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Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris

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Chemical Research in Chinese Universities 2008年第3期24卷 316-321页

作者： ZHAO Li-hui YU Xiang-hui JIANG Chun-lai wu yong-ge SHEN Jia-cong KONG Wei Changchun University of Science and Techinology Changchun 130022 P. R. China College of Life Science for Supramolecular Structure and Materials Jilin University Changchun 130012 P. R. China State Key Laboratory for Supramoleeular Structure and Materials Jilin University Changehun 130012 P. R. China

To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes（850 amino acids） from Guangxi in China was inserted into Pichia pastoris expression vector pPICZαB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAge and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pastoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA.

关键词： Pichia pastoris env gene Glycoprotein Epitope Immunogenicity

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration(hALR), Expression and Purification of hALR

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Chemical Research in Chinese Universities 2006年第2期22卷 201-204页

作者： SUN Tian-xu wu yong-ge YU Xiang-hui JIANG Chun-lai JIN Ying-hua CHENG Yue KONG Wei Vaccine Research Center College of Life Science Jilin University Changchun 130012 P. R. China

Experimental evidence has been presented to suggest that the human augmenter of liver regeneration （hALR） serves as a hepatotruphic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by means of Western Blotting. The protein was purified by Ni-NTA affinity chrumatography and this FAD-dependent sulthydryl oxidase activity was measured.

关键词： Augmenter of liver regeneration Prokayotic expression plasmid FAD-dependent sulthydryl oxidase

Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

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Chemical Research in Chinese Universities 2007年第5期23卷 567-570页

作者： HE Kan CHEN Yue LI Jing-hua ZHAN Zhuo wu yong-ge KONG Wei JIN Ying-hua Vaccine Research Center College of Life Science Key Laboratory for Supramolecular Structure ＆ Materials of Ministry of Education School of Public Health Jilin University Changchun 130012 P. R. China

Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various *** determine a specific substrate of Cyclin A-Cdk2 for apoptosis,in this study,we carried out an in vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source,and recombinant protein GST-Bad as a *** study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in *** examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo,we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn,a dominant negative version of Cdk2,in Hela cells and determined the phosphorylation status of protein *** test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock ***,etoposide also caused the phosphorylation of endogenetic *** conclusion,here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.

关键词： Apoptosis Bad Cyclin A-Cdk2 Phosphorylation Substrate

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

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Chemical Research in Chinese Universities 2008年第6期24卷 767-770页

作者： ZHANG Hai-hong ZHANG Xi-zhen ZHAO Dong-hai SHI He-liang YU yong-hui wu yong-ge YU Xiang-hui KONG Wei Vaccine Research Center College of Life Science Jilin University Changchun 130012 P R. China

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in *** BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAge and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.

关键词： Clone Survivin Expression Polyclonal antibody

Construction of Recombinant Modified Vaccinia Ankara(MVA) Expressing Hepatitis B Virus Surface Antigen

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Chemical Research in Chinese Universities 2006年第2期22卷 213-216页

作者： CHEN Yu YANG Xiao-song YU Xiang-hui JIANG Chun-lai wu yong-ge JIN Ying-hua CHEN Yue CHEN Yan LI Wei KONG Wei Vaccine Research Center College of Life Science Jilin University Changchun 130012 P. R. China

The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections. Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system. Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

关键词： Recombinant MVA Therapeutic vaccine T lymphocyte response

Peptide Inhibitors of HIV-1 Virus Infection Based on Cullin-5

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Chemical Research in Chinese Universities 2008年第3期24卷 338-343页

作者： ZHU Ke-tong ZHANG Xi-zhen LOU Chao-ping GUO Bo DU Juan WANG Xiao-dan wu yong-ge KONG Wei YU Xiang-hui College of Life Science Jilin University Changchun 130012 P. R. China

Virion infectivity factor（Vif） is one of the six accessory proteins of HIV-1 and is necessary for viral infectivity. Human Apolipoprotein B editing complex protein 3G（h-APOBEC3G） is a cytidine deaminase only expressed in ＂nonpermissive＂ cells and exhibits virus suppressive activity. With the aid of a Cullin-5 E3 ligase, Vif induces h-APOBEC3G degradation and with the destruction of this ligase, Vif is functionally inactive. Therefore, it is expected that blocking this E3 pathway would be a new therapeutic strategy against HIV-1 infection. In this article, the authors＇ took sequence alignment of the N-termini of Cullin-5 and three other members of the Cullin protein family, respectively. A set of small peptides has been synthesized based on the sequence comparison results and possible Vif-Cullin-5 interaction domains. Moreover, it has been demonstrated that several peptides can reduce virus infectivity in ＂nonpermissive＂ cells with a dose-responsive manner, but not in ＂permissive＂ cells. The results also indicate that the loss of viral infectivity may be because of the increase of APOBEC3G amount in the peptide-treated cells. It is concluded that peptides derived from Cullin-5 can block the APOBEC3G degradation induced by Vif and suppress HIV-1 infectivity. Therefore this study starts a novel strategy for the development of a new HIV-1 inhibitor.

