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Preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1

A NY-ESO-1, Mycobacterium smegmatis technology, applied in the fields of biology and genetic engineering, can solve problems such as patient harm and tuberculosis infection

Inactive Publication Date: 2012-12-26
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] BCG has a long history as an immune adjuvant therapy for superficial bladder cancer, but live BCG is still toxic and may cause Mycobacterium tuberculosis infection, especially for immunodeficiency patients.

Method used

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  • Preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1
  • Preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1
  • Preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1

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Experimental program
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preparation example Construction

[0095] The preparation method of recombinant Mycobacterium smegmatis of the present invention may comprise the steps: (1) providing a plasmid that can be used to express the recombinant protein of the present invention; (2) transforming Mycobacterium smegmatis with said plasmid to obtain the recombinant Mycobacterium smegmatis of the present invention Mycobacteria; and (3) optionally culturing, expanding and / or isolating the obtained recombinant Mycobacterium smegmatis.

[0096] Transformation can be carried out by conventional transformation methods of the present invention such as electroporation and protoplast transformation. The fusion protein produced by the recombinant Mycobacterium smegmatis of the present invention can stimulate the immune system of mammals to produce antibodies against NY-ESO-1.

[0097] In a preferred embodiment of the present invention, a Mycobacterium smegmatis bacterial strain selected from the following group can be used to construct the recombin...

Embodiment 1

[0136] Example 1. Plasmid (pLA71NY) for cell wall-localized expression of NY-ESO-1 in Mycobacterium smegmatis or pLA73NY recombinant plasmid) construction

[0137] The human testis tissue mRNA was reverse-transcribed into cDNA according to conventional methods, and the NY-ESO-1 gene was amplified by PCR using the human testis tissue cDNA (GenBank number: AJ003149.1, purchased from Shanghai Shenergy Biology Co., Ltd.) as a template. The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The PCR reaction system is as follows (in μl):

[0138] template

forward primer

reverse primer

10×Taq buffer

10mMdNTP

wxya 2 o

Taq enzyme

1

1

1

5

1

40

1

[0139] The PCR reaction conditions are: 96°C for 5 minutes → (94°C for 30 seconds → 60°C for 30 seconds → 72°C for 50 seconds) for 30 cycles → 72°C for 5 minutes

[0140] The target fragment...

Embodiment 2

[0142] Example 2. Construction and verification of recombinant Mycobacterium smegmatis

[0143] 1. Mycobacterium smegmatis mc 2 155 Competent Cell Preparation

[0144] a. inoculate the Mycobacterium smegmatis strain into 3ml of M7H 9-Tween80 medium, and cultivate overnight at 37°C and 250rpm;

[0145] b. Inoculate 150 μl of bacterial liquid into 150 ml of M7H 9-Tween80 medium, and culture overnight at 37°C and 250 rpm;

[0146] c. Measure OD 600 , when it reaches 1.2-1.6, centrifuge at 2500rpm at 4°C for 5min;

[0147] d. Discard the supernatant, resuspend in 150ml ice water, and centrifuge at 2500rpm at 4°C for 5min;

[0148] e. Discard the supernatant, resuspend in 75ml of ice water, and centrifuge at 2500rpm at 4°C for 5min;

[0149] f. Discard the supernatant, resuspend in 10ml 10% glycerol, and centrifuge at 2500rpm at 4°C for 5min;

[0150] g. Discard the supernatant and resuspend in 1.5ml of 10% glycerol to obtain competent cells.

[0151] 2. Electroconversion ...

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Abstract

The invention relates to preparation and application of recombinant Mycobacterium smegmatis expressing NY-ESO-1. Specifically, the invention provides a plasmid for expressing a recombinant cancer-testis antigen NY-ESO-1 protein; and the plasmid comprises a beta-galactosidase signal peptide coding sequence or a beta- galactosidase coding sequence, and an NY-ESO-1 coding sequence. The invention also provides a recombinant Mycobacterium smegmatis containing the plasmid provided by the invention and its composition, and application thereof. The recombinant Mycobacterium smegmatis provided by the invention has significant killing effect on tumor cells expressing NY-ESO-1; therefore the recombinant Mycobacterium smegmatis can be used in effective prevention and / or treatment of the tumors.

Description

technical field [0001] The invention belongs to the fields of biology and genetic engineering, and in particular relates to a Mycobacterium smegmatis expressing tumor / testis antigen NY-ESO-1, its preparation and the use of the recombinant Mycobacterium smegmatis in immunotherapy of tumors application. Background technique [0002] Mycobacterium smegmatis (M.smegmatis) is an atypical mycobacterium. Compared with the typical mycobacterium BCG, Mycobacterium smegmatis grows rapidly and is harmless to humans, mice, guinea pigs and other animals. Virulence, as a bacterial vaccine carrier has broad application prospects. [0003] BCG has a long history as an immune adjuvant therapy for superficial bladder cancer, but live BCG is still toxic and may cause Mycobacterium tuberculosis infection, especially for immunodeficiency patients. Preliminary studies have shown that the acellular Mycobacterium smegmatis vaccine has a good immune regulation effect, has obvious protective effect...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/11C12N15/56C12N15/12C12N15/74C12N1/21A61K48/00A61K38/17A61P35/00C12R1/34
Inventor 陈丹丹徐帆洪高丹
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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