The Construction Of Recombined HBV Vector With Fluorescence Labeling

Objective: To construct recombined HBV vectors carrying Fluorescence labeling, which can visual the HBV replication and expression.Methods:1 Using PCR and molecular clone technology to construct plasmid psu CMV-GFP1-10 that express Split-Green fluorescent protein 1-10 fragment, and to construct plasmids p CH-GFP11-Core, p CH-GFP11-Bsd R and p CH-GFP11-S2 that express split GFP11 fragment.2 psu CMV-GFP1-10 was respectively cotransfected hepatic carcinoma cell line Hep G2 with the vector plasmids p CH-GFP11-Core, p CH-GFP11-Bsd R and p CH-GFP11-S2 to observe the intensity and character of the expressing fluorescent.3 The vector plasmids p CH-GFP11-Core, p CH-GFP11-Bsd R 和p CH-GFP11-S2 were transfected into Hep G2 cells separately, using southern blot to analyze the abstracted DNA in the lysate at the fifth day.4 The vector plasmid p CH-GFP11-Core and control plasmid p CH-9/3093 which expressed wild-type HBV were transfected into Hep G2 cells separately, using native western blot to analyze the HBV Core particles formed in the lysate at the fifth day.5 Using psu CMV-GFP1-10 and p CH-GFP11-Core to cotransfect Hep G2 cells, nucleocapsid inhibiting drug Bay41-4109 adding after 24 h, In contrast, inactive drug Bay41-4842 was adding at the same concentration. observing the changes of fluorescent after 24 h.6 Using plasmid psu CMV-GFP1-10 to cotransfect Hep G2 cells with p CH-GFP11-Core、p CH-GFP11-Bsd R 和 p CH-GFP11-S2 separately, with or without p BS-sh HBV6 which expressed Short hairpin RNA, using microscope to observe changes of fluorescent inside the cell after 48 h.Results:Part.1 The study of split GFP11 fused with the N terminal of HBV core protein1 The construction of recombined plasmid p CH-GFP11-Core. The plasmid p CH-9/3093 which expressed wild-type HBV as vector. The GFP11 sequences were inserted to the N terminal of HBV core protein. GFP11 sequences CGG GAC CAC ATG GTG CTG CAC GAG TAC GTG AAC GCC GCC GGC ATC ACA were inserted into the HBV core protein N terminal ninth nucleotides later, linkered them with a short Hydrophilic peptide GGC GAC GGC GGC AGC GGC GGC GGa tc C GGT to form GFP-Core fusion protein. The vector plasmid p CH-GFP11-Core was confirmed by restriction endonuleases digestion and sequence determination.2 The recombined HBV Fluorescent expression: plasmids psu CMV-GFP1-10 cotransfected Hep G2 with the vector plasmids p CH-GFP11-Core, observed strong green fluorsence inside the cell Under the fluorescence microscope, mainly in nucleus. This result was consistent with that recognized HBV core protein normal distribution.3 The form of recombined HBV Core particles: The vector plasmid p CH-GFP11-Core and control plasmid p CH-9/3093 which expressed wild-type HBV were transfected into Hep G2 cells separately, agarose gel electrophoresis by cell lysates directly, transgenic PVDF membrane, detected recombined HBV Core particles using mc312 PO antibody. The electrophoretic position were was similar in two groups, which demonstrate GFP-Core fusion protein can form integrated core particles.4 The replicate capability of recombined HBV: The vector plasmid p CH-GFP11-Core and control plasmid p CH-9/3093 which expressed wild-type HBV were transfected into Hep G2 cells separately, using southern blot to analyze the abstracted DNA in the lysate. There was no HBV replicative intermediate in the vecter plasmid p CH-GFP11-Core, the control plasmid p CH-9/3093 could be detected HBV DNA.5 The influence of Bay41-4109 on HBV core protein by fluorescent traking: plasmids psu CMV-GFP1-10 cotransfected Hep G2 with the vector plasmids p CH-GFP11-Core, nucleocapsid inhibiting drug Bay41-4109 adding after 24 h with concentrate 1μM, In contrast, inactive drug Bay41-4842 was adding at the same concentration. In Bay41-4109 group, fluorescence express in nuclear membrane, no change in contrast group, still in nuclelus.6 The inhibition effect of sh RNA: plamid p BS-sh HBV6 that express Short hairpin RNA constructed and preservative in our laboratory was cotransfected Hep G2 cell with plasmids psu CMV-GFP1-10 and p CH-GFP11-Core. The intensity of fluorescent significantly decreased along with less fluorescent cells compared with no p BS-sh HBV6 group.Part.2 The study of split GFP11 fused with Blasticidin resistance gene to express HBV core protein and P protein1 The construction of recombined plasmid p