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Objective To determine whether Morphine has the ability to enhance HIV-1 replication in MT2 and Macrophage in vitro and assess the influence of Naloxone on Morphine2s effect.Methods MT2 cells were randomly assigned into 4 groups: (1) Morphine treatment for MT2 group, (2) Morphine+Naloxone co-treatment for MT2 group, (3) Naloxone treatment for MT2 group and (4) MT2 Control;Macrophages were also randomly assigned into 4 groups: (5) Morphine treatment for Macrophage group, (6) Morphine+Naloxone co-treatment for Macrophage group, (7) Naloxone treatment for Macrophage group and (8) Macrophage Control. Group (2), (3), (6) and (7) were pre-treated with 10-8 mol/L Naloxone for 0.5 h, and then group (1) and (2) were treated with 10-12, 10-10 and 10-8 mol/L Morphine for 24 h;group (5) and (6) were disposed of 10-10 mol/L Morphine for 24 h.All 8 groups were added in HIV-1 viral strain with 50% tissue culture infective dose(TCID50).P24 antigen in MT2 cells culture supernatant at day 3, 4, 5 and 6, and in Macrophages culture supernatant at day 4, 6, 8, 10 and 12 after infection were determined with ELISA.Student2s t-test and ANOVA were used to compare the differential expression in different groups, and repeated measures ANOVA was used to compare the increasing or decreasing expression of p24 antigen in morphine treatment groups than that in the control group at different time points.Results On the 3rd day of infection with HIV-1 in MT2 cells, the expression of p24 antigen in 10-12, 10-10 and 10-8mol/L dose of group (1) were (4.44?.30), (5.59?.25) and (4.60?.24) ng/ml respectively, compared to control[(1.93?.05) ng/ml, t= 14.15, 24.74 and 19.14, all P<0.01].On the 4th day, 10-12, 10-10 and 10-8mol/L dose of group (1) resulted in a significant increase of p24 antigen expression [(24.30?.66), (31.73?.17) and (26.02?.37) ng/ml]in culture supernatants compared to control[(8.03?.09) ng/ml, t=10.59, 34.92 and 81.2, all P<0.01].On the 5th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (56.30?.26), (81.77?.49) and (63.66?.57) ng/ml respectively, compared to control [(15.30?.91) ng/ml, t= 45.83, 43.51 and 30.07, all P<0.01].On the 6th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (150.70?.97), (243.09?.93) and (173.72?.73) ng/ml respectively, compared to control [(41.01?.84) ng/ml, t= 21.09, 39.02 and 29.55, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of morphine treatment group compared to control increased with HIV-1 infected MT2 cells time, trend analysis of repeated measurements showed statistically significant time effect (F=842.18, P<0.01). On the 4th day of infection with HIV-1 in Macrophage cells, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (0.68?.15), (0.87?.41) and (0.75?.09) ng/ml respectively, compared to control [(0.60?.01) ng/ml, t= 7.27, 11.06 and 3.02, all P<0.05]. On the 6th day, 10-12, 10-10 and 10-8 mol/L dose of group (5) resulted in a significant increase of p24 antigen expression[(1.64?.57) , (2.07?.12 ) and (1.75?.17) ng/ml]in culture supernatants compared to control [(1.16?.07) ng/ml, t=8.93, 11.3 and 5.45, all P<0.01].On the 8th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (6.31?.17), (8.81?.34) and (7.19?.11) ng/ml respectively, compared to control [(3.84?.45) ng/ml, t=8.83, 15.11 and 12.42, all P<0.01]. On the 10th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of Morphine treated group were (32.30?7.55), (50.74?7.55) and (39.74?.56) ng/ml respectively, compared to control [(17.55?.86) ng/ml, t= 13.65, 17.84 and 36.69, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of group (5) compared to control increased with HIV-1 infected Macrophage cells time, trend analysis of repeated measurements showed statistically significant time effect (F=135.58, P<0.01).Conclusions Morphine has the ability to enhance HIV-1 replication in MT2 cell and Macrophage. This Morphine-mediated increase of p24 antigen expression can be blocked by Naloxone.
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Objective To construct recombinant plasmid with p24 and gp41 gene, express fusion protein in E.coli. Methods To design primer with restriction endonuclease position,and amplify p24 and gp41 by RT-PCR, link both into pMD18-T vector.To choose correct clone with target gene.Then p24 fragment will be cleaved and linked into pMD18-T vector within gp41 gene. Both post-linked gene will be cleaved and linked into pET21a vector. The vector will be transformed into E.coli. And protein is highly effective expressed in E coli. Western blotting proved that the expressed product could react with 6?his antibody. Result Fusion protein p24-gp41 is highly effective expressed in E.coli. Conclusion Fusion protein p24-gp41 is highly effective expressed in E.coli in pET21a vector.
RESUMEN
Se estudió la presencia de anticuerpos contra la proteína de 24 kd del VIH mediante el empleo paralelo del Western Blot DAVIH BLOT y del DAVIH AC P24 en muestras de suero de 176 pacientes en diferentes estadíos de la infección por VIH-1. Los resultados se correlacionaron con la clasificación clínica del paciente al momento de la toma de muestra y con la evolución posterior durante 6 meses. El 57 % de los pacientes con infecciones oportunistas menores y el 96 % de los enfermos de SIDA presentaron títulos bajos de anticuerpos. Los fallecidos no mostraron reactividad o presentaron títulos muy bajos en muestras tomadas antes del fallecimiento. Se observaron diferentes titulaciones en grupos de sueros con reactividad aparentemente uniforme en el Western Blot. Los resultados indican una adecuada correlaciòn clínico serológica; por lo que el ELISA DAVIH AC P24 pudiera ser útil en el seguimiento clínico de personas infectadas por el VIH-1.
The presence of antibodies against the HIV protein of 24 kd was studies by the parallel use of the DAVIH BLOT Western Blot and of the DAVIH AC P24 ELISA in serum samples from 176 patients at different HIV-1 infection stages. The results were correlated with the clinical classification of the patient at the moment of taking the sample and with the further evolution during 6 months. 57 % of the patients with opportunistic minor infections and 96 % of AIDS patients had low antibodies titres. Dead pattients showed no reactivity or presented very low titres in samples taken before dying. Different titrations were observed in serum groups with an apparently uniform reactivity in the Western Blot. The results show and adequate clinical and serological correlation. Therefore, the DAVIH AC P24 ELISA could be useful in the clinical follow-up of HIV-1 infected persons.