Construction of attenuation salmonella bacterin with Vp3 and TRAIL co-expression plasmid

A Salmonella, double-expression technology, applied in the construction of attenuated Salmonella, the application field in the treatment of tumors, can solve the problem of not being able to meet the nutritional needs of bacteria, and achieve the effects of being suitable for in vivo experiments, less toxic and side effects, and simple production processes

Inactive Publication Date: 2009-01-07
向廷秀
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Pawelek et al. believe that the substances required by the auxotrophic strains used, such as aromatic amino acids, are very low in normal cells, which is not enough to meet the nutritional needs of bacterial growth, but they are abundant in the "necrotic area" of tumors where cells are constantly dividing. Causes a relative concentration of bacteria; it is also possible that the presence of certain adhesion molecules leads to the targeting of bacteria to tumor cells

Method used

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  • Construction of attenuation salmonella bacterin with Vp3 and TRAIL co-expression plasmid
  • Construction of attenuation salmonella bacterin with Vp3 and TRAIL co-expression plasmid
  • Construction of attenuation salmonella bacterin with Vp3 and TRAIL co-expression plasmid

Examples

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Effect test

Embodiment 1

[0050] This example describes the construction process and expression in mammalian cells of the attenuated Salmonella SL7207 strain (pBud-Vp3-TRAIL-EGFP / SL7207) provided by the present invention, and includes the following steps:

[0051] 1. Construction of pBud-EGFP

[0052] (1) Digest pEGFP and pBudCE4.1 with XbaI, recover the 757bp EGFP fragment and the large fragment of the linearized vector, and then connect them to construct the recombinant plasmid pBud-EGFP;

[0053] Both the vector and EGFP were digested with XbaI, and the reaction system was as follows:

[0054] vector EGFP

[0055] DNA 10uL 10uL

[0056] KpnI 1uL 1uL

[0057] 10×buffer 2uL 2uL

[0058] wxya 2 O 7uL 7uL

[0059] total volume 20uL 20uL

[0060] Mix well, and place in a 37°C water bath for enzyme digestion for 6 hours.

[0061] (2) Recovery and purification of digested products (Omiga DNA Gel Recovery Kit)

[0062] The operation was carried out according to the instructions, and the 757bp EGFP ...

Embodiment 2

[0171] This example describes the inhibitory effect of the attenuated Salmonella SL7207 strain (pBud-Vp3-TRAIL-EGFP / SL7207) provided by the present invention on tumor cell growth, and includes the following steps:

[0172] Observation indicators:

[0173] 1. Experimental study on apoptosis of gastric cancer cells induced by SL7207 / pBud-Vp3-TRAIL strain

[0174] 1) Detection of cell growth state MTT method was used to detect the changes in the growth state of gastric cancer cells after the expression product of pBud-Vp3-TRAIL-EGFP / SL7207 strain was acted on at different concentrations and at different times.

[0175] Take the cells in the logarithmic growth phase, digest with 0.25 trypsin and 0.02% EDTA, make a single cell suspension, and use 5.0×10 4 / ml concentration was inoculated in a 96-well plate, 200 μl per hole, and the experiment was divided into 3 groups, namely the control group, the empty bacteria group (SL7207), the experimental group (pBud-vp3-TRAIL-EGFP / SL7207),...

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Abstract

The invention relates to an attenuated salmonella of a double expression plasmid which carries Vp3 and TRAIL as well as the applications thereof in constructing and preparing a gene treatment medicine for curing or preventing knub. Not only the body of the invention can restrain the knub from growing, but also can be used as a foreign gene excellent expression carrier to submit the characteristic of a pharmaceutical protein in a knub cell to insert the Vp3 and the TRAIL into the double expression plasmid by combining the tumoraffin of an invasion bacterial with the knub characteristics of the Vp3 and the TRAIL for inducing apoptosis, forms a strain of pBud-Vp3-TRAIL / SL7207 by taking the attenuated salmonella as a carrier and researches the effect and mechanism for resisting the knub inside and outside a body by a mode of gastric cancer. Test inside and outside the body shows that the co-expression of the Vp3 and the TRAIL can play the better coordination effect for restraining the growing of the knub.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the construction of an attenuated Salmonella carrying pBud-Vp3-TRAIL-EGFP double expression plasmid and its application in treating tumors. Background technique [0002] For nearly half a century, tumors have become a common disease that seriously threatens human health. According to estimates by the World Health Organization (WHO) and the American Society of Clinical Oncology (ASCO), there are 10 million new cancer patients worldwide every year, and 6-7 million patients die from cancer, accounting for 12% of the deaths. . The fight against cancer has been intensified over the past few decades using surgery, radiation therapy and chemotherapy, but the results have so far been disappointing. Therefore, people continue to explore new methods of tumor treatment. With the rapid development of molecular biology research, the mechanisms of cell growth, differentiation, and apoptosis are...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K35/74A61P35/00C12R1/42
Inventor 向廷秀曹红丹李彦王丕龙刘少宁吕琳王丽娟赖国旗姜政
Owner 向廷秀
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