0206创新课题组二期创新总结报告.doc

1、Part I. IntroductionGroup: Molecular VirologyResearch area: Molecular mechanism of HBV T-cell immunity and enveloped virus-cell fusionGroup leader: Po Tien, Virologist. He graduated from Beijing Agricultural University in 1954. He is a professor of Institute of Microbiology of CAS.Research team:Geor

2、ge F. Gao, Ph.D, guest professorHuaiyi Yang, Ph.D, associated professorChangmei Liu, Ph.D, assistant professorRong Wang, B.S.Part II. Background and SignificanceHBV infection is a serious medical problem worldwide (over 350 million persons) and especially in China (over 120 million). Immune response

3、 failure is the major reason for persistent HBV infection, which in turn, causes chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) in some patients. Stimulate and restore the cytotoxic T lymphocytes reaction (CTL) of chronic HBV infected patients plays vital roles in immunotherapy towa

4、rds HBV related diseases, while dendritic cells (DC cells) are one of the main regulating factors in the process. My lab is focusing on the study of new therapeutic vaccines. Meanwhile, some tentative efforts of HBV entry to primary human liver cells are also now in process.Enveloped viruses cause i

5、mportant human pandemic diseases such as human immunodeficiency virus type 1 (HIV-1), SARS-coronavirus (SARS-CoV) and human respiratory syncytial virus (hRSV). The fusion proteins of enveloped viruses promote viral infection by mediating the fusion of viral membrane with target cell membrane. Two he

6、ptad-repeat (HR1 and HR2) regions of class I fusion proteins play a very important role in this process and most of the peptides derived from HR1 and HR2 exhibit inhibitory activity for membrane fusion and can potently inhibit viral infection. However, synthesis of these peptides is at high cost and

7、 these HR1 and HR2 peptides are unstable. We have used a number of strategies to develop recombinant protein inhibitors for membrane fusion.Part III. Major AchievementsIn our previous work, we isolated an HLA-A11 restricted 7 mer peptide of HBV core Ag associated with heat shock protein (HSP) gp96 i

8、n liver tissues of patient with HBV-induced hepatocellular carcinoma (HCC). Both gp96 and its N terminal fragments augment peptide specific CTL expansion and HBV antigen specific antibody production in BALB/c mice. In vitro experiments also indicated that gp96 and both of its N and C terminal fragme

9、nts can stimulate HCC patients derived DC maturation and HBV eptitope CTL expansion in HLA-A2 transgenic mice. Those results clarified a future of utilizing gp96 in HBV diseases immunotherapy.Our previous studies showed that the two HR peptides of hRSV had membrane fusion inhibitory activity and the

10、 HR2 peptide of SARS-CoV also had such activity. On the other hand, crystallization and preliminary crystallographic analysis of the trimer core from the fusion proteins of Mumps, New Castle, SARS-CoV, Measle and Menangle viruses showed they all exhibited a six-helix bundle structure. In addition, w

11、e designed several multi-HR proteins three helix (HR121 and HR212) and five helixes (HR12121) for HIV-1, hRSV and SARS-CoV, respectively. All these proteins could be abundantly expressed as soluble proteins and could be easily purified at low cost of production. Moreover, all these proteins inhibite

12、d their corresponding viral entry.Publication:1 Songdong Meng, Fu Gao, and Po Tien (2001) Isolation of a specific peptide associated with heat shock protein gp96 from Hepatitis B virus induced hepatocelluar carcinoma. The Lancet, 357: 528-529, 2001.2 Song-Dong Meng, Jian Song, Zihe Rao, Po Tien, Geo

13、rge F. Gao, Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides, Journal of Immunological Methods, 264: 29-35, 2002.3 Minghai Zhou, Yanhui Xu, Zhiyong Lou, David K. Cole, Xiaojuan Li, Yiwei Liu, Po Tien, Zihe Rao and George F. Gao. Complex ass

14、embly, crystallization and preliminary X-ray crystallographic studies of MHC H-2Kd complexed with an HBV-core nonapeptide. Acta Crystallographica Section D Biological Crystallography. 60, 1473-1475, 2004.4 Xiao-Dong Zhu, Wei-Hua Zhang, Cheng-Lin Li, Yang Xu, Wei-Jiang Liang, Po Tien. New serum bioma

