Construction And Expression HIV Multi-epitope DNA Vaccine Containing Replicon Of Semliki Forest Virus

Acquired Immunodeficiency Syndrome (AIDS) is a seriously disease, which is caused by the infection of Human Immunodeficiency Virus(HIV).Since it was firstly found in American in 1981, there have been more than 65 million infectiouscases now. AIDS is still overrunning at remarkable speed, seriously threatens human health, and influences global economic development. The HIV gene is highly variable and can avoid immune supervision, which made traditional vaccine be difficult to produce effective immune protection. Nucleic vaccine which had become a promising AIDS candidate vaccine is a newly immune preventive and curable, which is developed from gene curable domain, and which can induce both cell immune and humoral immune response, and which is safe and flexible in constructing.The vector pSFV1 which is an eukaryotic expression vector was derivatized from the eukaryotic expressionvector of SFV (Semliki forest virus) RNA replicon. But when the system was used. the plasmid DNA should be transcripted into RNA in vitro. For this reason, the manipulation is not convenience, and the extensive usage is restricted. Therefore, we reformed the vector by the technology of molecular cloning, and the nonstructural gene of SFV was inserted into the position, which is downstream of CMVie and upstream of Poly(A)of pIRESneo, which is immediate early promoter and adenylic acid transcriptionstop signals, respectively. Replication form DNA expressing vector pCS was constructed which contained SFV replicon and could be prepared, immuned, used as regular DNA vaccine way, and overcome all of the abuses that the origin vector pSFV1 transcripted and prepared RNA in vitro. pCS-EGFP, which was constructed by inserting EGFP into pCS, was transfected into the BHK-21 cells. The result indicated that EGFP was effective]y expressed in the cells by observed under fluorescent microscope, and the new system can be used in the expression of the exogenous gene.For researching the possibility that developing anti-Virus vaccine with new-style vector pCS, inour research, dominant antigen epitopes of HIV-1 was selected as foundation, and antigen gene was designed by artificial molecular, and the MEG (multipte-epitope gene)was gomphosised with HIV giant molecular granules. Gomphoisi multi-epitipes gene MEGp24 which was cut from constructed and conserved pVAX1-MEGp24, and cloned to expressional carrier vector which in replication form, was successfully constructed HIV multi-epitipes gene DNA vaccine pCS-MEGp24 which contained SFV duplicon. Then, pCS-MEGp24 gene was effectively expressed in transfected BHK-21 cells by IFA test, and the transcript mRNA of which was detested by RT-PCR.In a word, our research proved the foundation of new anti-HIV DNA nucleic vaccine, which confirmed to express objective gene in eukaryocyte, and made a foundation for going on researching and supplying in next step.