Transformation of lactic acid bacteria with tim3 human single chain antibody fusion gene and its application

A technology of fusion gene and TIM-3, applied in the direction of antibodies, applications, antibacterial drugs, etc., can solve the problems of high production cost and inconvenient medication, and achieve the effect of improving the level of cellular immunity in the body

Active Publication Date: 2021-08-03
广州维生君生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the traditional production method is used to prepare TIM-3 antibody, there are still two insurmountable problems of high production cost and inconvenient medication

Method used

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  • Transformation of lactic acid bacteria with tim3 human single chain antibody fusion gene and its application
  • Transformation of lactic acid bacteria with tim3 human single chain antibody fusion gene and its application
  • Transformation of lactic acid bacteria with tim3 human single chain antibody fusion gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1T

[0065] Preparation, screening and identification of embodiment 1 TIM3Fv and CTB-TIM3Fv fusion gene transformed lactic acid bacteria

[0066] 1. Synthesis and recombination of TIM3Fv and CTB-TIM3Fv fusion genes

[0067] (1) According to the amino acid sequence of the SPK1 signal peptide, the codons were optimized to make it suitable for high-efficiency expression in lactic acid bacteria, and the cDNA sequence of the SPK1 gene (SEQ ID NO: 1) was designed.

[0068] (2) Design the complementary DNA gene sequence according to the amino acid sequence and functional region structure of the TIM3 human antibody, and connect the heavy chain variable region (VH) and light chain variable region (VL) through a connecting peptide (Linker) and Flag tag sequence The connection forms the TIM3Fv gene, and the codon is optimized to make it suitable for high-efficiency expression of lactic acid bacteria (SEQ ID NO: 2).

[0069] (3) According to the CTB gene sequence, its codons were optimized to...

Embodiment 2

[0080] Example 2 Application of TIM3Fv and CTB-TIM3Fv transformed lactic acid bacteria live bacteria in the prevention and treatment of subcutaneous transplanted tumors of renal adenocarcinoma in mice

[0081] (1) Establishment of mouse kidney cancer RAG xenograft model

[0082] Select 8-week-old healthy male BABL / c mice with a body weight of 18-20 g, and in vitro cultured RAG mouse renal adenocarcinoma suspension (about 1×10 6 1) injection and inoculation into each mouse subcutaneously, and the bacteria powder was fed for treatment after the mouse transplanted tumor grew to a diameter of 0.5 cm.

[0083] (2) Cultivation of transformed bacteria

[0084] Use EM medium to resuscitate TIM3Fv and CTB-TIM3Fv to transform lactic acid bacteria strains (obtained in embodiment 1), after the bacteria reach a certain concentration, inoculate in the EM medium according to a certain ratio (1:100-500), wait for the bacterial density to reach OD 600 Bacteria were collected at 0.6-1.0.

[...

Embodiment 3

[0100] Example 3 Application of TIM3Fv and CTB-TIM3Fv Transformed Live Lactic Acid Bacteria in the Prevention and Treatment of Primary Colorectal Cancer

[0101] (1) The methods of TIM3Fv and CTB-TIM3Fv transformed lactic acid bacteria recovery, cultivation and preparation of viable bacteria powder are the same as those in Example 2.

[0102] (2) Construction of colorectal cancer APCmin / + model

[0103] APCmin / + male mice and C57BL female mice were selected and housed together in a ratio of one male and three females per cage. After 2 weeks after the birth of the suckling mouse, cut the toe marks, and cut off a small piece of tail or a small piece of ear. Genomic DNA was extracted by conventional methods, and the target DNA fragment was amplified by polymerase chain reaction (PCR) according to the method of Nanjing Model Animal Resource Bank to identify APCmin / + heterozygotes. Only 700bp band in the PCR product is wild-type mouse; only 300bp band is mutant homozygous mouse; ...

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Abstract

The present invention discloses the transformation of lactic acid bacteria with TIM3 human single-chain antibody (TIM3Fv) fusion gene, including the transformation of TIM3Fv gene or CTB-TIM3Fv fusion gene into lactic acid bacteria. The TIM3Fv gene or CTB-TIM3Fv fusion gene is inserted into lactic acid bacteria expression vector pLN and transformed The lactic acid bacteria are obtained, and the application of the transformed bacteria in preparing the oral live bacteria medicine with the effect of disease immunotherapy is also disclosed. In the present invention, the TIM3Fv and CTB-TIM3Fv fusion genes transformed into lactic acid bacteria can be prepared into viable bacterial preparations such as viable bacterial suspensions, dairy products, live bacterial powder, bacterial tablets or capsules, which can be used as immunotherapy live bacteria after oral administration and can proliferate in the human intestinal tract It is used in the prevention and treatment of tumors, viral infections or intracellular bacterial infections, primary and secondary immunocompromised diseases.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to transformation of lactic acid bacteria with TIM3 human single-chain antibody fusion gene and application thereof. Background technique [0002] In recent years, immunotherapy targeting immune checkpoint molecules has become a hot spot in anti-tumor research. A variety of immune checkpoint inhibitor drugs, such as PD-1 and PD-L1 humanized antibodies, have been approved for clinical use. application, and achieved good results. [0003] The main reason why malignant tumors can evade immune surveillance is that both malignant tumor cells themselves and immune cells express some co-inhibitory molecules, also known as immune checkpoint molecules, which produce immune suppression and escape from the body. immune surveillance. This is also the main obstacle that tumor treatment needs to break through. After activation, T cells express a series of immune checkpoint m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/13C12N15/62A61K39/395A61P35/00A61P31/12A61P31/04A61P37/04C12R1/01C12R1/23C12R1/25
CPCA61K39/39566A61K2039/523A61K2039/53A61P31/04A61P31/12A61P35/00A61P37/04C07K16/2818C07K2319/02C07K2319/55
Inventor 曾位森陈泽荣范宏英白杨孟晓静
Owner 广州维生君生物科技有限公司
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