| HIV, human immunodeficiency virus is the pathogen of acquired immunodeficiency syndrome, AIDS. Until now, there are no effective vaccines to prevent HIV infection and no treatment to cure AIDS. There are 60,000,000 people in the world who had infected HIV by the end of 2002 and human kind suffer more seriously by this kind of disease as time goes by. During HIV transcriptation, not only structural proteins are coded by HTV, but also some regulatory proteins including trans - activator protein Tat which is made up of 86 -130 amino acids and responsible for regulating HTV transcription are coded. Tat protein functions at TAR, transactivation response element while other cytokines can also cooperate to promote transcription. Although the mechanism is not clear, the theory accepted by most of the researchers is that transcription is promoted by increased activity of UNA polymer-ase II strengthened by the protein - RNA component which is made up by the combining of Tat, TAR and other cytokines. Some research show that the replication level of HTV -1 determines the disease progression of HTV -1 infected individuals. Viral load and viral replication efficacy is low in some no symptom HIV -1 infected individuals while viral replication is continuous and viral replication level is high in some rapid progressing individuals who develop into AIDS later with quick CD4 + T cells depletion. As high level viral replication determines disease progression and the effective cooperation of Tat protein and TAR can increase the viral replicating level, the tat gene variation which affects the function of Tat protein will change the viral load andCD4 + T cells so as to affect the disease progression. Our study is going to amplify , detect and analyze the gene sequences of the Tat exonl in the HIV -1 provirus which is integrated in the genome of 22 HIV -1 infected individuals confirmed by HIV confirming lab in our hospital. The nucleotides and amino acid variations are characterized and the relationship between the variation of HIV -1 Tat exonl and disease progression is discussed.Materials and methodsEpidemiological and clinical data and whole blood sample of Chinese HIV infected individuals are collected. HIV - 1 Tat exonl gene sequencing: Qiaquick Spin kit is used to extract genome DNA including integrated HIV -1 provirus DNA from whole blood samples. Tat gene specific primers NP7/ENV - 1 is used as outer primers to process the first round nested - PCR and tat - B/NP11 is used as inner primers to process the second round nested - PCR on the extracted DNA. 1% agarose gel electrophoresis is used to detect target amplified DNA fragment, then the gel with the target amplified DNA were excised compared with the marker. Then QIAquick Gel Extraction Kit is used to purify the PCR product within the gel. LI - COR Global IR2 System DNA analyzer is used to detect the gene sequences. Analyzing sequences of HIV -1 tat gene nucleotides and amino acid in the HIV -1 infected individuals with different disease progression: HIV - 1 infected individuals are grouped by different disease progression stage. CLUSTAL W program in the BIOEDIT software is used to do the alignment to produce consensus sequences and the gaps are induced into the sequences to keep complete translation. DNADIST(Kimura's twoparameters method)program is used to compute nucleotides and amino acids variation distances between different sequences and based on it NEIGHBOR (neighbor -joining algorithm) program is used to produce phelogenetic tree. Position and type of the HTV - 1 tat exonl variations are analyzed and compared with that of foreign HIV - 1 strains searched from HIV Sequence Database. The relationship between HTV -1 tat gene variation and disease progression is discussed based on comparing the variations and number of CD4 + T cells.Results1. Comparing Tat amino acid sequences between AIDS patients and asymptomatic HTV -1 infected individuals:B' subtype in 15 cases compared with B' consensus; position 7 N substitutes R; position 20 P substitut... |
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