The Construction Of HIV-1 VLP Vaccines And The Study Of It's Immunogenicity

As the AIDS epidemic continues to spread across the world,it isbecame the focus that the treatment strategies of it,s infection and theprevention of it,s spreading. We currently have no effective treatmentto reverse the disease process or vaccines to prevent infection. It isnow thought that an ideal HIV-1 vaccine should prime bothcross-neutralizing antibodies and long-lasting cytotoxic CD8+ Tlymphocytes (CTL)The CTLs that are capable of lysing target cellsexpressing HIV proteins have been detected in HIV-infectedpatients,andthese HIV-spectific CTL can inhibit HIV replication in peripheral bloodlymphocytes.Accordingly,CTL appear to play a critical role in thecontrol of HIV infection. But the CTL failed to confer protection fromvirus infection.Recently studies have shown that neutralizingantibodies against HIV can prevent infection and can accelerateclearance of cell-free virions from the blood .But now it is difficultyin eliciting such neutralizing antibodies.VLP can give fresh impetus to the path of HIV-1 vaccin. Previousstudies have shown that immunization with the recombinant virus-likeparticle(VLP) may elicit powerful serum neutralizing antibody titersand promoted both T helper cell and CTL response.Recent studieshave shownthat the gag-encoded precursor of HIV-1 contains the minimalparticle-forming unit of the virus,and partical formation is independentof the other protein.The VLP vaccine has the following advantage: highersafty and stronger immunogenicity ,the dose of inoculation islittle.Formerly,the researcher have assembled HIV-1 VLP used differentsystem such as virus and yeast.But they are failed for some reason. In this study, stable cell line were constructed to express humanimmunodeficiency virus-like particles (VLP) to elicit broad-spectrumimmune responses to multiple HIV-1 antigens. This stable cell line canefficiently secreted VLP into the supernatant that banded within asucrose gradient at densities similar to infectious virions. The VLPcarrying the considered antigenic domain such as gag,gagpol and env,it could be expressed inlarge quantities and is esay for purificationand provide a safe ,efficient and stable strategy for presenting multipleHIV-1 antigens. In order to construct a system of VLP which is more safer,effectiveand stable.In this paper,the following studies we have been finished: (1)D-GPEi plasmid and pc-DNA3.1(-) plasmid cotransfection into 293cell by lipofectmine 2000. After transfection 48 h, trypsinize and serisdilute cells with medium added 1mg/ml G418. Cells of antiG418 wereattached to the substratum. After 1 to 2 weeks, harvest cells of monoclonewere harvested and detamined by Western-blot to select the strongestmonoclone cells.(2)We harvested the surpernant that we have selected the monoclonecells to be determained the optional density to purified VLP by thesucrose gradient centrifuge at 26000rpm,4 ℃,4h. Furthermore weconfirmed that the pininacle and concentration of the VLP secreted tothe surpernant by lowery and coomassie blue assay.The morphology of the...