| Background: AIDS (acquired immunodeficiency syndrome) is a devastating disease. Since 1980s, there has been more than 68 million people infected with human immunodeficiency-1 (HIV-1) and more than 28 million people have died. Highly active antiretroviral therapy (HAART) is an effective therapy against HIV, and has led to profound decreases in morbidity and mortality rates in HIV-1-infected persons. Many infected persons have plasma levels of HIV-1 RNA that are less than the limits of detection of most clinical assays due to combination antiretroviral therapy. Nonetheless, HIV-1 has not been eradicated by HAART. Therefore, vaccine is a fundamental method to get rid of HIV-1.Currently, candidates of recombinant HIV gp120 protein vaccine are unable to generate antibodies capable of neutralizing infectivity of primary isolates from patients. Here, "fusion-competent HIV vaccine" immunogenes were generated to capture the transient envelop-CD4-coreceptor structures that arise during HIV binding and fusion. Development of these fusion-dependent immunogens may lead to a wide-range effective HIV vaccine. Construction of vector of HIV-1 p24 and gp41 fusion gene and expression, purification of p24-gp41 fusion protein in E.coli lays ground for therapeutic vaccine.Part one Sequence variation of p24 coding region in gag gene of human immunodeficiency virus type 1,Acquisition of HIV-1 p24 protein region encoding gene and gp41 protein region encoding gene Objective To acquire HIV-1 p24 protein region encoding gene and gp41 protein region encoding gene using by RT-PCR. To comprehend the variation of p24 coding region in p24 protein of HIV-1 in China.Method Total RNA in plasma of HIV-1 infected human were extracted,and RNA were retranscripted into cDNA,the primer with restriction endonuclease site were designed ,target gene segment were amplified by nest PCR.The sequence of p24 region,680 nucleotides were determined,then phylogenetic analyses were performed.Result Of 28 specimens,25 were B subtype,and 3 were A subtype. Comparing to the consensus sequence, nucleotide variation in B subtype was 0.4%~4.8%, with an average of 1.4%. The transition changes of A-to-G and G-to-A were 20.5% and 17.3% respectively. While the G-to-A hypermutation was not observed. The intrasubtype distance for B subtype was 2.9%, and the substitution for predicted amino acid was 0.4%~5.2%. Because of the small number, the variation in A subtype was much lower than that of B subtype. Phylogenetic analyses implied that most of HIV-1 B subtype infections in Henan province were close to Thailand isolate. Acquisition of gene fragment of HIV-1 region encoding p24 protein and region encoding gp41 protein is corresponding to p24 and gp41 of HXB2. The changes of p24 coding region of HIV-1 patients in this study was still relatively conservative. And gp41 coding region of HIV-1 was highly conservative.Part two Ligation of p24 and gp41 gene and construction of expression vectorObjective To ligate p24 and gp41 gene and to construct expression vector of p24 -gp41 fusion protein. Method Both of p24 and gp41 gene were ligated into pGEM-T easy vector and pMD18—T vector. The target DNA fragment amplified from p24 and gp41 gene were sequenced after T-A cloning. The post-linked gene were cleaved and linked into pET21a vector. The correct sequence was proved by enzyme incision and sequence test and open reading frame was correct.Result Expression vector pET21a of p24-gp41 fusion protein were constructed successfully. The correct sequence was proved by enzyme incision and sequence test and open reading frame was correct.Part three Expression of p24-gp41 fusion protein and proven by Western blottingObjective To express p24-gp41 fusion protein in E.coli and then proved by Western-Blot.Method The vector pET21a was transformed into E.coli.p24-gp41 fusion protein induced by IPTG (isopropyl-beta-thiogalactopyranoside) was examined by SDS-PAGE. That the expressed product could react with serum of 6×his antibody were proved by Weste... |
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