Construction Of The Primary Medicine Screening Technique Based On Human Promoter Of Cxcr4

Background and Objective:Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). The entry of HIV-1 into target cell critically depends on two cell surface components, CD4 and a chemokine coreceptor. Usually the chemokine coreceptor refers to CXCR4 or CCR5 (CC Chemokine Receptor 5). The chemokine receptor CXCR4, a member of the superfamily of G-protein-coupled receptors (GPCRs) like CXCR1-CXCR6,CCR1-CCR11,XCR1 and XCR2 etc., is the first to be discovered HIV coreceptor, and mainly found on the surface of immune cells. The HIV-1 envelope (Env) consists of gp120 and gp41. gp120 contains the CD4 binding site and a hydrophobic fusion peptide directly involved in membrane fusion respectively. During the infection, CD4 binding induces conformational changes in gp120 that exposes the coreceptor binding determinants. The gp120 interaction with the coreceptor then induces a further conformational change in Env which results in insertion of the fusion peptide into the target cell membrane. Then HIV-1 RNA integrates into the infected cell genome. The study of anti-AIDS drugs targeting on HIV-1 membrane fusion and virus entry becomes a hotspot of research interests nowadays.Traditional Chinese medicines are the peculiar drugs in China. Their pharmacology has a wide variety of different functions. Some of these traditional Chinese medicines, whether they could interact the CXCR4 promoter and cause CXCR4 expression decline, are worthy of further research.We cloned the DNA fragment of CXCR4 promoter into pGL4 vector, then transfected the recombinant into Jurkat cells, and then select the transfected cell by G418. Thirteen kinds of traditional Chinese medicines were given to rats by gastric gavage once a day. 7 days later, the serum samples were separated. After being stimulated by the thirteen medicated serums, the luciferase activity inside the transfected Jurkat cells were analyzed in order to know the activity of CXCR4 promoter. Through analyzing the effects on the CXCR4 promoter transcriptions by the thirteen traditional Chinese medicines, we established an anti-HIV/AIDS drugs screening technique targeting on CXCR4 promoter.Methods:Design primers to amplify the promoter of CXCR4, then amplify the gene from plasmid pUC-CXCR4. Insert the gene of CXCR4 promoter into the vector PGEM-T Easy, then subclone the gene of CXCR4 promoter into the reporter vector pGL4; Identify the luciferase reporter plasmid pGL4-CXCR4 by double restriction endonuclease,PCR and DNA sequencing analysis. The luciferase reporter vector pGL4-CXCR4 was transfected into Jurkat cells (the cell line of acute T lymphocyte leukemia) by bangosome. The transfected cell was screened by G418. The adult Wistar rats were administered intragastrically with 13 kinds of Chinese traditional medicine decoction (Radix Scutellariae, Rhizoma Coptidis, Cortex phellodendri chinensis, Flos lonicerae japonicae, Fructus forsythiae, Herba lobeliae chinensis, Herba houttuyniae, Folium isatidis, Radix isatidis, Ganoderma, Herba andrographis, Polyporus, Radix et rhizoma glycyrrhizae). Seven days later, blood was obtained and the separated serum were termed as medicated serum. After stimulated by the 13 kinds of medicated serum, the stable transfected Jurkat cells were analyzed luciferase activity in order to know the activity of CXCR4 promoter. The result is analysised by the statistical software SPSS13.0. Results:1. Amplify the gene fragment of CXCR4 promoter by PCR, we can see a bright straps located in 777bp by electrophoresis, conform to the design.2. Conjuncted the gene fregment CXCR4 promoter with T-vector, then transform into DH5a, screened to identify the positive recon of pGEM-T-CXCR4.3. Subclone the gene fragment of CXCR4 promoter into the luciferase reporter vector pGL4, obtained luciferase reporter plasmid pGL4-CXCR4. Double restriction endonuclease,PCR certificate confirmed the inserted sequence. DNA sequence analysis conforms to the design.4. The stable transfected Jurkat cell line with pGL4-CXCR4 was obtained by G418 screening.5. Detection of the luciferase activity shows: Cortex phellodendri chinensis (406175.2±35146.2) and Herba houttuyniae (612186.8±38890.9) can down-regulate the activity of CXCR4 promoter(P<0.05); Herba lobeliae chinensis ( 1146839±106138.3 ) can enhance the activity of CXCR4 promoter remarkably (P>0.05).Conclusions:1. Constructed the luciferase report vector of CXCR4 Promoter, and set up a medicine screening technique in vitro targeting at the promoter of CXCR4 using serum pharmacology method.2. Cortex phellodendri chinensis and Herba houttuyniae can significantly depress the activity of CXCR4 Promoter of in Jurkat cells cultured in vitro, may have potential anti-AIDS effect.