Thymopolypeptides combined with matrine type alkaloids suppress HBV replication / 中国中药杂志

To investigate the antiviral effect of thymopolypeptides combined with 4 kinds of matrine type alkaloids on HepG2.2.15 cells, oxymatrine, sophocarpidine, sophocarpine, and sophoridine (at concentration of 0.2 mmol•L⁻¹ respectively) were respectively combined with thymopolypeptides (0.025, 0.1 g•L⁻¹), and after 48 h and 72 h treatment on HepG2.2.15 cells, the cells and supernatants were collected. The cells activity in various groups was determined by CCK-8 method to evaluate the toxic effects of the drugs on HepG2.2.15 cells. Enzyme linked immunosorbent assay (ELISA) was used to determine HBeAg and HBsAg levels in cellular supernatants. HBV DNA levels in cellular supernatants andcells were quantified with fluorogenic quantitative PCR method; and the expression level of IFN-α in supernatants was detected with CBA method. The results indicated that single thymopolypeptides at 0.025-0.4 g•L⁻¹ had no toxicity to cells. Thymopolypeptides in this concentration range combined with 0.2 mmol•L⁻¹ matrine type alkaloids also had no toxicity to cells. Anti-HBV activity of drug combination was better than that of alkali or thymopolypeptides alone. Thymopolypeptides at 0.025 g•L⁻¹ had better inhibitory effect than thymopolypeptides at 0.1 g•L⁻¹ on intracellular HBV DNA expression, but the inhibitory effect on supernatant HBeAg level was on the contrary. Anti-HBV activity was similar between alkaloids combined with 0.1 g•L⁻¹ and alkaloids combined with 0.025 g•L⁻¹. There was no statistical difference in anti-HBV effect between various combined groups (P<0.05). In general, 72 h anti-HBV effect was better than 48 h anti-HBV effect (P<0.05). The expression of IFN-α was increased after drug combination, with positive correlation to the changes of other four indicators (P<0.05). In conclusion, oxymatrine, sophocarpidine, sophocarpine and sophoridine combined with thymopolypeptides could inhibit HBsAg and HBeAg secretion in HepG2.2.15 cells and HBV DNA replication, and further promote the antiviral effect by promoting the expression of IFN-α.

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 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 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>

Estimation of liver inflammation and necrosis in patients with chronic hepatitis B through different biochemical items / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To establish a new grading system to evaluate liver inflammation and necrosis in patients with chronic hepatitis B through clinical biochemical assays.</p><p><b>METHODS</b>Clinical and pathological data were collected from 255 cases with chronic hepatitis B. 19 biochemical items were analyzed and 5 items were selected for our grading system. Each of the five items was scored 0 to 4 based on the different values. The extent of liver inflammation and necrosis was evaluated according to the total score.</p><p><b>RESULTS</b>ALT, AST, ChE, GGT and TBA were selected for our grading system. The grade of liver inflammation and necrosis was considered less than 2.0 if total score lower than 6, and higher grade was considered with higher total score. The estimated results shared an identity of 82.8% with the real grades of liver inflammation and necrosis. When this grading system was applied to patients with liver inflammation and necrosis equal to or higher than grade 2.0, it exhibited a sensitivity of 83.8%, a specificity of 81.2%, a positive prediction value of 88.6%, a negative prediction value of 74.2% in all cases, and 88.0%, 84.7%, 90.6%, 82.4% respectively in patients older than 12 years.</p><p><b>CONCLUSION</b>Our data suggest that the grading system can be used to evaluate the extent of liver inflammation and necrosis in patients with chronic hepatitis B.</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>

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>

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>

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>

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>

Prokaryotic expression and purification of human immunodeficiency virus p24 antigen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express and purify the HIV p24 gene in E. coli cells, and identify p24 antigen activity.</p><p><b>METHODS</b>The full length gene fragment of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E. coil, the purified p24 protein was prepared by metal-ligand affinity chromatography (IMAC). The accuracy of inserted gene and activity, specificity of HIV p24 proteins were detected by two enzymes digestion technology, SDS-PAGE, Western Blot (WB) and ELISA.</p><p><b>RESULTS</b>The length of the HIV p24 gene fragment was 690 bp after digesting the recombinant plasmid T-p24 and pRSET-p24 with BamH I and Hind III. The expressed proteins had a single expected band of about 24 x 10(3) in SDS-PAGE. The specificity and activity of p24 protein were tested by WB and ELISA.</p><p><b>CONCLUSION</b>The HIV p24 sequence from HIV-1 gene plasmid hasbeen expressed in E. coil. This protein possessed good specificity and activity.</p>

Study on the response of specific antibodies against SARS-CoV in patients infected with SARS / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the response of specific antibodies against severe acute respiratory syndrome (SARS)-CoV in patients infected with SARS.</p><p><b>METHODS</b>IgM-capture, indirect and antigen-sandwiched enzyme linked immunosorbent assay (ELISA) were used to detect the SARS-CoV specific IgM, IgG and total antibodies in sera of clinical SARS patients or non-SARS individuals.</p><p><b>RESULTS</b>The positive rates of IgM, IgG and total antibodies to SARS-CoV in 146 sera of SARS patients collected in different phases of the disease were 61.64%, 53.43% and 69.86%, respectively. The earliest detectable days after onset of the disease for IgM and IgG to SRAS-CoV were 7 and 12 days, respectively. The specific IgM disappeared as early as 42 days after the onset of SARS. Of 70 sera from hepatitis A patients, 2 showed false positive results, while 127 sera from other patients were all negative, detected by the 3 methods. Serum from one medical worker who had been close contact to SARS patients was positive for anti-SARS-CoV IgG and total antibodies. These 3 methods used for detection were all not influenced by rheumatoid factor (RF).</p><p><b>CONCLUSION</b>All of the three methods were specific and sensitive for the detection of specific antibodies to SARS-CoV, and useful for epidemiological research and clinical diagnosis, but not for early diagnosis of SARS.</p>