15、rkers for detection of HBV-induced liver cirrhosis using SELDI protein chip technology. World Journal of Gastroenterology, 10(16): 2327-2329, 2004.5 Xiao-Dong Zhu, Cheng-Lin Li, Zhen-Wei Lang, George F. Gao*, Po Tien*, Significant correlation between expression level of HSP gp96 and the progression

16、of hepatitis B virus induced diseases. World Journal of Gastroenterology, 10(8):1141-1145, 2004. 6 Hongtao Li , Minghai Zhou , Jinle Han, Xiaodong Zhu, Tao Dong, George F. Gao, and Po Tien. Generation of murine CTL by a hepatitis B virus-specific peptide and evaluation of the adjuvant effect of heat

17、 shock protein glycoprotein 96 and its terminal fragments. The Journal of Immunology. 174(1):195-204, 2005.7 Ming Yu, Enxiu Wang, Youfang Liu, Dianjun Cao, Ningyi Jin, Catherne W.-H. Zhang, Mark Bartlam, Zihe Rao, Po Tien and George F. Gao, Six-helix bundle assembly and characterization of heptad re

18、peat regions from the F protein of Newcastle disease virus. Journal of General Virology, 83, 623-629, 2002.8 Jieqing Zhu, Catherine W.-H. Zhang, Yipeng Qi, Po Tien, George F. Gao, The fusion protein core of measles virus forms stable coiled-coil trimer, Biochemical and Biophysical Research Communica

19、tions. 299, 897-902, 2002.9 Enxiu Wang, Xiaoou Sun, Yuan Qian, Linqing Zhao, Po Tien, and George F. Gao, Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion, Biochemical and Biophysical Research Communications, 302: 469-475, 2003.10 Jieqing Z

20、hu, Yi Ding, Feng Gao, Tinghe Wu, Catherine W.-H. Zhang, Po Tien, Zihe Rao and George F. Gao. Crystallization and preliminary crystallographic analysis of the trimer core from Measles virus fusion protein. Acta Crystallogr D Biol Crystallogr. 59:587-590, 2003. 11 Jieqing Zhu, Pengyun Li, Tinghe Wu,

21、Feng Gao, Yi Ding, Catherine W.-H. Zhang, Zihe Rao, George F. Gao, Po Tien. Design and analysis of post-fusion 6-helix bundle construct of heptad repeat regions from Newcastle disease virus F protein. Protein Engineering. 16(5):373-379, 2003.12 Jieqing Zhu, Catherine W.-H. Zhang, Zihe Rao, Po Tien a

22、nd George F. Gao. Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus. Archives of Virology 148(7):1301-16, 2003.13 Pengyun Li, Jieqing Zhu, Beili Wu, Feng Gao, Po Tien, Zihe Rao and George F. Gao. Crystallization an

23、d preliminary crystallographic analysis of post-fusion 6-helix bundle core structure from Newcastle disease virus F protein. Acta Crystallogr D Biol Crystallogr. 59:1296-1298, 2003.14 Jieqing Zhu, Gengfu Xiao, Yanhui Xu, Fang Yuan, Congyi Zheng, Yueyong Liu, Huimin Yan, David K. Cole, John I. Bell,

24、Zihe Rao, Po Tien and George F. Gao. Following the rule: formation of the 6-Helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors. Biochemical and Biophysical Research Communications, 319(1): 283-288, 2004. 15

25、 Yueyong Liu, Jieqing Zhu, Mingguang Feng, Po Tien, and George F. Gao. Six-helix bundle assembly and analysis of the central core of mumps virus fusion protein. Archives of Biochemistry and Biophysics, 421: 143-148, 2004.16 Ling Ni, George F. Gao, Po Tien (2005) Rational design of highly potent HIV-

26、1 fusion inhibitory proteins: Implication for developing antiviral therapeutics. Biochemical and Biophysical Research Communications 332: 831-836.17 Ling Ni, Jieqing Zhu, Junjie Zhang, Meng Yan, George F. Gao, Po Tien. Design of recombinant protein-based SARS-CoV entry inhibitors targeting the hepta

