Recombinant Expression And Anti-HIV-1 Study Of Soluble DC-SIGN And Viral Chemokine VMIP2

There are three chapters in this thesis.Chapter one is a general introduction of research on HIV infection of human cells, including global epidemic of AIDS, overview of progress in HIV co-receptors and attachment receptor DC-SIGN.Chapter two is a report of expression, purification, and biological analysis of soluble DC-SIGN. DC-SIGN cDNA was amplified from mRNA of dendritic cells by RT-PCR and subsequently cloned into pcDNA3.1. Using recombinant plasmid pcDNA-DC-SIGN as template, the DC-SIGN lectin cDNA was amplified and subcloned into prokaryotic expression vector pET-32a(+). Following transformation and induction in E.coli AD494(DE3), the recombinant DC-SIGN fusion protein was purified using Ni2＋-NTA agarose. Western blotting demonstrated that the fusion protein had specific immunological activity with mouse anti human DC-SIGN antibody. HIV binding assay indicated that the soluble DC-SIGN fusion protein blocked the binding of both R5 and X4 HIV to DC-SIGN. This research provides basis for further investigating the mechanisms of interaction between DC-SIGN and HIV gp120. It also gives implications for developing novel strategies to prevent HIV and other pathogens that bind DC-SIGN.Chapter three is the expression, purification, and anti-HIV study of viral chemokine-vMIP2. vMIP2 gene was cloned into pET-32a(+) expression vector, which allows production of the desired protein along with a thioredoxin fusion tag. The vector containing the sequence encoding the mature form of vMIP2 was transformed into AD494(DE3). After induction, TrxA- vMIP2 fusion protein was purified using Ni chelating column. Cleavage of the thioredoxin fusion tag was subsequently carried out with enterokinase. The cleaved protein was further purified by ion exchange column.Western blotting indicated that purified vMIP2 had specific immunological activity with vMIP2 antibody. In vitro infection demonstrated that vMIP2 potently inhibited the replication of R5 and X4 HIV in human peripheral blood mononuclear cell (PBMC). This study provides basis for development of effective prevention strategies against HIV. Further investigation may help to define the role of vMIP2 in HHV8 pathogenesis.