| More than 350 million people are persistently infected with HBV, making HBV one of the most hazardous viral pathogens for humans and a global public health concern. Chronically infected individuals are at a risk of 15-25% of dying from HBV-related complications such as end-stage liver cirrhosis or hepatocellular carcinoma, accounting for over one million deaths annually. So it is important to treat of patients with HBV chronic infection.In chronic HBV infection, the CTL response is weak and has a limited repertoire. In mice and inhuman primate animal experiments have demonstrated that DNA vaccines can stimulate CTL response. But using as therapy vaccine, the clinic effects are not observed.Human Flt3 ligand (FL) stimulates early hematopoiesis by activating a type â…¢ tyrosine kinase receptor on primitive bone marrow stem cells. Which is found and cloned in 1993. It is one of critical factors inducing dendritic cell (DC) to different and proliferation. Because DC is a professional antigen presentation cell (APC) in procession of immune response, the role of FL in immune response was attention. Current research has showed that cytokines, such as IL-2, IL-12, GM-SCF et,have immunomodulatory effects, which has been demonstrated in HCV, HIV, tumor vaccines.Reviewing current research mentioned above, we presumed that the DNA vaccine of HBcAg combined with FL as adjuant should have a better immune response. So we propose to construct bicistronic DNA vaccines containing hepatitis B virus core antigen and extracellular fragment of Flt3 ligand genes. Using polymerase chain reaction (PCR) to introduce cloning sites to target genes, we insect HBcAg and FL genes into pIRES vector which is a bicistronic plasmid including internal ribosome entry site (IRES) sequences. Then IRES and gene fragments from digested bicistronic plasmids were cloned to pJW4303, another vector, to construct bicistronic DNA vaccines. After screening and identifying destination DNA vaccines, 293T cells were transfected in order to examine HBcAg and FL genes expression levels. Results are showed brefly as below:1. Designed primes according multi-cloning sites in pIRES vector and target genes sequences using computer. Then amplified gene fragments using those primes and cloned PCR product to T vector in order to sequence amplified genes. Got gene fragments by digesting correct clones and cloned HBcAg, FL genes to upstream and downstream IRES of pIRES vector. After screening and identifying destinationplasmids, we successfully constructed bicistronic plasmids which named after pIRES/FL/C and pIRES/C /FL.2. Using computer, we designed primes according cloning sites in pJW4303 vector and HBcAg and FL genes sequences. Then PCR product amplified gene fragments using those primes were cloned to T vector in order to sequence target genes. Got gene fragments by digesting correct clones and cloned HBcAg, FL genes to pJW4303 vector between cloning site of HindIII and BamHI. By screening and identifying destination DNA vaccines, we successfully constructed bicistronic DNA vaccines which named after pJW4303/FL/C and pLW4303/C /FL.3. After quantifying the destination DNA vaccines, which were constructed by methods above mention, we transfected 293T cells with DNA vaccines by liposome that is transient expression system in order to examine HBcAg and FL genes expression levels. After 293T cells were transfected with DNA vaccines, we collected supernatant of cell culture and 293T cells. The supernatant of cell culture was used to examine expression level of FL by ELISA kits. The lytic products of 293T cell were centrifuged and HBcAg was examined by western blot method. The results showed thatbicistronic DNA vaccines expressed both HBcAg and extracellular FL fragment in vitro and expression levels of gene located upstream IRES were higher than downstream. |
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