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ABSTRACT: To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 01/2013; 29(1):7-11.
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ABSTRACT: The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 05/2012; 28(3):231-6.
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ABSTRACT: To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.
M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.
The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.
These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 08/2011; 25(4):254-7.
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ABSTRACT: To construct vectors expressing M2 and NA genes of H5N1 influenza virus.
Based on the human H5N1 avian influenza virus (A/Anhui/1/2005) isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids.
Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA.
Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 06/2011; 25(3):167-9.
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ABSTRACT: In order to study the anti-obesity ability and inhibition towards fatty acid synthase (FAS) of the extract, a 70% ethanol extract of Acer truncatum Bunge (AT) leaves was further extracted with ethyl acetate. FAS is a very significant lipogenic enzyme, participating in energy metabolism in vivo; it has also been observed that FAS inhibitors might be potent anti-obesity agents. Experimental results on animals showed that the extract significantly reduced food intake and adipose, and effectively controlled weight evolution. Lipogenesis inhibition might be regarded as one of the reasons for the weight control and adipose reduction by AT. The extract was further isolated using a series of column chromatography that yielded 10 known compounds. 1,2,3,4,6-Penta-O-galloyl-β-D-glucose was found to be one of the major active constituents in the extract of AT.
Natural product research 02/2011; 25(4):422-31. · 1.01 Impact Factor
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ABSTRACT: To describe the epidemiologic characteristics and clinical manifestations of 59 persons recruited via an internet chat group who complained of AIDS-like symptoms, so as to formulate effective intervention strategies and measures.
Case was defined as onset of any three of the following self-reported AIDS-like symptoms in a member of relevant "internet chat groups": persistent low grade fever, rash, swollen lymph node, fatigue, diarrhea, weight loss and low CD4(+)T count. We administered an internet-based questionnaire, and invited 59 of the 88 case-persons for voluntary physical examination and laboratory testing.
The 59 case-persons came from 22 provinces; 54 (91.5%) were men; the median age was 34 (range: 22-53) years; 84.7% of them had high-risk sexual behaviors before the onset of self-reported symptoms. The median time interval from exposure to onset was 15 d (range: 1-365 d). Blood specimens for all the 59 case-persons were tested negative for HIV and syphilis antibodies. There was also no evidence of Xenotropic murine leukemia virus-related virus infection. One case-person was tested positive for hepatitis C virus antibody. The average CD4(+)T lymphocyte count was 707/µl. Of the 59 case-persons, 57 (96.6%) sought medical care from multiple providers; 40 were diagnosed to have no physical disorders.
None of the 59 case-persons had any evidence of infection with HIV or any other infectious agents that could explain their self-reported symptoms.
Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 12/2010; 31(12):1379-82.
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ABSTRACT: M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 05/2010; 26(3):189-94.
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ABSTRACT: To prepare monoclonal antibodies specific for M1 protein of H5N1 subtype human influenza virus, this work may provide new tool in rapid diagnosis and study of type A influenza virus.
BALB/c mice were immunized with purified recombinant H5N1 (A/Anhui/1/2005)/M1 protein expressed in E. coli. Spleen cells of the immunized mice were fused with sp2/0 cells to produce hybridoma cell lines. ELISA was performed to identify the monoclonal antibody against M1 protein of H5N1. Immunofluorescence assay (IFA) were applied to identify the specificity of these antibodies.
Three hybridoma cell lines steadily secreting anti-H5N1/M1 McAb were obtained, and their cross reactivity was confirmed by cross-reaction test and IFA.
Monoclonal antibodies immunized with H5N1 subtype influenza virus M1 protein are cross-reactive, which can be used to detect different subtype of influenze virus type A.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 12/2009; 23(6):412-4.
