Study On The Gene Sequences Variations And The Second Structure Of HIV-1 Tat Exonl And The Relationship With Disease Progression

ObjectiveOur 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 40 cases of HIV - 1 infected individuals confirmed by HIV/AIDS confirming lab in our hospital. The nucleotides and amino acid variations and the second structure 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 reserved at -80℃ of China 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 excise the gel with the target amplified DNA fragment 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 differ-ent 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. NNPRIDICT program (http://ncbi. nlm. nih. gov) predict and compare the second structure of Tat protein. NEIGHBOR( neighbor - joining algorithm) program is used to produce phelogenetic tree. Position and type of the HIV - 1 tat exonl variations are analyzed and compared with foreign HIV - 1 searched from HIV Sequence Database. The relationship between HIV - 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 among LTNPs, asymptomatic HIV -1 infected individuals and AIDS patients:33 cases of B' subtype compared with their according international consensus sequences HXB2; (1)LTNPs: position 19 R substitutes K; position 21 S substitutes A; position 58 S substitutes A; position 65 N substitutes H; position 67 V substitutes A; position 69 V substitutes L. (2)asymptomatic HIV - 1 infected individuals and AIDS patients-, position 17 L substitutes Q; position 19 N substitutes K; position 20 P substitutes T; position 58 T substitutes A; position 70 P substitutes S. These variations don' t appear in LTNP group. (3)AIDS group: position 21 L or R substitutes A; position 26 F or C substitutes A; position 39 T substitutes I; position 58 P substitutes A; position 64 N substitutes T; position 67 G or D substitutes A; position 71 E substitutes K. These variations appear only in AIDS group.7 cases of BC subtype compared with their according international consensus sequences 07 BC. CN. 97.97CN001: (1)asymptomatic HIV -1 infected individuals: position 60 P substitutes R; position 67 D substitutes N. (2) AIDS patients : position58 T substitutes A; position 62 R substitutes S.2. Comparing the second structure of Tat protein among LTNPs, asymptomatic HIV - 1 infected individuals and AIDS patients: There is not big differ-...