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Recombinant BCG and its preparation method and application

A technology of recombinant BCG and BCG, which is applied in the field of medicine, can solve the problems of insufficient capacity of bladder tumor cells and epithelial cells, low immune response, and aggravated adverse reactions, so as to enhance local innate immune effect and adaptive immune effect, high Adhesion internalization force, beneficial to the effect of inhibiting or eradicating tumors

Active Publication Date: 2022-02-08
XUZHOU HAIRUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Increasing the dose of BCG can enhance its anti-bladder tumor effect, but it will also cause local inflammatory reactions, resulting in significant local irritation symptoms such as frequent urination, urgency, and painful urination. The severity of adverse reactions is positively related to the dose of BCG.
At present, the biggest problem with infusion of BCG in the treatment of bladder cancer is that the ability of BCG to adhere to bladder tumor cells and epithelial cells is insufficient and the selectivity is low, which may lead to a low immune response that is not conducive to anti-tumor. If the dose is simply increased, the adverse reactions may be significantly aggravated. Including frequent urination, urgency, painful urination, fever and even systemic infection

Method used

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  • Recombinant BCG and its preparation method and application
  • Recombinant BCG and its preparation method and application
  • Recombinant BCG and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] A recombinant BCG can express FimH protein on its surface, so as to use the FimH protein expressed on its surface to specifically bind to an object that has the property of binding to the FimH protein. Therefore, the recombinant BCG can theoretically play a positive and significant role in any occasion that requires the specific binding of the BCG to the target through the FimH protein.

[0063] The recombinant BCG can express FimH protein on the surface, so that it can better bind bladder cancer specifically. Such as figure 1 As shown, the surface of bladder cancer cells is rich in mannose residues, and the FimH protein expressed on the surface of recombinant BCG can bind to mannose residues, thereby enhancing the specificity of recombinant BCG binding to bladder cancer cells and improving the immune efficacy. figure 2 It is the protein co-crystal structure of FimH and mannose residues, and the part in the circle is the domain of FimH recognizing mannose residues.

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Embodiment 2

[0079] To construct rBCG-S.FimH expressing FimH protein on the surface of the capsule, select the pMV261 plasmid with kanamycin resistance, insert the 19ss anchor protein behind the promoter HSP60, and construct pMV261-S.EGFP and pMV261-S. FimH-EGFP, pMV261-S.FimH and other plasmids were constructed by electrotransfection to construct rBCG-S.EGFP, rBCG-S.FimH-EGFP, rBCG-S.FimH. Kanamycin resistance screened the best monoclonal strain, and observed the fluorescence of the recombinant plasmid under a green fluorescence microscope; verified the transcription of the recombinant plasmid at the cDNA level by RT-PCR experiments; Verify the protein expression of recombinant BCG. see figure 2 , 4 and 5, Figure 5A Under 488nm fluorescence excitation, both rBCG-S.EGFP and rBCG-S.FimH-EGFP can emit green fluorescence; Figure 5B In order to extract the BCG capsule protein, it was found that most of the recombinant proteins could be expressed on the membrane, which indirectly proved th...

Embodiment 3

[0082] Incubate BCG-S.EGFP and recombinant BCG-S.FimH-EGFP with human bladder cancer cell line 5637 for 4 hours, or add D-mannose competitive inhibition to rBCG-S.FimH-EGFP for 4 hours Afterwards, the culture medium was discarded, the nuclei were fixed and stained with DAPI, and confocal photographs were taken. The recombinant BCG rBCG-S.EGFP and rBCG-S.FimH-EGFP were obtained according to the method in the summary of the invention, and the human bladder cancer cell line 5637 was obtained from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Such as Figure 6A and 6C as shown, Figure 6A and 6C It is the fluorescence diagram of the binding status of BCG rBCG-S.EGFP and recombinant BCG rBCG-S.FimH-EGFP respectively incubated with human bladder cancer cell line 5637 for 4 hours, or after adding D-mannose for competitive inhibition. From Figure 6A and 6C It can be seen that recombinant BCG-S.FimH-EGFP has stronger fluorescence and adhesion, and w...

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Abstract

The invention discloses a recombinant BCG and its preparation method and application. The invention constructs a shuttle plasmid capable of expressing FimH protein on the surface of BCG, and transforms the gene fragment expressing FimH protein into wild-type BCG, thereby obtaining recombinant BCG. It has been verified that the recombinant BCG can express FimH protein on the surface of BCG, so the BCG can specifically bind to objects that selectively bind to the FimH protein, and can enhance the local innate immune effect and adaptive immune effect induced by BCG. Anti-tumor effect of blood mononuclear cells, and significantly improve the effect of BCG against bladder tumors, and treat bladder tumors.

Description

technical field [0001] The invention belongs to the technical field of medicine and relates to a recombinant BCG, in particular to a recombinant BCG capable of expressing FimH protein after recombination, a preparation method and application thereof. Background technique [0002] Bladder urothelial carcinoma is one of the common malignant tumors in the urinary system. Bladder cancer is divided into non-muscle-invasive bladder cancer (superficial) and muscle-invasive bladder cancer, of which superficial bladder cancer accounts for about 75% -80%. At present, the clinical standard treatment for bladder cancer is transurethral resection followed by bladder infusion therapy. Because surgical resection has a high recurrence rate, late bladder infusion is the key to prolonging the patient's life. The most clinically effective immunotherapy is infusion of BCG, which can kill tumors by activating immune cells. Bacillus Calmette-Guerin is the preferred bladder infusion drug for med...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/62C12N15/74A61K39/04A61P35/00C12R1/32
CPCC07K14/245C12N15/74A61K39/04A61P35/00C07K2319/02A61K2039/523Y02A50/30C07K2319/60C12R2001/32C12N1/205A61K2039/585A61P13/10
Inventor 张杨李鹏举
Owner XUZHOU HAIRUN BIOTECHNOLOGY CO LTD
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