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Construction and application of TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) recombinant bacille calmette guerin (rBCG)

A technology of recombinant BCG vaccine and BCG vaccine, which can be applied in recombinant DNA technology, medical preparations containing active ingredients, drug combinations, etc., and can solve the problems of reducing clinical BCG dose, side effects, toxic side effects, etc.

Inactive Publication Date: 2012-01-25
沈周俊
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cytokine TRAIL can effectively inhibit the formation and growth of bladder cancer cells, induce tumor cell apoptosis, and cause tumor shrinkage or even disappear, but it has no killing effect on most normal cells. The factor "no distinction between friend and foe" and strong toxic and side effects are incomparable. Therefore, TRAIL is one of the most effective exogenous genes for constructing rBCG. rBCG-TRAIL is expected to become an effective biological agent for treating bladder cancer and reducing clinical BCG dosage and side effects
At present, there is no effective construction of BCG shuttle expression vector pMV261-Ag85B-TRAIL

Method used

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  • Construction and application of TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) recombinant bacille calmette guerin (rBCG)
  • Construction and application of TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) recombinant bacille calmette guerin (rBCG)
  • Construction and application of TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) recombinant bacille calmette guerin (rBCG)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The flow chart of the present invention to construct the BCG shuttle expression vector pMV261-Ag85B-TRAIL can be found in figure 1 .

[0040] 1. Experimental method

[0041] 1. Obtain the target gene: mouse TRAIL gene

[0042] 1.1 Extraction of total RNA in tissues

[0043] 1.1.1 Total RNA extraction

[0044] (1) Take the mouse spleen stored in the -80℃ refrigerator, add 1ml TRIZOL to every 50-100mg of mouse spleen tissue, and homogenize it with a homogenizer; let it stand at room temperature for 5min; (2) add 0.2 ml chloroform, cover the lid, shake fully for 15 sec, let stand at room temperature for 2-3 min, centrifuge at 12000 rpm for 15 min at 4°C; (3) suck the upper aqueous phase into a new tube, add 0.5 ml of isopropanol for every 1 ml of TRIZOL reagent, mix Evenly, let stand at room temperature for 10 min, centrifuge at 4°C, 12000rpm for 10min, pour off the supernatant; (4) wash the RNA pellet with 75% ethanol, add 1ml of 75% ethanol for every 1ml TRIZOL, shak...

Embodiment 2

[0167] Example 2 Construction and identification of recombinant BCG rBCG-TRAIL

[0168] 1. Main reagents and materials

[0169] The BCG shuttle expression plasmid pMV261-Ag85B-TRAIL constructed in embodiment 1; 300 mesh nylon filter screens, produced by Shanghai Industrial Filter Factory; primary antibody (mouse anti-mouse TRAIL monoclonal antibody), secondary antibody (HRP-goat anti-mouse IgG) all Purchased from R&D Company; cell lysate purchased from PIERCE Company of the United States: PVDF membrane from Amresco Company; glycine and SDS from Huashun Company: Mouse TRAIL ELISA kit purchased from ADI Company; BCG liquid medium Middlebrook 7H9 broth, BCG solid culture The base Middlebrook 7H 10 agar and nutritional additives ADC and OADC were purchased from Difco; the rest of the reagents and materials were the same as in Example 1.

[0170] 2. Main experimental instruments

[0171] 96-well and 6-well flat-bottom cell culture plates purchased from Nunc Company of Denmark; ul...

Embodiment 3

[0266] Example 3 Research on Recombinant BCG-TRAIL Immunotherapy

[0267] 1. Main experimental reagents and materials

[0268] Experimental animals: BTT739 mice, 8-10 weeks old, weighing 18-20g, purchased from the Experimental Animal Center of Cancer Hospital, Chinese Academy of Medical Sciences; cell line: mouse bladder tumor cells BTT is the bladder derived from the BBN-induced BTT739 mouse bladder cancer tumor model Cancer cell lines were donated by the Central Laboratory of Shanghai First People’s Hospital, and were passaged in 10% FCS-RPMI 1640 culture medium; human recombinant TRAIL protein was purchased from Sigma Company in the United States; Dimethyl Sulfoxide (DMSO) was purchased from Jiangsu Hong Produced by the Acoustic Chemical Factory; RPMI1640 culture solution: powder, purchased from Hyclone Company of the United States, dissolved in three-distilled water to form 1×RPMI1640, adjusted to pH 7.3, and then sterilized by filtration with a filter membrane (pore diame...

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Abstract

The invention provides construction of TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) recombinant bacille calmette guerin (rBCG), and relates to a shuttle expression vector comprising a signal peptide fragment of a major secretory antigen Ag85B of BCG and a gene fragment of a TRAIL and a construction method thereof. The obtained shuttle expression vector pMV261-Ag85B-TRAIL is used for constructing rBCGTRAIL, and can be applied to preparation of TRAIL rBCG for treating superficial bladder tumors, preventing postoperative recurrence thereof and preventing tuberculosis. The rBCG has dual functions of TRAIL and BCG, so that cooperative and synergistic actions of the TRAIL and BCG can be better brought into play; rBCG-TRAIL can secrete TRAIL, and the using amount of the rBCG-TRAIL can be lower than that of the BCG under the condition that the same or better immune effect is achieved, so that the toxic or side effect is reduced; and the rBCG-TRAIL can directly secrete TRAIL efficiently on a certain part, so that tumor cells can be killed in cooperation with the rBCG-TRAIL, and high cost caused by the use of a foreign cell factor is avoided.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a BCG shuttle expression vector pMV261-Ag85B-TRAIL that secretes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its construction method, namely a The construction and application of TRAIL recombinant BCG, specifically related to a shuttle expression vector comprising the main secretion antigen Ag85B signal peptide fragment of BCG and a TRAIL gene fragment and its construction method, and the obtained shuttle expression vector pMV261-Ag85B-TRAIL is used to construct recombinant BCG rBCGTRAIL , rBCGTRAIL can be used in the treatment of superficial bladder tumors and the prevention of postoperative recurrence and the prevention of tuberculosis. Background technique [0002] Bacille Calmette-Guérin (BCG) intravesical infusion has been recognized as the most effective treatment for superficial bladder tumors and prevention of postoperative recurrence. I...

Claims

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Application Information

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IPC IPC(8): A61K39/04A61K39/00A61P13/10A61P31/06A61P35/00C12N15/74C12N15/66C12R1/32
Inventor 沈周俊陈善闻周卸来章国卫金晓东
Owner 沈周俊
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