Research Of Vaccine About Human Immunodeficiency Virus Genes Recombinant With Poliovirus

Acquired immune deficiency syndrome(AIDS),which is caused by human immune deficiency virus(HIV),is one of the infectious disease infected through sexual contact,blood transfusion.It is characteristic of the functional destruction of human immune system,more and more pandemic in the world.In spite that some medicine is effective on such a disease,it is usually expensive,hard to solve the viral recurrence and resistance.On condition that most of the AIDS patients live in the developing contries,the cheap,convenient,effective HIV vaccine is more urgently needed for them.So many kinds of HIV vaccines have been invented now,including the traditional inactived,attenuated vaccine,and some modern developed vaccine such as nucleic acid vaccine,synthetic peptide vaccine,viral vector vaccine,etc.However,none of them produce the good effect in clinical trial.Such a result perhaps owes much to the highly diversity of HIV,unclear AIDS pathogenesis and a low antibody titer induced by the vaccine.In that most of AIDS is transmited through mucosal surface,the vaccine which could induce the stronger mucosal immune in human body perhaps is a better choice to hinder its popularity.Polivirus is kind of positive single strand RNA virus ,competent in self-replication.The attenuated subtype Sabin strain is proved safe and effective in preventing the poliomyelitis.The vaccine is conveniently taken orally, can induce a good mucosal immune reaction.The research purpose is to develop a practical HIV vaccine based on Poliovirus Sabin I strain integrated with antigen gene of Chinese popular HIV.In the study, the gagprotease gene fragment with appropriate enzyme cleavage site at its both ends is amplified through PCR technique based on gagpol gene cDNA of HIV-1 CN54 strain as the template.After the enzyme cleavage reaction for a eukaryotic vector with poliovirus cDNA, part of the structural gene fragment between two unique restriction enzyme site is removed. Then through enzyme ligation reaction,HIV-1 gagprotease gene fragment is inserted into the cDNA of Poliovirus between two cohesive terminus with its right genetic reading frame.The result is verified later by double digestion that the antigen gene is orientedly placed into the vector genome.The recombined vector then is cotransfected with the other eukaryotic vector with poliovirus capsid genes into the cultured Hela cells,so that the poliovirus would be generated in it.Under the electronic microscope, the new assembly virus within the cells is visible.For a continual observation by light microscope,no cytopathic effect is visible,which indicated that the new recombinant virus is biologically safe,perhaps for no progency virus is produced.In expanded cultured human diploid cell,the recombinant poliovirus HIV vaccine was produced on a large scale.The virus is separated and purified with sucrose density gradient centrifugation technique.Also they were identified using RT-PCR,Western blot method with the results that the recombinant virus carrying the HIV-1 antigen gene and poliovirus capsid protein.The infection capacity of the virus was tested when it was put into Hela cells,and the specific antigen protein was detected in the cells.In addition, HIV-1 DNA vaccine was developed in the research,in which the gagprotease-gene was inserted into the multiple cloning site of another eukaryotic expression vector.After it transfected the cells with liposome,fluorescence immunoassay was adopted to validate the expression of antigen gene.In several randomly allocated transgenic mice with polivirus receptor,the recombinant poliovirus HIV-1 vaccine and DNA vaccine were intranasally inoculated monthly into them,for three times altogether.Finally,humoral immune reaction antibody of IgG and IgA induced by the vaccine was examined by enzyme-linked immunosorbent assay,and the cellular immune reaction of cytotoxic T lymphocyte was also detected by lactate dehydrogenase resleas test. All the data support the fact that recombinant virus vaccine is superior to DNA vaccine in immunogenicity,especial for some indicator which is statistically significant.It is probablely related to the viral competent self-replicating ability,which benefit to express more antigen protein.No toxic effect was found in the tiral animal. Conclusion : The recombinant defective poliovirus with HIV-1 antigen genes entails a very good immunogenicity, will have a better practical prospect as a prophylactic vaccine for pandemic AIDS.