-
[show abstract]
[hide abstract]
ABSTRACT: OBJECTIVE: To develop and evaluate a novel system for detecting and quantifying hepatitis A virus (HAV) nucleic acid. METHODS: Real-time TaqMan® reverse transcription-polymerase chain reaction (PCR) procedures were established, based on amplification of the highly conserved 5'-non-coding region. Synthetic single-stranded RNA transcripts synthesized in vitro were used as the quantification standard. Ten-fold dilutions were prepared from HAV strain stock suspension to determine precision, accuracy, sensitivity and specificity. In addition, serum specimens from patients with acute HAV underwent clinical evaluation. RESULTS: The novel assay had a detection limit for HAV RNA of 10 TCID50/ml (where TCID50 is median tissue culture infective dose). It was more sensitive and specific than the commercial quantitative PCR kit manufactured by Shanghai Zhijiang Bio-Tech. However, the Artus HAV RT-PCR kit (Qiagen) had the best performance of the three assays and had a detection limit of 5 TCID50/ml. The new HAV real-time PCR detection system was also successfully applied in 90 serum specimens from patients with confirmed acute HAV infection. CONCLUSION: Considering its high reproducibility, sensitivity, specificity and simplicity, this novel amplification system may be suitable for wide clinical application as a diagnostic tool, and for the surveillance and investigation of infectious diseases in developing countries where HAV is endemic.
The Journal of international medical research 03/2013; · 0.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering.
Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software.
SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene.
Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 10/2012; 26(5):335-7.
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the seroprevalence of hepatitis D virus in Foshan of Guangdong province, to provide the data for the study about it in China.
ELISA kits from two different companies were used for detecting anti-HDV IgG of all the serum samples, and then RT-PCR was carried out about the selected serum to ensure the results. All the serum samples were collected in 2011 in The First People's Hospital of Foshan.
The results from two ELISA kits and RT-PCR were identical. Eight samples were positive.
The seroprevalence rate of HDV in Foshan is higher than that in China. It has no statistically significant difference between female and male. Morever, the older with HBsAg are susceptible to HDV.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 10/2012; 26(5):382-3.
-
Changhong Huang,
Feng Qiu,
Minzhuo Guo, Yao Yi,
Liping Shen,
Feng Wang,
Zhiyuan Jia,
Jingchen Ma,
Yuliang Zhao,
Shuang Zhang,
Yong Zhang,
Shengli Bi
[show abstract]
[hide abstract]
ABSTRACT: Illegal commercial plasma and blood donation activities in the late 1980s and early 1990s caused a large number of hepatitis C virus (HCV) infections in rural areas of China.
A cross-sectional survey was carried out in 2008, in which all residents in a former blood donation village in rural Hebei Province were invited for a questionnaire interview and testing for HCV antibodies. Questionnaires were administered to collect information about their personal status and commercial blood donation history, and HCV antibodies were tested by enzyme immunoassay.
Of 520 villagers who participated in the interviews, 236 (45.4%) reported a history of selling whole blood or plasma. HCV seropositivity was confirmed in 148/520 (28.5%) interviewees and 101/236 (42.8%) former commercial plasma and blood donors. Selling plasma was the strongest independent predictor of HCV seropositivity (p=0.0037). Past history of an operation was also independently associated with HCV infection (p=0.0270).
Unsafe practices during illegal plasma donation led to a high risk of HCV seropositivity for donors during the late 1980s and early 1990s. Many infected people suffered chronic hepatitis from that time onwards and urgently needed treatment and care.
International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases 07/2012; 16(10):e731-4. · 2.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Background/Aims: Ras homologue A (RhoA) plays a crucial role in the proliferation, apoptosis, adhesion and migration of gastric cancer cells. Rho associated kinase (ROCK) is an effector protein of RhoA. Methodology: In the present study, RhoA activity was inhibited by siRNA targeting RhoA and Y-27632, an inhibitor of ROCK, and the role of RhoA/ROCK signaling pathway in the apoptosis of gastric cancer cells was investigated. Results: RNAi of RhoA inhibited the survival and promoted the apoptotic of AGS cells. RhoA RNAi caused an obvious decrease of ROCK1 expression but an increase of caspase-3/cleaved-caspase-8. Inhibition of ROCK by Y-27632 inhibited the activity of RhoA and promoted the apoptosis. Conclusions: We speculate that RhoA/ROCK signaling pathway plays an important role in the regulation of apoptosis of gastric cancer, and to inhibit this pathway may promote the apoptosis of cancer cells. Thus, inhibition of RhoA/ROCK signaling pathway may become a novel target in the treatment of gastric cancer.
Hepato-gastroenterology 05/2012; 59(120). · 0.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 05/2012; 28(3):201-6.