27、d-repeat regions of the spike protein S2 domain. Biochemical and Biophysical research communications, 330: 39-45, 2005.18 Ling Ni, Linqing Zhao, George F. Gao, Yuan Qian, Po Tien. The antibodies directed against N-terminal heptad-repeat peptide of hRSV fusion protein and its analog- 5-Helix inhibit

28、virus infection in vitro. Biochemical and Biophysical research communications, 331:1358-1364, 2005.19 Shao-Man Yin, Yi Zheng, and Po Tien. On-column purification and refolding of recombinant bovine prion protein: using its octarepeat sequences as a natural affinity tag. Protein Expression and Purifi

29、cation, 32：104-109，2003.20 Shao-Man Yin, Man-Sun Sy, Huai-Yi Yang, and Po Tien. Interaction of Doppel with the full-length laminin receptor precursor protein. Archives of Biochemistry and Biophysics 428: 165-169, 2004.21 Shao-Man Yin, Man-Sun Sy, and Po Tien, An engineered PrPSC-like molecule from t

30、he chimera of mammalian prion protein and yeast ure2p prion-inducing domain. Acta Biochimica et Biophysica Sinica 36(2): 42-46, 2004. 22 Canhui Liu, Ruozhi Cheng, Lun-Quan Sun, Po Tien. Suppression of platelet-type 12-Lipoxygenase activity in Human erythroleukemia cells by an RNA-cleaving DNAzyme. B

31、iochemical and Biophysical Research Communications, 284(4): 1077, 2001.Patents:1. Hepatitis B virus (HBV) antigenic polypeptide-heat shock protein complex and use thereof (No.: ZL 01 1 04060.2), 2003.2. A recombinant BSE PrP fusion protein with PrPSC-like information and its application, 2003. 3. Pe

32、ptide drug and its derivative for inhibition of SARS-CoV infection and their application, 2003.4. One class of immuno-adjuvant and their usage in anti-virus vaccine or medicine manufacture, 2004.5. Multi-helix proteins and their corresponding encoding genes for inhibition of enveloped virus infectio

33、n and their application, 2005.Part IV. Future Research Plan1HBVProtein based, DNA based, and cellular based vaccine studies are now under extensive studies in my lab. In detail, DNA vaccine and virus based vaccine by using the adjuvant functions of gp96 and its functional fragments are now under int

34、ensive study in our laboratory. New approaches of using dendritic cells, antisense RNA techniques and HBV transgenic mice are also now under exploration for the clarification of HBV infection and HBV tolerance.From our observation, we noticed the high dose antigen specific suppression functions of g

35、p96. While this suppression function was not noticed in its N terminal domain. This intriguing aspect of gp96 may rely in its stimulating differences of its functional domains in cell signaling pathways, of which ERK pathway is our special interest. Further efforts are going to be put on the segment

36、al analysis of the adjuvant functions of gp96 and its stimulation abilities to the related cell signaling pathways. Viral life cycle study is essential for effective therapeutic designs in virus research. However, little is known of HBV infection. This is very much due to the lack of animal model an

37、d the limitation of virus host. My lab is now trying to study the virus entry by utilizing pseudovirus system mediated infection combined with primary liver cell cDNA library screening techniques. Understanding the process of virus entry and exploring more effective therapeutic vaccines are the long

38、 goals of my lab.2 The pre-clinical research on the entry inhibition of HIV-1 by the multi-helix proteins.3Misfolding of prion protein and its association with prion diseasesOur research will focus on two following aspects using mouse PrP (mPrP). (1) The role of associated mutation and unfolded N-te

39、rminal of mPrP in its misfolding in neuroblastoma cell. (2) The ability of in vitro self-propagation of PrPSC-like conformation generated in yeast cytosolic environment and its infection in vivo. We have generated Flp-in system in N2a cell, detected the expression of different PrP mutants in N2a-Flp-in cell, and have expressed mPrP in yeast cytosol, in which PrP was aggregated, resistant to proteinase K, and this cytosolic PrP aggregate can convert its conformation to normal cellular PrPC.