Spectrum of produced GAG gene viral proteins in lymphocytes infected HIV-1 NL4-3 strains in vitro
V P Koksin , I G Mustafin , S V Boichuk
Kazan medical journal ›› 2016, Vol. 97 ›› Issue (3) : 377 -380.
Spectrum of produced GAG gene viral proteins in lymphocytes infected HIV-1 NL4-3 strains in vitro
Aim. To study spectrum of viral proteins secreted into the culture medium by the healthy donor peripheral blood lymphocytes infected by HIV-1 NL4-3 strain in vitro.
Methods. We used a primary lymphocytes culture obtained from healthy donors. For synchronous infection of primary lymphocytes culture in vitro NL4-3 strain of human immunodeficiency virus (HIV) (NIH AIDS Research & Reference Reagents Program, USA) was used. The HIV-1 titer in the supernatants was determined by enzyme immunoassay (EIA p24gag Coulter). In addition, electrophoresis of clarified culture medium proteins in 12.5% polyacrylamide gel under denaturing conditions followed by electron transport onto a nitrocellulose membrane; immunoblotting for detection of virus-specific proteins using positive control serum to HIV-1 («Bio-Rad») were performed; protein markers kits LMW 94-14.4 kD, HMW 212-53 kD («Pharmacia Biotech») were used to determine the seroactive fractions molecular weight.
Results.. HIV antigens spectrum in the culture medium was studied on the 4th, 9th and 13th days after lymphocyte culture infection with HIV. Within 13 days of virus reproduction both non-structural and structural proteins of HIV-1 gag gene are detected in the culture medium, spectrum and quantitative range of which varies with time. After 4 hours of HIV reproduction in primary lymphocytes culture in vitro large number of gag gene nonstructural proteins (p40, p55) are exported from cells into culture medium. Further, gag gene non-structural proteins may be cleaved into the structural proteins (p24/25, R18) under the cellular proteases action, which is caused by the proteolytic enzymes accumulation in the culture medium.
Conclusion. In HIV reproduction in primary lymphocytes cultures soluble forms of the HIV-1 gag gene proteins are secreted into culture medium; in the initial stages of the infection HIV proteins spectrum in the culture medium of lymphocytes activated with phytohemagglutinin in vitro consists of only the HIV-1 precursor proteins (p55, p40) and at a later stages - also of the gag gene viral capsid proteins (p24/25, R18).
HIV-1 / proteins spectrum analysis / immunoblotting / enzyme immunoassay
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Koksin V.P., Mustafin I.G., Boichuk S.V.
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