| The human immunodeficiency virus-1 (HIV-1) is a single chain RNA retrovirus belonging to the lentivirus family that selectively infects and kills CD4+ T-cells and macrophages resulting in immune system failure. After the immunity system was destroyed by the AIDS virus, the human body lose the ability of resistance to each kind of pathogen, thus it is easy caused many kinds of infections or the tumor, finally caused the death. Because there does not have the special effect treatment, the mortality rate is extremely high, therefore AIDS is called 20th century plagues. In developing countries, behavioural changes are difficult to implement in an effective manner and the high cost of anti-viral drugs places them beyond the reach of thepeople. At present, there does not have effective control its popular measure.The ultimate solution for the control of the AIDS epidemic will require the development of an effective vaccine strategy.The role of CD8 T cells in the control of immunodeficiency virus replication has been demonstrated. Soon after primary infection, activated HIV-specific cytotoxic T lymphocytes, mediated primarily by CD8+ cells, were detected in patients and so the viral load was controlled. To the chronic AIDS patient who infects HIV-1 for a long time low virus load capacity in vivo and the condition slow development also corresponds. Widely CTL responses have been detected aims at Gag, Pol, Nef and so on different conservative region, but once loses these CTL reaction, the patient rapidly changes over. In the monkey AIDS model, removes CTL which CD8 T cell mediate causes to be unable to control the load capacity of monkey HIV (SIV) and rapidly to be taken bad. All these proved that, effectively induces CTL reaction aims at conservative region as Gag, Pol and so on has the possibility to develop the effective AIDS vaccine completely.Currently, there have been several definite vaccine forms, which produce cellular immunologic response, such as the DNA vaccine, recombinant virus carrier vaccine etc.The studies for VLP vaccine provide a new reference for the AIDS vaccine. A little bit of the former researches has indicated that immunised VLP can not only induce strongly humoral immunity titer, but also can make the helper T cell and cytotoxic T-lymphocyte immunologic response. There is the minimum formation unit of virus in the core protein gag pre-body of HIV-1and it can assemble into VLP by itself outside body. So it can according to our demands insert exogenous gene or produces VLP together with other structural protein in its not- essential area. The VLP vaccine has the followed advantages: more security, more antigenicity, less dosage and no limitation of immunity path. People once used different express system to produce VLP such as virus, yeast and so on. But some defect was showed such as less antigenicity, less security, and the most just express in transient.How to construct a system to form vlp safely, efficiently and persistently? First, we make the VLP include enough antigenic determinant and chose eukaryotic cell to express plasmid D-GPEi which can express the HIV-1 main structural protein Gagpol andEnv in eukaryotic cell .Gag is not only necessary for the formation of virus particle, it is but also conservative in sequence where contain a lot of essential antigenic determinant. Enzymes coded by Pol gene is needed blend expression with gag gene; Although Env gene is not necessary to form virus grain, it included mainly neutralization antibody determinant group and parts of T cells determinant group which induced person and animal to produce neutralization antibody and CTL respond. Based on the above- mentioned, VLP is very similiar to natural virus particle and is not contained virus nucleic acid, so it is hopeful to induce stronger neutralization antibody and respond to CTL. It is instantaneous expression if only uses D-GPEi alone to transfect into eukaryotic cell. We chosed pcDNA 3.1 and D-GPEi co-trasfected into eukaryotic cell and neomycin is marker for resistance screening.G418 resistance was carried on adding press to sieve the cell line which efficiently and stablly expressed HIV-1 main structure protein gagpol and env. This thesis mainly carried on several following works:(1) To make use of cotransfection method for the first time, taking neomycin as resistance screening marking, cotransfected D-GPEi and pcDNA 3.1of eukaryotic cell expression plasmid into 293 cells. The expression most strong single clone cell was finded by examining the expression of protein under the pressure of resistance with culturing medium contained the G418.(2) VLP is purificated after sucrose density gradient centrifugalization. It is proved that Gagpol and Env has been assemblied into VLP by the Westernblot, Elisa, and electron microscope. We also detected high peak of VLP secretion and examined the stable expression of cell line.(3) Taking the BALB/c small rat as the model carried on animal immunological reaction experiment and analyzed humoral immunity and cellular immunity. Result proved that the VLP injected to the small rat by different dose without adding an adjuvant can induced particularity immunological reaction. The CTL, ELISPOT experiment proved that VLP has better cell immunity immunogenicity. Neutralization antibody experiment confirmed that VLP can induce neutralizing antibody respond. (4) To establish the quality standard of the final product. The initial safety evaluation was accomplished according to corresponding guide and research method in order to provide reference for the VLP applicationIn brief, the completed thesis provided a new reference for the development and usage of ideal AID vaccine. There are too much works have to be done to prove it is a ideal HIV vaccine, including to combine applying different formal HIV vaccine, to definite reasonable"prime-boost"immunization strategy, to improve immunization level by using immunological adjuvant and more consummate animal experiment to inspect its safty. All this will provide detailed and complete experiment datas for the immunity procedure amendment and development of HIV vaccine research...
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