CH-GFP11-Bsd R. The plasmid p CH-Bsd R that our laboratory prophase constructed as vector. split GFP11 fused with Blasticidin resistance gene to express between HBV core protein and P protein. The sequences GFP11 and linker were the same as part.1. The vector plasmid p CH-GFP11-Bsd R was confirmed by restriction endonuleases digestion and sequence determination.2 The recombined HBV Fluorescent expression: plasmids psu CMV-GFP1-10 cotransfected Hep G2 with the vector plasmids p CH-GFP11-Bsd R, observed strong green fluorsence inside the cell. The fluorescence was distributed equally between cytoplasm and nucleus.3 The replicate capability of recombined HBV: The vector plasmid p CH-GFP11-Bsd R and control plasmid p CH-9/3093 and p CH-Bsd R were transfected into Hep G2 cells separately, using southern blot to analyze the abstracted DNA in the lysate. Quantify all the total HBV DNA signal and using as 100%, comparing other transfection group with p CH-9/3093 to get the relative signal intensity(% of co). In p CH-GFP11-Bsd R transfection group, the HBV DNA strip distribution trend is similar to p CH-9/3093 group. After quantifying the graph data by computer,in the total DNA level, p CH-Bsd R group’s HBV DNA is p CH-9/3093 group’s 40-45%, p CH-GFP11-Bsd R group’s HBV DNA is decrease slightly, but still up to p CH-9/3093 group’s 20%.4 The inhibition effect of sh RNA: plamid p BS-sh HBV6 that express Short hairpin RNA constructed and preservative in our laboratory was cotransfected Hep G2 cell with plasmids psu CMV-GFP1-10 and p CH-GFP11-Bsd R. The intensity of fluorescent significantly decreased along with less fluorescent cells compared with no p BS-sh HBV6 group.Part.3 The study of split GFP11 fused with HBV pre S2 protein1 The construction of recombined plasmid p CH-GFP11-S2: The plasmid p CH-9/3093 which expressed wild-type HBV as vector. The GFP11 sequences were inserted HBV pre S area. The sequences GFP11 and linker were the same as part.1. The pre S1,S2 initiation codon ATG mutated ACG, GFP11 and pre S2 formed fused protein inserted Pre S2 starting site. The vector plasmid p CH-GFP11-S2 was confirmed by restriction endonuleases digestion and sequence determination.2 The recombined HBV Fluorescent expression: plasmids psu CMV-GFP1-10 cotransfected Hep G2 with the vector plasmids p CH-GFP11-S2, observed week green fluorsence inside the cell. The fluorescence was diffused distribution in cytoplasm.3 The replicate capability of recombined HBV: The vector plasmid p CH-GFP11-S2 and control plasmid p CH-9/3093 were transfected into Hep G2 cells separately, using southern blot to analyze the abstracted DNA in the lysate. Quantify all the total HBV DNA signal and using as 100%, comparing other transfection group with p CH-9/3093 to get the relative signal intensity(% of co). In p CH-GFP11-S2 transfection group, the HBV DNA strip distribution trend is similar to p CH-9/3093 group. After quantifying the graph data by computer,in the total DNA level,p CH-GFP11-S2 group’s HBV DNA is p CH-9/3093 group’s 95%. The result suggested that the HBV vector does not affect HBV replication.4 The inhibition effect of sh RNA: plamid p BS-sh HBV6 that express Short hairpin RNA constructed and preservative in our laboratory was cotransfected Hep G2 cell with plasmids psu CMV-GFP1-10 and p CH-GFP11-S2. The intensity of fluorescent significantly decreased along with less fluorescent cells compared with no p BS-sh HBV6 group.Conclusions:By green fluorescent protein complementation technology, insert the 48 bp small GFP11 fragment to different regions of HBV genome and construct three recombinant HBV vectors:1 By inserted into HBV core protein N terminal, the GFP11 was fused with HBV core protein, and could express high intensity fluorescence, form the core protein particles, but lose the ability of HBV replication. The vectors can be used to observe the polymerization of HBV core protein and research some drugs to inhibit HBV m RNA.2 The GFP11-Bsd R fused protein, inserted between HBV core protein and P protein, could express high intensity fluorescence and the HBV retain about 20% replication capacity. The vectors can be used to research inhibiting drugs of HBV replication.3 The fluorescent express of GFP11 fused with HBV pre S2, which inserted into HBV pre S2 area is weak, but the HBV retain about 95% replication capacity. The vector can be used to observe HBV replication inhibiting drug.