samples of healthy people were compared with Abbott EIA kit, the coincidence was 100%. 12 051 sera from normal persons and patients were examined, the sensitivity of 100 %and specificity of 99.62 %, respectively.</p><p><b>CONCLUSION</b>The anti-HIV1/2 antibody and HIV P24 antigen can be measured at the same time using this EIA kit, while the examination window period of HIV infection is shortened. Thus, the method is suitable for laboratory diagnosis and epidemiological investigation.</p>

Expression, purification, and characterization of an anti-human RBC ScFv-HIV gp160 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct and express anti-human RBC and HIVgp160 fusion protein for rapid detection of antibody to HIV.</p><p><b>METHODS</b>The gene of the anti human RBC ScFv and HIV antigen were constructed together into expression vector. The fusion protein was expressed in E. coli.</p><p><b>RESULTS</b>The fusion protein was proved to be able to bind both anti-RBC and HIVgp160. It could cause agglutination of human RBC when HIVgp160 was present.</p><p><b>CONCLUSION</b>The fusion protein has the potential in rapid detection of HIV.</p>

Preliminary study on determination of specific cellular immunity of patients with hepatitis B / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To evaluate an enzyme linked immunospot (ELISPOT) method for testing the specific cellular immunity of patients with hepatitis B and preliminarily investigate into the difference of cellular immunity in patients with various types of hepatitis B.</p><p><b>METHODS</b>The patients with acute hepatitis B, chronic hepatitis B liver cirrhosis, healthy persons with HBV vaccine immunization, healthy persons with past HBV infection and HBV naive persons were enrolled in this study. Their peripheral blood mononuclear cells were tested by ELISPOT to determine the number of gamma-interferon secreting cells.</p><p><b>RESULTS</b>The number of gamma-interferon secreting cells was significantly different between the patients with acute hepatitis B and those with chronic hepatitis B, and between the patients with acute hepatitis B and those with liver cirrhosis (P=0.0209 and P=0.0211).</p><p><b>CONCLUSION</b>The specific cellular immunity in the patients with hepatitis B could be evaluated by determining the number of gamma-interferon secreting cells with the method of testing their peripheral blood mononuclear cells by ELISPOT. The specific cellular immunity was stronger in the patients with acute hepatitis B than in those with chronic hepatitis B and liver cirrhosis.</p>

Identification and typing of human enteroviruses using an RT-PCR assay / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To establish a molecular detection and typing assay for identification and typing of human enteroviruses (HEV) which is suitable for clinical detection and epidemiologic research.</p><p><b>METHODS</b>Using both primers specific for HEV genus and HEV typing primers and reverse transcription polymerase chain reaction (RT-PCR) the authors detected preliminarily HEV by agarose gel electrophoresis and then identified serotype through nucleotide sequence analysis of RT-PCR amplicons. The monospecific antisera neutralization was applied to validate the typing results.</p><p><b>RESULTS</b>The serotype of 18 suspicious HEV samples was identified: 4 Coxsackievirus type A24 (CVA24), 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3 (E3), 1 E6, 1 E9, 1 E11, 1 E14, 1 E33 and 1 Rhinovirus type 9. The result was validated by monospecitive antisera neutralization.</p><p><b>CONCLUSION</b>This RT-PCR assay for HEV detection and typing may be suitable for clinical detection and epidemic research since this method is sensitive and specific for direct identification and typing of HEV.</p>

Follow up study on viruses associated with SARS among the SARS patients / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the existence status of the SARS-CoV, retrovirus, and the poliovirus in the bodies of the patients with SARS and the possible relationship between the three viruses and SARS.</p><p><b>METHODS</b>The clinical specimens of the nasopharyngeal swabs, sputum (or saliva), urine, fecal specimens were collected on three consecutive days from 8 patients with SARS 2 years after the recovery from SARS. SARS-CoV, reovirus and poliovirus RNA was detected by using reverse transcription (RT)-PCR; IgG antibody to the poliovirus type 1 and 3 and the antibody to SARS-CoV were determined using enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>All the specimens were negative for SARS-CoV and reovirus by RT-PCR, but the fecal specimens from 4 persons were positive for poliovirus. The sequences of these poliovirus were highly homologous to that of human poliovirus type 1 strain sabin 1 genome at nucleotide level, but back mutations have occurred in the primary attenuating mutation sites at nucleotide position 480 (G --> A) in the 5' UTR and the nucleotide position 2795 (A --> G). No SARS-CoV, reovirus, and poliovirus were found in the normal controls. Three serum specimens were positive for the antibody to SARS-CoV. The IgG antibody to poliovirus were detected in 4 SARS patients and 23 healthy persons. No positive results for antibody to SARS-CoV were detected in the 25 healthy persons.</p><p><b>CONCLUSION</b>The positive rate of the poliovirus antibody in the serum of SARS patients 2 years after recovery was significantly different from that of the normal controls, and the positive rate of poliovirus in the fecal specimens was still very high, and more importantly back mutations have occurred in the attenuating mutation sites at nucleotide position which plays an important role in the poliomyelitis.</p>