-
[show abstract]
[hide abstract]
ABSTRACT: Hepatitis C virus (HCV) is an important causative agent of acute and chronic hepatitis worldwide. We prepared a fusion protein in the vector of pET-11d that included three conserved broadly neutralizing B-cell epitopes and a series of T-cell epitopes located in the HCV NS3 region. In vivo administration of this fusion construct resulted in specific CD8+ cytotoxic lymphocytes in both PBMCs and splenocytes that could recognize specific antigen sites that could be detected by FACS. An HCVcc system was established and applied to detect HCV-specific neutralizing antibodies. These results suggest that the multi-epitope fusion protein is immunogenic and can elicit both humoral and cellular immune responses. In particular, this fusion protein is able to elicit HCV-specific neutralizing antibodies, which are critical for viral clearance. This construct may be significant for vaccine development and could be a potential candidate to be included in the design of a prophylactic and therapeutic vaccine against HCV.
International Journal of Molecular Medicine 09/2011; 29(1):12-7. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Obtain the hepatitis C virus high purified subunit fusion protein and detect its immunogenicity.
With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21 (DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein. At the same time, the sera were collected and prepared from the immunized experimental animals in order to investigate the immunogenicity of the protein by EIA.
High purified hepatitis C virus subunit fusion protein was obtained successfully. The EIA indicated that the fusion protein could elicit specific antibodies in the animals with very high titers.
The hepatitis C virus subunit fusion protein expressed in prokaryotic system was proved to have strong immunogenicity. It could provide some helpful and useful information to the hepatitis C virus prophylactic and therapeutic vaccine development.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 04/2010; 24(2):113-5.
-
[show abstract]
[hide abstract]
ABSTRACT: To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface.
Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody.
The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA.
The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 02/2010; 24(1):30-2.
-
[show abstract]
[hide abstract]
ABSTRACT: To determine the main genotype of hepatitis B virus (HBV) in Xinjiang.
200 HBsAg positive serum specimens were detected from more than 2000 serum of Xinjiang inhabitants, and HBV S gene was detected by using nPCR amplifying, and compared with the standard S region HBV nucleotide sequences of genotypes A-H retrieved from GenBank, then analyzed and drawn the polygenetic tree by MEGA3 software.
Gene in 127 (63.5%) serum specimens was detected from 200 samples. Among 127 serum specimens, 10 (7.8%) was genotype B, 58 (45.7%) was genotype C, and 59 (46.5%) was genotype D.
Genotype B, C and D have been found in Xinjiang.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 10/2009; 23(5):346-8.
-
[show abstract]
[hide abstract]
ABSTRACT: Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.
Biochemical and Biophysical Research Communications 10/2009; 390(4):1126-8. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To prepare monoclonal antibodies (McAbs) against VP1 capsid protein of Enterovirus 71.
Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, were synthesized respectively. Immunized BALB/c mice with the synthetic peptides to establish the hybridoma cell strains secreting specific McAb to VP1. After the specific McAbs were prepared, identified and analyzed the titer by indirect ELISA assay. The positive clones were selected and their neutralization titer were determined by neutralization test.
Two high titered anti-VP1 antibodies secreted by the hybridoma cells showed good neutralization reaction with enterovirus 71 on RD cells, and the neutralization titer were 1:8 and 1:16 respectively.
Two high titered anti-VP1 antibodies, with good neutralization activity, secreted by the hybridoma cells, which lays the foundation for further study.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 02/2009; 23(1):62-4.
-
[show abstract]
[hide abstract]
ABSTRACT: To construct the programmed cell death 1 ligant 1 (PD-L1) recombination expression vector, express the fusion protein in prokaryotic and analyze the biological action of express product.
The whole PD-L1 gene sequence was synthesized after codon optimized. Construct the thioredoxin-(PD-L1) recombination expression vector and express the fusion protein in E. coli. Purified the target protein and analyze the conjugated ability of protein by ELISA.
The PD-L1 recombinant expression vector has been constructed correctly. The target protein has been obtained with which expressed in high efficiency and production. The target protein can conjugate specifically with the PD-1, its specific receptor.
We have obtained the PD-L1 recombinant protein success with high biological activity. The result provide the basic condition for further study on antibody and mutually action between PD-L1 and chronic virus infectious.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 02/2009; 23(1):5-7.
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate whether tacrine and donepezil can prevent apoptosis induced by Lipopolysaccharides.
Phase-contrast microscopes was used to observe the morphological changes of Vero cells. Cell counting kit-8 was used to measure cell survival vitro. DNA fragment was analyzed by agarose gel electrophoresis to determine the apoptotic biochemical changes.
