Study On CCR5Gene Expression And Its Promoter Region59029G/A、59353C/T Mutation From The Population Living The Area With Hepatitis B Virus Infection In Dong Minority Of Guizhou

[Abstract] Objective To determine the expressions of CC chemokine re ceptor5(CCR5) at mRNA and protein level in peripheral blood leukocytes fro m the Dong minority people who live in an area with hepatitis B virus infecti on.meanwhile,analyse the influence of CCR5gene promoter region59029G/A、59353C/T mutation, so as to investigate the relationship between CCR5and sus ceptibility to hepatitis Bvirus infection fortherly. Method The investigated subje cts were selected from the area of hepatitis B virus infection in Congjiang cou nty, Guizhou province of China, and divided into HBV infection group and no n-infection group, meanwhile, the population in control group were selected in an area with non-infection in same place,each group contained100Venous bio ods from adults, leukocytes separated and RNA in the cells extrated. The mRN A expressions of CCR5in leukoxytes from HBV infection group and control g roups were detected by Real-time PCR.The protein expressions of CCR5in leu koxytes from HBV infection group and control groups were detected by enzym e linked immuno sorbent assay.Further more,DNA specimens were selected fro m the minority nationalities gene pool in our laboratory.The CCR5promoter se gments that including59029G/A、59353C/T separately were replicated by PCR method and then inserted them into pGL3-Basic vector. The luciferase reporter gene plasmid pGL3-Basic containing human CCR5gene promoter segments w ere constructed by gene recombination technique and sequencing.the electransfec tion parameters of HepG2cell were optimized, The recombinant vectors, pGL3-G、pGL3-A、pGL3-C、pGL3-T, were transiently co-transfected into HepG2cells with control vector pRL-TK respectively by FuGENE6transfection reagent s and electroporotion. The influence of sudden change of CCR5gene promoter on the gene transfection activity was evaluated by adopting dual luciferaserepo rter gene system. The statistical analyses were taken with statistical software S PSS18.0, for of these results concerning mRNA expressions and protein express ions of CCR5in peripheral blood leukocytes in each group and their correlatio ns to HBV infection. Results significant change of the mRNA was found betw eenHBV infection group and control group (P<0.05),Decreased expression of CCR5mRNA in peripheral blood leukocytes in control group as compare to H BV infection group was detected; the expression level of CCR5protein in H BV infection group was lower than control group (P<0.05).The promoter frag ment (412bp) containing CCR559029G/A site was replicated by using PCR method, at the same time,The promoter fragment (437bp) including CCR559353C/T site was replicated by using PCR method too,the two gene promoter seg ments recombined luciferase reporter gene vector were successfully constructed, and the results of sequencing and double digesting of recombined plasmid we re completely corrected.the transfection rate would be improved to (60.68±1.87)%by improving electroporation buffers and others factors.After transient tr ansfection by FuGENE6transfection reagents and electroporotion respectively. the activity of dual luciferase of pGL3-G was increased to28%as compared with pGL3-A.,the activity of dual luciferase of pGL3-C was creased to16%as compared with pGL3-T. Conclusion CCR5expression will be closely associated with hepatitis B virus infection in Dong minority.high level of CCR5may be propitious to its i nhibition and clearance even recovery.CCR559029G and CCR559353C allele may be pro tective factors to HBV infection.