A Study On The Expression Of Proteins Composed Of Two Subunits Using Recombinant Adeno-associated Virus Vectors

A study on the expression of proteinscomposed of two subunits usingrecombinant adeno-associated virus vectorsMaster candidateHao XiaomengSupervisorShu Siyun Hou YundeCo-mentorWu XiaobingAbstractGene therapy is a new technology aimed at correcting the deficiency of human genes or curing human diseases by supplying targeted cells with normal or therapeutic genes. The success of gene therapy will depend on the ideal combination of therapeutic genes, gene delivery system and expression of the fUnctional genes. In vivo expression of protein that consists of several subunits is a problem in the field of gene therapy. The conventional methods for expressing such kind of proteins include placing the two subunits genes under the control of two different promoters or combing the two genes by the internal ribosome entry site (IRES). However, there are some defects in these methods, which need to be improved.Adeno-associated virus-2 (AAV-2) vector represents a class of small, non-enveloped, replication-defective DNA viruses that are being developed for human gene therapy. Recombinant adeno-associated virus(rAAV) has been used successfully in establishing long-term gene expression without substantial immune response or toxicity in both dividing and non-dividing cells in vivo. In this study, using human interliukin-12(h]IL-12) and epidemic hemorrhagic fever (EHIF) as the models, we tried to explore the feasibility of expressing proteins that composed of two subunits by two separate rAAV vectors.rAAV vectors plasmids carrying either the 1L12 p35 or p40 subunit gene, were constructed by inserting the genes into the multiple cloning sites of pSNAV. Transfection of BI-IK-21 cells with pSNAV-IL-12-p35 or pSNAV-LL-12-p40 and selection of the cells using G418 lead to the generation of neomycin resistant rAAV vector cell lines. Infecting the celllines with an rHSV helper virus resulted in the generation of rAAVs carrying 1Ll2-p35 or 1L12-p40 expression cassette, designated rAAV-1L12-p35 and rAAV-1L12-p40 respectively. For expression of functional LL-12 protein, I3HIK-2 I cells were co-infected with the two rAAVs. 4Shr later, the supernatants were collected for ELISA and biological analysis for IL-12. The data showed that 10.l85pgIml of hIL-12 p70 were produced as measured by ELISA. Biological active hIL-12 proteins could also be detected by stimulation the secretion of interferon- I in vitro. rAAV-1L12-p35 and rAAV-IL 12p40 obtained in the experiment have potential use for the treatment of tumors by local injection.Similarly, rAAVs carrying the genes of light and heavy chains of EFH virus NP antibody were obtained respectively. 48hrs after the co-infecting BHK-2 ~ cells with the two rA.AVs, the media was collected for immunot]uorescent staining assay. The results confirmed the formation of integrate EFI-1 virus NP antibody.Compared with the conventional methods for expressing such kind of proteins, the strategy in this study provides some advantages. First, it mimics the natural expression fashion of such proteins in that the two- subunit genes were constructed into two different vectors, which avoided the mutual interference between the promoters. Second, the proportion of the two subunits can be adjusted by adjusting the proportion of input rAAVs, so that the optimal yield of functional proteins is readily achieved.In this study, hIL-12 and EFH virus NP antibody were successfully expressed by using two separate rAAV vectors, which further confirmed the feasibility for the formation of functional proteins by separate expression of their subunits The firstly established method to express hIL-12 has its potential clinical value. The strategy to express antibody by separate expression of heavy and light chains was also firstly reported and provided the experimental basis for clinical use.