Construction and expression for tumor immunity synergistic carrier pBVIM
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A carrier and tumor technology, applied in the fields of genetic engineering and immunology, to avoid degradation, simple and easy process, and increase translocation
Inactive Publication Date: 2007-10-17
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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The mature peptide of 111 amino acids contains 4 cysteine residues and has 21%-33% homology with other h
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example 1
[0037] Example 1, construction of expression vector pBVIM
[0038] 1. Determine the molecular order of immune potentiating elements
[0039] In the present invention, active small molecules such as SLC, PADRE, penetrating peptide, phagocytic release agent and cationic peptide are selected as the backbone gene of the carrier, and at the same time, multiple cloning restriction sites are inserted into the gene sequence combination, and the linked gene is inserted into the carrier pBV220 A schematic diagram of the structure (see Figure 1).
[0040] 2. Gene synthesis of vector immune potentiating elements
[0041] The tetrapeptide sequence is placed at the starting position of the N-terminal of the vector for MARV, which can improve the expression ability of the vector itself. The gene of SLC was retrieved from GENEBANK on the Internet. The signal peptide sequence of 69 bp was removed, and the mature peptide sequence of 333 bp was selected as the selected gene. The multiple cloni...
example 2
[0059] Example 2. The expression of the antigen gene after it is inserted into the vector
[0060] 1. Obtain antigen gene by PCR method
[0061] In order to verify whether the designed vector of the present invention can efficiently express genes, we selected an antigen gene expressed in our laboratory to insert into the vector pBVIM to verify the expression. The gene is a core streptavidin gene with a full length of 387bp. Design primers with Xho I and Xba I restriction sites at both ends:
[0062] F1 GGT GGT GGA TCT GGTGCTGCTGAAG
[0063] R1 ACC TCC ACC ACT GGAGGCGGCGGAC
[0064] The PCR amplification system is: 10×Buffer 5μl, primers (20pmol / μl) R1, F1 1μl each, 4dNTPs (2.5mM) 4μl, Taq DNA polymerase (5U / μl) 0.5μl, add water to 50μl. (Taq DNA polymerase, Buffer, and dNTPs were all purchased from Tacara Biotechnology Co., Ltd.). PCR amplification reaction: 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, a total of 30 cycles.
[0065] The PCR product was purifie...
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Abstract
Disclosed is a chimeric expression vector pBVIM with full length of 4338bp comprising most of the sequences of plasma pBV220 and some of small molecular immunologic adjvant gene, wherein SLC (secondary lymphoid chemokine) is the secondary lymph chemotactic factor, PADRC is the universal DR allclomorphic gene epitope, the cationic peptide can combine with the nucleic acid, membrane-penetrating peptide and phagosome releaser by electrostatic adsorption so as to increase the property of transfer of exogenous gene to the interior of tumor cell and resistance of degradation. Moreover, a tetrapeptide sequence for enhancing the expression of exogenous gene expression and a multi-clone enzyme cutting sites to facilitate the insertion of exogenous gene are also incorporated. The vector is used mainly in expression of tumor specific antigen, it can upgrade the immunogenicity of the antigen, promote the presentation and recognition of tumor antigen, and collect active cell in the local part of the tumor, create suitable immune micro-circumstances thereby promoting the immunity utilized bio-treatment on tumor.
Description
Technical field: [0001] The present invention relates to multiple fields such as genetic engineering and immunology, and specifically includes an expression vector pBVIM with immune enhancing function and its construction and expression. function in. Background technique: [0002] Malignant tumor is a deadly killer that seriously threatens human health in today's world. At present, although surgery can effectively remove tumors visible to the naked eye, it cannot guarantee that all tumor cells are removed, nor can it change the internal environment in which tumors occur; in addition to serious toxic and side effects, radiotherapy and chemotherapy are not suitable for many tumors. Sensitive or resistant. In addition, tumor recurrence and metastasis are important factors affecting the prognosis of patients, and they are also the most dangerous stage in the occurrence and evolution of malignant tumors. It involves a series of complex and multi-step changes, which make the cur...
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IPC IPC(8): A61K48/00C12N15/85A61P35/00
Inventor 于继云阎瑾琦修冰水刘荷中贾锐黄悦陈兴刘国栋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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