关键词： HIV-1 inhibitor Peptide Vif Cullin-5 APOBEC3G

Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Model

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Chemical Research in Chinese Universities 2007年第3期23卷 329-332页

作者： ZHANG Yi-zhe JIANG Chun-lai YU Xiang-hui LOU Chao-ping ZHAO Dong-hai wu yong-ge JIN Ying-hua LIU Cheng-shan KONG Wei Vaccine Research Center College of Life Science Jilin University Changchun 130021 P. R. China

Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and administration, the lyophilization as a good option could be resorted to; through screening, the right stabilizer composition and its production procedure were obtained. The final moisture content of freezing-dried recombinant MVA-HIV vaccine was lower than 3%. It can be reconstituted quickly and shows regular physical appearance and stable potency. In vivo functional experiment, mice were divided randomly into the liquid vaccination group, the lyophilized vaccination group, and the control group. Having been DNA vaccine priming, the mice were boosted with a dose of 10^7 pfu MVA- HIV vaccine, which produced indistinguishable antibody titer and cytotoxic T-lymphocyte（CTL） level compared with those of liquid vaccination group （ P 〉 0.05 ）. These results demonstrate that lyophilized MVA vaccine can induce high immunogenicity in mice.

关键词： Recombinant MVA Vaccine Lyophilize Immunogenicity Stability

Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein(HPV16 L1) in Pichia pastoris

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Chemical Research in Chinese Universities 2007年第2期23卷 200-203页

作者： LIU Da-wei ZHANG Yu YU Xiang-hui JIANG Chun-lai CHEN Yue wu yong-ge JIN Ying-hua NIU Jun Qu Ning LIU Ming KONG Wei Vaccine Research Center College of Life Science Jilin University Changchun 130023 P. R. China

In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expression induced by methanol was screened by using sodium dedecyl sulfate-polyacrylamide gel electrophoresis （SDSPAge） and Western blotting. The results indicate that the HPVl6 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into vires-like particles（ VLPs）, which show a good immunogenicity and induces high-titer antibody in mice.

关键词： Human papillomavirus Major capsid protein Recombinant Pichia pastoris Vires-like particles

Development and Validation of a Reverse-phase High Performance Liquid Chromatography Method for Determination of Exenatide in Poly(lactic-co-glycolic acid) Microspheres

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Chemical Research in Chinese Universities 2010年第1期26卷 33-37页

作者： LIU Bin DONG Qing-guang SHI Lin WANG Meng-shu LI Chun wu yong-ge YU Xiang-hui SHAN Ya-ming CHEN Yan KONG Wei SHEN Jia-cong AIDS Vaccine National Engineering Lab State Key Laboratory of Supramolecular Structure and Material Jilin University Changchun 130012 P. R. China

Exenatide（synthetic exendin-4）, which has been approved by the Food and Drug Administration（FDA） for the adjunctive treatment of patients with type 2 diabetes, is an incretin mimetic agent. The development and validation of a RP-HPLC method for the quantification of the exenatide in poly（lactic-co-glycolic acid）（PLGA） microspheres is described. Separation was performed on a C4 column via a mobile phase consisting of ACN：KH2PO4（0.02 tool/L, pH=2.5） gradient elution from 30：70 to 45：55（volume ratio） in 30 min. Multi-diode array detection（DAD） appears to be most appropriate to evaluate the spectral purity of exenatide. The limits of detection and quantification of exenatide were 0.4 and 1.2 μg/mL, respectively. The calibration curve of exenatide was linear in a range of 0.025--0.2 mg/mL with a correlation coefficient of 0.9995. The results of validation study show that this method is specific, accurate（recovery〉95%）, precise（RSD〈2.0%） and robust.

关键词： Exenatide Quantification RP-HPLC-DAD PLGA Microsphere