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ABSTRACT: Virus-like particles (VLPs) structurally mimic the authentic virus whereas contain no viral genome. VLPs technique plays an important role in basic research such as virus assembly and virus morphology diversity. With special immunology properties, VLPs vaccine can induce immune response effectively. VLPs can act as adjuvant by regulating dendritic cells. Other adjuvant or polypeptide can be integrated into VLPs to construct chimeric vaccines. With the capability of packaging nucleic acid or other small molecules, VLPs can be used for vehicles to deliver these substances under suitable conditions. VLPs can substitute natural virus in immunology assay.
Zhongguo ji hua mian yi = Chinese journal of vaccines and immunization 04/2009; 15(2):167-73.
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ABSTRACT: Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 04/2009; 25(2):107-12.
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ABSTRACT: To optimize a technical process for extracting the polyphenols that can inhibit fatty acid synthase by ethanol from the leaves of Acer truncatum.
The extracting time, extracting temperature, extracting solvent and the ratio of solvent to raw material were studied by L16 (4(5)) orthogonal experiments. The extraction rate of polyphenols and the inhibitory efficiency on the fatty acid synthase were utilized as evaluating criteria, and the optimum conditions for the extraction were obtained through summing up the above factors.
The efficient technological conditions were as follows: the concentration of ethanol was 60% (v/v), the ratio of solvent to raw material was 1 g: 30 mL; and the stirring time was 1.5 h at 50 degrees C.
The polyphenols that extracted from the leaves of Acer truncatum have inhibitory activity against fatty acid synthase and some of them have stronger inhibitory effect.
Zhong yao cai = Zhongyaocai = Journal of Chinese medicinal materials 03/2009; 32(2):283-6.
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Li-Fang Huan,
Li-Hong Yao,
Ai-Jun Chen,
Cong-Sheng Cheng,
Run-Qing Jia,
Qing Tian,
Hong Bo, Jian-Qiang Guo,
Min Wang,
Yue-Long Shu,
Zhi-Qing Zhang
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ABSTRACT: Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 10/2007; 23(5):366-70.
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ABSTRACT: The gene encoding a extremely thermostable and acid-stable alpha-Amylase was amplified by PCR using hyperthermophilic archaebacterium pyrococcus furiosus genomic DNA as template. Then the gene was cloned into the vector of pPIC9K. The recombinant vector pPIC9K-amy was then transformed into E. coli DH5alpha strain. Sequencing test showed that the a-amylase gene cloned consisted of 1305 base pairs and the mature protein encoded by the gene consisted of 435 amino acids. The recombinant vector was transformed into chromosome of methylotrophic yeast Pichia pastoris GS115 strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant a-Amylase was expressed and excreted out of the cells. The expression of the recombinant alpha-amylase was strictly induced by methanol. As induction time increased, the activity of amylase per milliliter medium went up accordingly. After 7 days induction, the activity of the amylase reached the max. The recombinant alpha-amylase exhibited maximal activity at 90 to approximately 100 degrees C and at pHranging from 4.5 to 5.0. The enzyme is so thermostable that after disposed at 100 degrees C for 5 hours over 60% of activity was retained.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2006; 22(2):237-42.
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ABSTRACT: Extremely thermostable and acid-stable a-amylase produced by Pichia pastoris GS115/pPIC9K-Amy-228 was purified to electrophoretic homogeneity by the steps of ultrafiltration and PAGE. Purification of about 11.7 fold was achieved with an overall yield of 29.8%. Its molecular weight was estimated to be about 55kD by SDS-PAGE. The isoelectric point was 5.0 (room temperature). Michaelis constant of the enzyme for soluble starch was 1.12g/L. The carbohydrate content was 15.4% by the phenol-sulfuric acid method. The optimum temperature and pH of the enzyme activity were 95 degrees C and 4.5 respectively. The enzyme activity was stable under room temperature in the pH rang of 4.0 - 7.0 for 48 hours. About 60% of the initial enzyme activity was measured after 1h of incubation at 110 degrees C. The activity was strongly inhibited by Fe2+, Cr2+ and Cu2+, While Ca2+ had no effect on it. DTT and EDTA had no effect on the activity.
ACTA MICROBIOLOGICA SINICA 09/2005; 45(4):547-50.
