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Method for integrating exogenous gene into sheep listeria genome

A technology of Listeria and exogenous genes, which is applied in the field of integrating exogenous genes into the genome of Listeria ovis, can solve problems such as no public reports, and achieve wide application range, easy large-scale production, and simple and easy screening methods Effect

Active Publication Date: 2014-07-30
南京颂悦生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to prepare a recombinant live bacterial vaccine using Li as a carrier, it is necessary to integrate the foreign gene into the Li genome, but there is no public report on the method of integrating the foreign gene into the Li genome

Method used

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  • Method for integrating exogenous gene into sheep listeria genome
  • Method for integrating exogenous gene into sheep listeria genome
  • Method for integrating exogenous gene into sheep listeria genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Preparation of the first recombinant plasmid pCW203

[0072] (1) Amplify the PRRSV envelope protein GP5 coding gene ORF5 gene fragment (GenBank: JX105430.1)

[0073] The recombinant plasmid pRK5-GP5 was used as a template for PCR amplification, and the reaction system was: 10×ultra pfu buffer 2.5μl, 4dNTPs (10mmol / L. species) 0.4μl, upstream primer (10μmol / L) (ORF5-f.5' -TATGTAAGCTTTGTT GGGGAAGTGCTTGACC-3') 0.8 μl, downstream primer (10 μmol / L) (ORF5-r5'-ATAACTCG AGGAGACGACCCCATT GTTCC-3') 0.8 μl, pRK5-GP5 0.75 μl, ultra pfu 0.25 μl, ddH 2 O19.5 μl. Reaction cycle conditions: 94°C for 3min → (94°C for 30s, 55°C for 30s, 72°C for 1min20s) × 20 cycles → (94°C for 30s, 66°C for 30s, 72°C for 1min20s) × 10 cycles → 72°C for 10min → 4°C . After the PCR product was subjected to 0.8% agarose gel electrophoresis, a specific band was seen at 620bp, which was in line with the expectation ( figure 1 ). The PCR amplification products were qualified by sequencing.

...

Embodiment 2

[0077] Embodiment 2: Preparation of the second recombinant plasmid pCW153 and the third recombinant plasmid pCW154

[0078] (1) Amplify Li orfXYZ (GenBank: AJ249805.1), Li mpl (GenBank: AY510073.1), Li orfBAldh (GenBank: AJ249805.1) and Ery (see the sequence in SEQ ID NO.2 (6642).. (8092)) gene fragment

[0079]Take the Li strain out of the -80°C refrigerator, melt it in a water bath at 37°C, and streak the BHI agar plate, culture at 37°C for 24 hours, pick the colony and transfer it to 5ml of BHI liquid medium, shake it at 220rpm at 37°C for 12-18 hours, use a general-purpose The column genome extraction kit extracts genomic DNA as a template to amplify Li orfXYZ, Li mpl and Li orfBAldh gene fragments. The reaction system for amplifying Li orfXYZ is: 10×ultra pfu buffer 2.5μl, 4dNTPs (10mmol / L. species) 0.5μl, upstream primer (10μmol / L) (Li orfXYZ-f: 5'-ATAAAGCTTGTCGACATACTAGTTTCCAGCAAAGCGATT C-3') 0.8 μl, downstream primer (10 μmol / L) (Li orfXYZ-r: 5'-AACTCTGCGGCCGCTTATTTT...

Embodiment 3

[0094] Example 3: Preparation of the first recombinant strain Li lacZ and the second recombinant strain LiΔactA lacZ

[0095] (1) Amplify the lacZ gene fragment (see (8192)..(12171) in SEQ ID NO.1 for the sequence)

[0096] Use the plasmid pHS-LV as a template to amplify lacZ by PCR. The reaction system is: 10×ultra pfu buffer 2.5 μl, 4dNTPs (10 mmol / L. species) 0.6 μl, upstream primer (10 μmol / L) (LacZ-f: 5'- ATAAGCGGCCG CTAGCTTTAAGGCTAAATGCCG-3') 0.8 μl, downstream primer (10 μmol / L) (lacZ-r: 5'-ATAAG-CGGCCGCCTAGAGTGACTTTATGTTGAG-3') 0.8 μl, pHS-LV plasmid sample 1 μl, ultra pfu 0.25 μl, ddH 2 O19.05 μl. The reaction cycle conditions are: 94°C 3min→(94°C 30s, 52°C 30s, 72°C 4min20s)×20 cycles→(94°C 30s, 70°C 30s, 72°C 4min20s)×10 cycles→72°C 10min→4 ℃. After the PCR product was electrophoresed on a 0.8% agarose gel, a specific band was seen at 4001bp, which was in line with expectations (see figure 1 ). The PCR amplification products were qualified by sequencing.

[00...

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Abstract

The invention relates to a method for integrating an exogenous gene into a sheep listeria genome. The method comprises the following steps of: electro-transforming a first target recombinant plasmid into a first recombined bacterium, and then performing single / double homologous recombination hybridizing cultivation on the bacterium which is obtained after transforming, thereby obtaining a first target bacterium, wherein the first target bacterium is a recombined sheep listeria, the exogenous gene is integrated with an orfBAldh gene in the genome, and a code protein of the exogenous gene is stably expressed and secreted; or electro-transforming a second target recombinant plasmid into a second recombined bacterium, and then performing single / double homologous recombination hybridizing cultivation on the bacterium which is obtained after transforming, thereby obtaining a second target bacterium, wherein the second target bacterium is a recombined sheep listeria, actA gene and plcB gene are not in the genome, the exogenous gene is integrated with the orfBAldh gene in the genome, and the code protein of the exogenous gene is stably expressed and secreted.

Description

technical field [0001] The invention relates to a method for integrating exogenous genes into the genome of Listeria ivanovii (hereinafter referred to as Li). Background technique [0002] Li belongs to the genus Listeria, which is only pathogenic to sheep and other animals, but not to humans. It has a biological similarity to another bacterium in the genus Listeria—Listeria monocytogenes (hereinafter referred to as Lm). academic characteristics. [0003] Due to the unique biological characteristics of Lm that can grow in the phagocytic cytoplasm of the infected host, it has become a good T cell immune activation adjuvant and has been widely used in vaccine preparation. Some recombinant live bacteria with Lm as the carrier Therapeutic tumor vaccines have entered clinical trials. As reported by Wood LM et al., two genetically recombinant live Lm vaccines (Lm-LLO-CD105A and Lm-LLO-CD105B) can significantly reduce the volume of primary and metastatic tumor tissue in mouse ani...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/01
Inventor 汪川沈海浅
Owner 南京颂悦生物科技有限公司
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