Expression, Purification And Primary Application Of HIV-1 Gp120s

Human Immunodeficiency Virus ( HIV) is the pathogen that causes acquired immunodeficiency syndrome in human. It can be categorized into two types: HIV-1 and HIV-2. HIV-1 is the main type. It's different from other viruses for its complicated genome related to the latency,activation,transcription and replication and for its variation in host cells.Envelope glycoproteins gpl20 is the main antigen of HIV-1 and important component of diagnosis kits for detecting HIV antibodies. It can be used to produce gp120 monoclonal antibodies. Considering its importance in applications, we have tried to choose part of the gpl20 gene (named gpl20s)to express in E. coli.DNA fragment 537bp was obtained by PCR from the plasmid pHXB2-gp160 and constructed into plasmid pET32a. The recombinant plasmid was transformed into E.coli BL21 (DE3) plys. After the induction by IPTG, the expressed protein was detected by SDS-PAGE and Western blot. It showed that the 40Kd fusion protein was mainly expressed in inclusion bodies with an amount that accounts to at least 43.35% of all E.coli proteins. We obtained very pure gp120s proteins (85%) by washing,dissolving and refolding the inclusion bodies. Primary applications of gp120 in detecting antibodies by indirect ELISA in HIV showed that it have good antigen.In this study we tried to express gp120s in E. coli, and succeeded in expressing gp120s in E.coli. This study has laid some ground work for further researches and development of HIV diagnosis reagents and subunit vaccines for AIDS. It's also important for studying the properties of the main antigens of HIV. Human cytochrome P450 2A6 plays an important role in the metabolism of drugs and xenobiotics. PHarmacogenetic studies have shown that genetic polymorpHisms in this gene are important determinants of inter-individual and inter-ethnic variation in drug metabolism and toxicity. To develop a broadly applicable assay system for studying human CYP2A6 polymorpHic enzymes, we cloned and expressed the DNA of CYP2A6 wild-type(WT) in budding yeast Saccharomyces cerevisiaee, which was already integreted with CYP450 Oxidoreductase(POR). Using these recombinant enzymes in real-time biochemical assays and high throughput drug screening, the kinetic constants as well as inhibition constants for the test compounds were measured. The results we got can provid valuable information for studying the effects of polymorpHic genes on drug metabolism and evluating new drugs in the early pHase of drug discovery.Tempalte was obtained from DNA designed before.The wild-type DNA of CYP 2A6 was obtained by site-directed mutagenesis from the template . Transformed yeasts produced large quantities of microsome-bound 2A6 enzymes as determined by Western analysis, a 55 kDa protein was observed.The P450 content was assayed by reduced CO difference spectrum.There was a 450-nm peak observed in 2A6-WT, and itscontent respectively was 23.8pmol/mg.The isolated microsomes were used to measure the kinetic constants of 2A6 enzyme in real-time assays using a fluorogenic substrate Coumarin. The results showed that the enzyme possess robust activity, the Vmax value,Km value and Clint value were close with the related reports. The inhibition of recombinat CYP2A6-WT enzyme by known inhibitor drug was tested by serial titration of drugs in the fluorogenic assays. The results showed that: CYP2A6 was strongly inhibited by its known inhibitor tranylcypromine, IC50 was about0.05 mM. Used fluorogenic high throughput technique to detect the inhibition of 15 drugs on the active enzymes,the results showed :all of them can not inhibit 2A6. We also used HPLC to measure the kinetic constants of 2A6 enzyme using the fluorogenic substrate Coumarin. The results showed that the enzyme possess robust activity, the Vmax value,Km value and Clint value were close with real-time assays.It showed that 2A6 enzyme have robust stability and the two techniques have good consisitence.The experiments results can be convinced even more.For the first time,we successfully constructed the Saccharomyces cerevisiae expressing system for CYP2A6 gene; The catalytic activity of yeast microsome protein was robust and stable; Established an in vitro detection system for biochemical analysis and drug high throughput screening of CYP2A6 polymorpHic enzymes,which were efficient, sensitive and convenient. This work can guide the further study of CYP2A6 related drug-drug interaction,as well as can aid the future reducing adverse reaction and enhancing drug efficiency in vivo.