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
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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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