Construction And Expression Of An Eukaryotic Vector Encoding PreS-Tat

Background and purposeHepatitis B virus (HBV) infection has become one of the main issues which threat the health of all mankind. It is estimated that there are about 350 million HBV carriers in the world, about 1 million people die each year for the reason of hepatocellular carcinoma, liver cirrhosis, liver failure and other related diseases caused by hepatitis B virus infection. The situation of HBV infection in China is serious, according to incomplete statistics, China's chronic asymptomatic HBV carriers (AsC) may be more than 130 million people, it is about 1/3 of the total number of AsC in the word and accounts for 10% of the total population in China. The incidence of HBV infection has been at the forefront in our country.HBV has an unique structure, it is the smallest DNA virus which infects humans. It consists of two chains which has different lengths to form a double-stranded DNA. HBV viral has a high rate of replication, it is strong adaptative and variable, there are a reverse transcription process in HBV replication, but the DNA polymerase involved in reverse transcription is lack of corrected effection.Thus HBV is prone to mutation. It is difficult and challenging for the prevention, diagnosis and treatment of the disease because of the variation of the virus. In vivo because the lack of spontaneous viral clearance mechanisms. HBV can not be cleared after infection, the most effective method to treat the disease is the use of antiviral drugs. However because restrictions of the drug targets and other factors, there are always drug resistance occur in the HBV.Chronic hepatitis could develop to inflammation and necrosis, fibrosis and cirrhosis and eventually liver failure and hepatocellular carcinoma gradually.It brings the family and society a heavy burden. In this study, we constructed the pEGFP-PreS-Tat chimeric vector and expressed in Hela cells which is the basis to study the specific targeted effecyion of the fusion protein.MethodExtract HB V from the serum of the patients who are clinically diagnosed of hepatitis B virus infection.The PreS sequence was amplified and then cloned into pEGFP-C3 vector through cloning technology.The synthetic Tat sequences were inserted into the downstream of the PreS to construct pEGFP-PreS-Tat chimeric vector. The successful connectted vector was transfected into competent cells, the Western blot method were used for check the expression of the purposed protein.ResultsAfter double digestion and sequencing, we found that we had successfully constructed pEGFP-PreS-Tat chimeric vector, the method of Western Blot was used to detect the purposed protein.ConclusionThe chimeric vector pEGFP-PreS-Tat is constructed and the fusion protein is expressed successfully,which lays the foundation for studying the effection of the functions protein.