Double promoter DNA vaccine expression vector pCMVnir and preparation method thereof

Abstract

The invention is an double-starter DNA vaccine expression carrier pCMVnir and its preparing method. The expression carrier pCMVnir is double stranded closed-loop molecule, a gene cloning carrier, and the length is 5.1Kb, used to express to prepare genetic engineering product and produce DNA vaccine, containing two starters, one is an eukaryoti cell starter (HCMV IE), and the other is an escherichia coli nitratase gene starter nirB, the two starters are activated in cell and prokaryotic cell to express the corresponding proteins, respectively, it contains polyclonal restriction site where the external source is inserted, and the carrier has the sequence shown in SEQ ID NO 1.It can be widely used to high-efficacy expression of external source gene and need not add inducer to the culturing system.

Description

(1) Technical field [0001] The invention relates to a DNA vaccine double-promoter expression vector pCMVnir matched with intracellular parasitic bacteria, also known as pCN and a preparation method thereof, belonging to the technical field of DNA vaccines in life science and medicine. (two) background technology: [0002] DNA vaccine is a eukaryotic expression plasmid carrying antigen gene constructed by DNA recombination technology. It is a third-generation vaccine with wide application prospects. A large number of documents have confirmed the feasibility and immunological mechanism of DNA vaccine from multiple angles and multiple directions. , 7 nucleic acid vaccines have been approved by the FDA to enter clinical trials. However, as a new technology, DNA vaccine still faces some problems that need to be solved. One of the important problems is that the induced immune response is not as strong as expected. Improving the immunity of DNA vaccine is one of the topics that nee...

AI Technical Summary

Problems solved by technology

However, the current DNA vaccine expression plasmids that use intracellular parasites as the immunization route still have certain defects, that is, the commonly used DNA vaccine vectors are all eukaryotic expression plasmids, which cannot express antigens in prokaryotic cells

Salmonella-carried DNA vaccines do not express antigens for a long time before entering intestinal cells, which undoubtedly affects the expression of antigens and the ability to induce immune responses

In order to solve these problems, it is necessary to construct a new double-promoter DNA vaccine vector that matches intracellular parasites, so that the antigen gene can be expressed in both bacteria and host cells without affecting their stability, while the commonly used prokaryotic cells Promoters T7, T5, Lac, Tac and other chemically induced and temperature induced promoters are not suitable for in vivo use, because intracellular parasitic bacteria do not contain the regulatory system of the E. coli operon, and the above promoters will continue to express uncontrollably after entering, seriously Affect the life of bacteria, so that the bacteria and the plasmids they carry are very bad and lost. The in vivo-inducible promoter can solve the above problems. According to the current research results of recombinant bacterial multivalent vaccines, the bacterial anaerobic inducible promoter nirB is anaerobic in bacteria. Oxygen environment can start the expression of antigen gene. Bacterial anaerobic environment-induced promoter nirB is isolated from Escherichia coli, and guides the synthesis of nitrate reductase gene NirB (nitrite reductase) in bacteria. Its inducible expression method is through Add nitrogen to the medium or change the oxygen saturation in the culture system. In the artificially synthesized NirB promoter, due to the artificial deletion of the nitrogen element, all NirB promoters are no longer affected by the nitrogen concentration in the medium. Influence, nirB is a promoter activated by the internal environment of bacteria, that is, an in vivo-inducible promoter, see Oxer MD et al., High-efficiency expression of exogenous genes by the anaerobic-inducible promoter nirB in Escherichia coli, Nucleic Acid Research, 1991; 19 (11): 2889-2892 (Oxer MD, et al. High level heterologous expression in E. coli using the anaerobically-activated nirB promoter. Nucleic Acid Research, 1991; 19(11): 2889-2892)

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