Establishment And Initial Application Of A Medicine Screening Technique Based On Ccr5 Promoter

Background and ObjectiveAcquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). The entry of HIV-1 into CD4~+T cells is mediated by the interactions between the viral envelope glycoproteins, the CD4 receptor and HIV-1 coreceptors. On primary infection, HIV-1 coreceptors are the chemokine receptors CCR5 (CC chemokine receptors 5), conjugated by HIV-1 R5 strain, during the later stage of infection, the CXC-chemokine receptor 4, CXCR4) used by HIV-1 X4 strain. The normal CCR5 protein works as a kind of channel protein of HIV endocytosis.Therefore, CCR5 is the most important co-receptor in the early stage of HIV infection.In recent years, many scholars are interesting in the research of CCR5 gene polymorphism. The research indicates that the homazygous individual of CCR5 A32 and CCR5m303 can resist of HIV-1 infection effectually. The influence of CCR5 gene polymorphism on HIV-1 invasion indirectly proofs the significance of CCR5 in course of HIV-1 infection. More and more research indicates that the individual of CCR5Δ32 has normal immune function and inflammatory reaction, moreover, he can resist of HIV-1 infection effectually. Therefore, HIV-1 infection can be obviated and treated through acting on the promoter of CCR5 and depress the expression of CCR5 gene. There has been much research on CCR5 antagonist in abroad, and much medicine has been in the second stage of clinical research.Chinese medical therapy has much good quality, such as abundant of medicinal herbs, cheap, little adverse reaction and being able to boost immune function of patients. Then, if traditional Chinese medicine could resist HIV-1 infection through acting on CCR5 promoter? Therefore, this experiment plans to construct a screening technique, which would screen larvaceous traditional Chinese drug against HIV-1/AIDS through acting on CCR5 promoter.First, insert the gene fragment of CCR5 promoter into the rebuilded reporter vector pGL3-neo; Then transfect luciferase reporter vector pGL3-neo-CCR5 into Jurkat cells(the cell line of acute T lymphocyte leukemia) liposome. The stably transfected cell line was obtained by G418 screening. After seven kinds of traditional Chinese medicine had acted separately on the stably transfected cells, detect the luciferase activity of luciferase reporter vector in the cells. The activity of CCR5 promoter would be reflected by the luciferase activity. Analysis the effection of Chinese drug on CCR5 promoter. Construct a medicine screening technique in vitro targeting at the promoter of CCR5.Methods:Design primers to amplify the promoter of CCR5, then amplify the gene from human genome DNA. Rebuild the luciferase reporter vector pGL3, design primers to amplify the screening marker neo. Amplify the gene neo from plasmid pcDNA3.1. Clone the unit neo into vector pGEM-T Easy, identify it by restriction endonuclease. Then, insert the unit neo into the vector pGL3. Insert the gene of CCR5 promoter into the vector PGEM-T Easy, then subclone the gene of CCR5 promoter into the rebuilded reporter vector pGL3-neo; identify the luciferase reporter plasmid pGL3-neo-CCR5 by double restriction endonuclease, PCR and DNA sequencing analysis. The luciferase reporter vector pGL3-neo-CCR5 was transfected into Jurkat cells(the cell line of acute T lymphocyte leukemia) by liposome. The transfected cell was screened by G418. After seven kinds of traditional Chinese medicine (Shuanghuanglian, Chuanhuning, Licorice root, Chuling, the Root of balloonflower, Milkvetch root, Baical skullcap root) had acted separately on the stably transfected cells for 16h, observe the activity of CCR5 promoter by detect the luciferase activity of luciferase reporter vector in the cells. The result is analysised by the statistical software SPSS13.0.Results1. Amplify the gene fragment of CCR5 promoter by PCR, we can see a bright straps located in 987bp by electrophoresis, conform to the design. 2. Amplify the unit neo by PCR, we can see a bright straps located in 1882bp by electrophoresis, conform to the design.3. Separately conjuncted the unit neo, the gene fregment CCR5 promoter with T-vector,then transform into DH5α, screened to identify the positive recon of pGEM-T-CCR5, pGEM-T-neo.4. Insert the unit neo into the luciferase reporter vector pGL3, obtained the reporter vector pGL3-neo with screening marker.5. Subclone the gene fragment of CCR5 promoter into the rebuilded reporter vector pGL3-neo, obtained luciferase reporter plasmid pGL3-neo-CCR5. Double restriction endonuclease, PCR certificate confirmed the inserted sequence. DNA sequence analysis conforms to the design.6. The Jurkat cell line stably transfected by pGL3-neo-CCR5 was obtained by G418 screening.7. Detection of the luciferase activity results shows: Shuanghuanglian may significantly depress the luciferase activity of the cell line transfected(P<0.05); Chuanhuning, Scutellaria, Milkvetch root may significantly boost the luciferase activity of the cell line transfected(P<0.01); There is no significant difference between Chuling group, Licorice root group, the Root of balloonflower group and control group(P>0.05).Conclusion:1. Constructed the luciferase report vector of CCR5 promoter, screen and obtained cell line stably transfected by pGL3-neo-CCR5 by G418, established a medicine screening technique in vitro targeting at the promoter of CCR5.2. Shuanghuanglian may significantly depress the activity of CCR5 promoter of in cells(Jurkat) cultured in vitro, may have larvaceous effect on the process of against HIV-1/AIDS; Chuanhuning, Scutellaria, Milkvetch root may significantly boost the activity of CCR5 promoter of the cell culture in vitro, hint that it is not suitable for the patient infected by HIV to use these medicine or the compound contained these element.