| The hepatitis B virus(HBV) is a double-stranded DNA virus which can causeacute or chronic hepatitis of human and made great harm to people.The mechanism of HBV infection the liver cells is not yet entirely clear,andvaccination is the most direct and effective measures to control the transmission HBV.During HBV infection, the liver cells secrete a large number of20nm outer membraneprotein particles, the same particles produced by cell lines or yeast is the vaccine. Thehepatitis B vaccine experienced a hepatitis B viral envelope protein recombinantvaccine; the main outer membrane protein of hepatitis B virus,protein and recombinantvaccines; HBV DNA vaccine process. These vaccines can effectively reduce theincidence of HBV mother-to-child transmission, however, there are still someproblems in the protections for human subjects rate,the level of antibody response, theimmune persistence. Therefore, looking for more of the immunogenicity of HBVvaccine to become a research focus.The outer membrane of HBV is synthesised by the open reading frame of the Sgene encodes, including three kinds of outer membrane components: a large protein(L), medium protein (M) and the main protein (S).They consist of an open readingframe encoding, respectively using the3initiation codon and a common stop codon.The outer membrane protein is the main protein HBsAg consisted by266amino acids,it is carry all the antigenic reactivity of structural proteins. M is the expansion plus55amino acids N-terminal from the S. pre-S2protein is the most immunogenic in theouter membrane of three protein components. L is the expansion plus108-109aminoacids N-terminal from the M. The most important parts of mediated is pre-S1proteinN-terminal21-47peptide when the virus attaches itself to the liver cells.The pre-S1/AA21-47section is more conservative, as long as this period is complete, thevariability of the virus will contagious. At present, the main strategy to improvevaccine immunogenicity in build HBV protein vaccine based on other protein components, such as pre-S1protein, pre-S2protein and C protein.This topic is divided into three parts, main contents and conclusions are asfollows:Part I: HBV/preS1-preS2-HBsAg eukaryotic expression vector and its expressionin293cells. According to the transmembrane features of outer membrane proteinartificially divided the outer membrane protein into six sections and six pairs ofprimers. After PCR amplification, amplification products in the former S1, pre-S2coding region of the5’end by adding urokinase original signal peptide, six histidine(HIS)respectively,3’end by adding the HA tag coding sequence and the EGFPfragment,these PCR products containing the coding sequence of the transmembraneregion. As the HBV pre-S1amino terminus anchored in the membrane, theexperimental design was amplified by PCR products do not contain pre-S1protein N-terminal11amino acid coding region. Constructed vector transfected into humankidney epithelial293cells, in order to observe the green fluorescence, Westernblotting and laser scanning confocal and other methods to detect protein expression ofcells inside and outside. Finally obtained higher expression of the target protein ofHBV/(the preS1-preS2-HBsAg) of pcDNA3.1/293cell lines, provides a reference forthe second part discusses the study of therapeutic vaccine.Part II: Construction of HBV/(preS1-preS2-FC)、HBV/(preS1-FC) eukaryoticexpression vector and expression in293cells and purified. According to the tips andresults of the first part of set overlap extension PCR method to connect the signalpeptide and the target gene, encoding HBV S1antigen, S2antigen and connected intocontaining the human IgG1Fc fragment of the pcDNA3.1expression vector. Theaddition of human IgG1Fc fragment can increase the immunogenicity. Use cationicliposome transfected into293cells, positive clones were screened by G418pressure.Finally obtained an higher expression of target protein HBV/preS1-preS2-Fc, HBV/(preS1-FC) pcDNA3.1/293cell lines. Lay the foundation for the next vaccineevaluation. |
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