Recombinant adeno-associated virus vector carrying human sperm protein 17 antigen gene and application value thereof

[0037] Example 1, Construction and identification of SP17 recombinant adeno-associated virus vector

[0038] 1. Materials and their sources:

[0039] 1. Four kinds of AAV type 2 (AAV-2) pBR322 plasmids (pBR-AAV2) with different promoters: the plasmids were successfully constructed by the inventors (see Chinese patent ZL201110125683.X, 0056-0059 section pBR-AAV2 plasmid reconstruction , PCR amplification of the promoter, inserting the amplified promoter into the rebuilt pBR-AAV2 plasmid, etc.). The four promoters are AAV p5 promoter (AAV p5), macrophage virus (CMV) promoter (CMVp), SV40 early promoter (SV40p) and human β-actin (β-actin) promoter ( β-actinp). The feature of this plasmid is the complete repeat terminal segment (TR) sequence at both ends, and a fragment CTGCGCTGG consisting of 9 nucleotides is inserted at the 75th nucleotide sequence of both ends TR, the purpose is to improve the recombinant AAV virus ( rAAV) and improve the replication efficiency of the virus,...

[0052]Embodiment 2, the preparation of the viral vector of recombinant adeno-associated virus (rAAV) and titer determination (such as figure 2 shown)

[0053] Materials and their sources:

[0054] A. The recombinant adeno-associated virus plasmid vector carrying the SP17 antigen gene constructed in Example 1.

[0055] B. The auxiliary plasmid pHelper containing the Rep gene and Lip / Cap gene of AAV: constructed by the inventors of this patent application (Liu, Y., Chiriva-Internati, M., Grizzi, F.Salati, E., Roman, J.J., Lim S., and Hermonat, P.L. Rapid induction of cytotoxic T cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector. Cancer Gene Therapy 8:948-957.).

[0056] C. AAVp cell line containing adenovirus genes (E1, E2A, E4, VAI and VAII genes) integrated in the cell chromosome and expressed: established by the Gene Therapy Center of the University of Arkansas...

[0072] Example 3. Experiment of lysing SP17 antigen-positive malignant tumor cells by introducing recombinant adeno-associated virus carrying SP17 antigen gene into monocyte-dendritic cell line

[0073] Materials and their sources:

[0074] A. Recombinant adeno-associated virus carrying the SP17 antigen gene.

[0075] B. AIM-V cell culture medium: purchased from Life Technologies, USA.

[0076] C. Cytokines: colony cell stimulating factor (GM-CSF), interleukin 2, 4 (IL-2, IL-4) and tumor necrosis factor (TNF-α) were purchased from American R&D Corporation.

[0077] D. Primary tumor cells positive for SP17 antigen: tumor cells isolated from tumor tissues of multiple myeloma and renal carcinoma respectively, and immunohistochemistry has confirmed that they are positive for SP17 antigen.

[0078] E. SP17 antigen-negative primary cells: tumor cells isolated from tumor tissues of skin epithelial cell carcinoma, lung adenocarcinoma, kidney cancer and multiple myeloma, immunohistoc...