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

A virus carrier and antigen gene technology, applied in the direction of virus/bacteriophage, application, carrier, etc., can solve the problems of insufficient ability, low efficiency, poor effect, etc., and achieve the effect of broad application prospects

Inactive Publication Date: 2020-03-17
深圳益世康宁生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a large amount of research data proves that neither the antigenic protein nor the antigenic peptide stimulates DC efficiently, and leads to the insufficient ability of DC to induce antigen-specific CTL responses, making the effect of anti-malignant tumor and viral infection poor.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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,...

Embodiment 2

[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...

Embodiment 3

[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...

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Abstract

The invention discloses a recombinant adeno-associated virus (rAAV) vector carrying a human sperm protein 17 (SP17) antigen gene and proves the application value of the recombinant adeno-associated virus vector. The rAAV vector is obtained by inserting an SP17 antigen gene into an AAV starting vector constructed by the inventor. The research proves that the virus vector of the rAAV can effectivelyconvey the carried SP17 antigen gene into a human mononuclear cell-dendritic cell line to stimulate effector cells of cellular immunity, that is, generation of SP17 antigen specific cytotoxic T lymphocytes (CTL). The experiment proves that the CTL induced by DC infected by the recombinant adeno-associated virus vector carrying the SP17 antigen gene can effectively split SP17 antigen positive malignant tumor cells. Therefore, the recombinant adeno-associated virus vector carrying the SP17 antigen gene or the related product thereof have practical values and can be used for preparing the SP17 antigen specific CTL; the SP17 antigen positive malignant tumor cells can be killed; and the targeted T cell immunotherapy on anti-SP17 antigen positive malignant tumors can be realized.

Description

【Technical field】 [0001] The present invention relates to vectors in the biological field and applications thereof, in particular to a recombinant adeno-associated virus vector carrying sperm protein 17 (spermprotein 17, SP17) antigen gene and its application in preparing anti-SP17 positive tumor drugs. 【Background technique】 [0002] The gene structure of adeno-associated virus (adeno-associated virus, AAV) has been identified. In 1983, Samulski et al. described the terminal repeat segment of AAV (upstream 5' segment, downstream 3' segment) (Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmamids: gene correction within the terminal repeats of AAV. Cell. 33:135-143.). In 1984, Hermonat et al. described the low infectious particle (lip) gene and envelope (cap) gene of AAV (Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary Characterization of adeno-associ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864A61K39/00A61P35/00
CPCA61K39/001184A61K2039/5256A61K2039/53A61P35/00C12N15/86C12N2750/14143C12N2800/107
Inventor 刘勇江孝青常爱全高雪王圆高洪吉张慧
Owner 深圳益世康宁生物科技有限公司
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