| The AIDS epidemic threatens the maintenance and development of the whole human. A prophylactic vaccine against HIV, the pathogen of AIDS, is deadly needed to stop the epidemic. Under the conditions that there is no effective vaccine and curable treatment for AIDS, it is an important strategy to detect the HIV antibodies among the high-risk population and blood donors, so as to prevent and control the spread of AIDS by restricting the source of infection and interrupting the ways of transmission. Rapid, sensitive, specific, and cost-effective screening assay of donated blood and confirmatory assay to prevent transmission of infectious become urgent.According to codon preference in E. coli and amino acid sequence of HIV envelope protein and integrase, the genes coding for envelope protein and integrase were synthesized. Then the fragments were inserted into the expression vector pET-HIS. Recombinant plasmids were transformed into E.coli (DE3) to obtain the genetic engineering strains. The expression strains were induced by IPTG and detected by SDS-PAGE and Western blot. The expressed products in a form of inclusion body showed that the recombinant proteins were recognized specifically by HIV-1 positive serum.Cultivation conditions of the genetic engineering E.coli P31 which expressed recombinant HIV integrase were investigated. The optimum experiment conditions were gained by determining different culture and induction conditions. The optimum experiment conditions were genetic engineering E.coli P31 cultured at 37℃until the OD600 0.8, then induced by 0.6 mmol/L IPTG at 32℃for 5 h.Fermentation of the genetic engineering E.coli P31 in 5L scale was studied. The kinetics curve of fermentation in 5L was established after three times of fed-batch fermentations, the technology suited for the industry production.The plasmid stability on generations was studied, the results showed that the genetic engineering E.coli P31 was stably.Preparation, washing, dissolving, purification and refolding by dilution inclusion body of recombinant HIV integrase P31 were studied, the conclusions showed that: combining the freeze-thaw method, enzyme and ultrasound methods, the inclusion bodies were broken effectively. The purity of inclusion bodies obtained after centrifugation broken increased about 10% compared with before thalli broken. Comparisons the washing effects of several detergents, 0.5% Triton X-100 could remove contaminants effectively, except for some LPS, nucleic acids, lipids, and miscellaneous proteins and other substances, but also the process of dissolution of host cell lysis released from membrane proteins and other substances, the purity of washed inclusion bodies was 89.6%. The refolding efficiency was high by dilution with the conditions of the redox ratio of GSSG / GSH = 0.15, pH 8.5, ionic strength of 0.15mol / L, 4℃and the use of sub-step-like operational methods.The refolding of integrase P31 using surfactant andβ-cyclodextrin (β-CD) was studied, an established method known as artificial chaperone. It was shown that the charge properties of surfactant affected the refolding performance significantly. The surfactants with the same charge of protein were preferred. The effect by the dosage ofβ-CD adding on renaturation of P31 was studied, the results showed that the chemical composition of SDS-denatured P31 complex was affected by the combining time, then refolding mechanism of artificial chaperone-assisted P31 refolding was presented. Refolding P31 was analyzed by fluorescence spectroscopy and circular dichroism spectroscopy, the results showed that P31 refolding assisted by artificial chaperone had more correct quaternary and secondary structures than dilution.According to several experiments, the preparation and protocol of immunoassay strip, the kinds of membranes, the concentration of recombinant antigens and fold times of enzyme, blocking buffer component and the dynamic control lines were confirmed. The line immunoassay methods based on recombinant antigens were established preliminary.The gene-specific primers of conservative integrase gene were designed. The factors of the Mg2+ concentration, the reaction temperature and time were optimized to determine the optimal RT-LAMP for detection of HIV RNA. The detection limit of RT-LAMP assay was 155copies/mL in detection HIV international standard RNA template.A total of 171 samples (18 HIV-1 confirmed positive samples by western blot and 153 serum specimens from healthy blood donors by ELISA) were tested by RT-LAMP, the results showed that 100% coincidence with WB in positive samples and 100% coincidence with ELISA in negative samples.The HIV line immunoassay and isothermal detection methods developed in this research both had high specificity and sensitivity. Furthermore, these methods had advantages on easiness in operation of reaction and determination of results. These detection methods offer promising alternatives for HIV detection.
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