Self-replicating vector for DNA immunication against HIV

A vector and recombinant vector technology, applied in the field of preparation of anti-HIV vaccine, treatment or prevention of HIV, can solve the problem that the virus cannot fully replicate due to the virus

Inactive Publication Date: 2001-05-16
OY FINNISH IMMUNOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Experiments with SIV have shown that viruses lacking one of the functions of the above-mentioned reg

Method used

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  • Self-replicating vector for DNA immunication against HIV
  • Self-replicating vector for DNA immunication against HIV
  • Self-replicating vector for DNA immunication against HIV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Cloning of HIV-1 gene REV and TAT into self-replicating pBNsr-α and pBNtk plasmids to produce pBNtkREV and pBNsrαTAT first stage:

[0070] Isolated BRU ( HIV-1 REV and TAT genes in LAI (also known as LAI) (Wain-Hobson et al., Cell 40: 9-17, 1985): REV: 5'-TTTTTCTAGAACCATGGCAGGAAGAAGCGGA-3'5'-TTTTCTCGAGCTATTCTTTAGTTCCTGG-3'TAT: 5 '-TTTTTCTAGAACCATGGAGCCAGTAGATCCT-3'5'-TTTTCTCGAGCTAATCGAACGGATCTGC-3'

[0071] The amplified gene and the pUE83 shuttle vector (Fig. 1) were digested with XbaI and XhoI (New England BioLabs, USA) overnight at 37°C to obtain compatible ends. The digested DNA fragments were analyzed on a 1.5% agarose gel and further purified using a Band Prep kit (PharmaciaBiotech, Sweden). Incubate overnight at 16°C to ligate each gene into the vector with T4 DNA ligase (NewEngland BioLabs, USA). The competent E. coli cells in the One Shot kit (Invitrogen, Netherlands) were transformed with the ligation product, and the transformed cells were plated...

Embodiment 2

[0074] Example 2 Cloning of HIV-1 NEF into a self-replicating pBNsrα plasmid produces the first stage of pBNsrαNEF:

[0075] The HIV-1 NEF gene was obtained from a plasmid pcNEF vector containing the NEF gene of an LAI isolate inserted into a pcTAT vector lacking the TAT gene. The 1.3 kb fragment obtained by digesting pcNEF with SpeI and HindIII can be used for further cloning of the NEF gene. In order to prevent the formation of HindⅢ sites again during ligation, after HindⅢ digestion, the fragments need to be treated with a mixture of Klenow enzyme and dATP, dCTP, dGTP nucleotides, and then digested with SpeI. The resulting fragments were separated by electrophoresis on a 1% agarose gel with markers of standard size. Bands of the correct size were excised and DNA was recovered using the Sephaglas Bandprep kit (Pharmacia Biotech) following the manufacturer's protocol.

[0076] The shuttle vector pNP177 shown in Figure 1B was first digested with XhoI, then treated with Kleno...

Embodiment 3

[0079] Example 3 illustrates the in vitro expression of HIV-NEF 3A. transfection

[0080] To test the expression of the pBN-constructs in Examples 1 and 2, COS-7 cells were transfected with them by electroporation. 10 µg of pBNβ-Gal used as a control, and 10 µg of pBN-NEF co-transfected with 1 µg of pCMVβ-Gal were each electroporated into 3 million cells using salmon sperm vector DNA. Electroporation was performed at a capacitance of 960 mF and a voltage of 260 V, and protein concentration and β-Gal activity were measured to control transfection efficiency and to calibrate the amount of lysate in Western blot experiments. 3B. Immunohistochemistry and Western Blot

[0081] The harvested cells transfected with the pBN-construct were lysed for Western blotting, after lysis, the protein samples were boiled in sample buffer, electrophoresed in a 12% SDS polyacrylamide gel, and then transferred to a 5- % milk in TBS and blocked on a 2 μm nitrocellulose filter. The primary antib...

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Abstract

A nucleotide sequence encoding the HIV regulatory protein NEF, REV or TAT or an immunologically active fragment thereof is inserted into a vector comprising papilloma virus nucleotide sequences necessary and sufficient for long-term persistence. The resulting vectors are self-replicating and have a high copy number. They express the HIV genes in high amounts for a long period of time. The vectors elicit both a humoral and cell-mediated immune response and are therefore potential DNA immunization vaccines against HIV. The invention is directed to said vectors and vaccines and to a method for preparing the vectors. The invention is further directed to a host cell comprising the vector, to the use of the vector in the manufacture of a vaccine and to a method of preventing or treating HIV.

Description

field of invention [0001] The present invention relates to a self-replicating recombinant vector for anti-HIV DNA immunization. The present invention also relates to a vaccine containing the vector, a method for preparing the vector and a host cell containing the vector. The present invention also relates to the use of the vector in The use of preparing the vaccine against HIV and the method of treating or preventing HIV. Background of the invention [0002] Interactions between vertebrates (and thus humans) and pathogenic microorganisms, such as bacteria, fungi and viruses, are regulated by the ability of vertebrates to mount an immune response to invading microorganisms. This response is based on the immune system's ability to distinguish self from non-self; normally, the immune response tolerates the organism's own structures, cells, and antigenic molecules, while attacking foreign antigens expressed by invading microorganisms. When a vertebrate, such as man, is infected...

Claims

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Application Information

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IPC IPC(8): C12N15/79A61K39/00A61P31/18C07K14/16C12N15/48C12N15/49C12N15/63C12N15/85
CPCC12N15/85A61K2039/51C12N2830/42C12N2800/108C12N2840/20C12N2740/16322A61K39/00C07K14/005A61P31/18
Inventor M·塔蒂恩P·彼得森K·克罗P·A·兰克
Owner OY FINNISH IMMUNOTECH
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