Vero cells treated with 400 microg/ml and 500 microg/ml Lipopolysaccharides exhibited cell apoptosis. 10 micromol/L tacrine provided protective effect to 500 microg/ml Lipopolysaccharides induced cell apoptosis measured by Phase-contrast microscopes, cell counting kit-8 and DNA fragment analyze. However, donepezil did not show any protective effect of the apoptosis induced by 500 microg/ml Lipopolysaccharides.
Lipopolysaccharides can induce apoptosis in Vero cells to built an apoptotic model in vitro. Tacrine rather than donepezil can inhibit Lipopolysaccharides induced apoptosis in Vero cells.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 01/2009; 22(6):458-60.
-
[show abstract]
[hide abstract]
ABSTRACT: To apply recombinant rubella virus envelope protein-1 (E1) to detect human rubella virus IgG antibody.
Rubella virus E1 protein was expressed in E. coli, purified E1 protein was used as the antigen for the detecting of anti rubella in human sera in the way of enzyme linked Immunosorbant assay (ELISA).
The antigenicity of the recombinant protein was checked by WHO rubella sera panel. We detected 200 sera samples, which came from Guangxi Guilin. 93% of these samples were positive.
The antigenicity of recombinant E1 is a satisfied candidate antigen for the detecting of human rubella virus antibody. The prevalence of anti rubella virus IgG in Guangxi is 93%. It is at the some level compared with other provinces in China.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 11/2008; 22(5):382-4.
-
[show abstract]
[hide abstract]
ABSTRACT: To determine the main genotype of hepatitis B virus (HBV) prevailed in Henan Luohe area.
Serum specimens were collected from 94 HBsAg positive individuals, and HBV S gene were obtained by PCR amplifying, and the gene sequences were analyzed and the polygenetic tree was drawn by the software MEGA3.
About 75.7% samples of HBV S gene clustered in genotype C, about 20% samples clustered at genotype B in the HBV polygenetic tree, about 4.3% samples clustered at genotype D in the HBV polygenetic tree.
The main genotype of hepatitis B virus prevalent in Henan Luohe is genotype C, genotype B is rarely seen, and genotype D is rarely seen.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 09/2008; 22(4):257-9.
-
[show abstract]
[hide abstract]
ABSTRACT: To study the antigenic properties of mutant hepatitis B virus surface antigen, to understand the sensitivity of the commercially available HBsAg assays to the variants and to reduce the undetectability of the variants.
Recombinant eukaryotic expression plasmids for HBsAg. The recombinant eukaryotic expression plasmids pSS1adr, pSS1adw2, pSS1adw2- 145Arg, pSS1adr-126 Asn and pSS1adr-126Ser were transfected into COS-7 cells. HBsAg in the supernatants of transfected cells was detected by using different commercial ELISA kits.
The absorbance value of pSS1adr-126 Asn and pSS1adr-126Ser plasmids were similar to that of the wild type HBsAg, the absorbance value of pSS1adw2-145Arg plasmids was lower than that of the wild type HBsAg.
It is estimated that the antigenicity of HBsAg mainly depended on the amino acid sequence of "a" antigen determinant and its conformation, so 145 amino acid substitutions led to the change of conformation and the antigenicity of variant HBsAg was lower than that of the wild type.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 03/2008; 22(1):48-50.
-
[show abstract]
[hide abstract]
ABSTRACT: To clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.
The human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing.
The PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing.
The human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 03/2008; 22(1):33-5.
-
[show abstract]
[hide abstract]
ABSTRACT: To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis.
Bicistronic 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.
Genetically 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.
rhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 10/2007; 21(3):235-7.
-
[show abstract]
[hide abstract]
ABSTRACT: To express P1B, P2A, P3AB and P3D cDNA gene fragments in prokaryotic system using thioredoxin fusion expression system; to investigate the antigenicity and application of recombinant protein.
By using PCR technique, P1B, P2A, P3AB and P3D gene fragments were cloned. Choosing M47 as the expressive vector, the recombinant plasmid P1B, P2A, P3AB and P3D was constructed and expressed in Escherichia coli after inducing by IPTG. By anion exchange and affinity chromatography, purified recombinant protein was obtained. By using Western Blot analysis and indirect ELISA to detect its antigenic activity.
Four recombinant plasmids was proved to be constructed successfully by sequencing and the correct molecular weight of their expression products. Recombinant proteins were obtained in BL21 (DE3) and purified after Ni2+ affinity chromatography. Western Blot analysis and indirect ELISA showed that P2a had specific antigenicity.
The P2a protein expressed in prokaryotic system was proved to have specific antigenicity. The indirect ELISA distinguished 24 positive sera from 24 negative sera. It is very likely that P2a can be an antigen to diagnose acute patients of hepatitis A and differentiate inactivated vaccine-induced immunity from an infection.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 04/2007; 21(1):50-2.
