Carrier capable of showing and expressing heterologous gene on surface of lactococcus lactis, and preparation method and application of carrier

A Lactococcus lactis, expression vector technology, applied in microorganism-based methods, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve problems such as unfavorable research and development of oral vaccines, affecting the application scope of lactic acid bacteria expression system, etc., Achieve far-reaching application prospects, great application value, and good immune effect.

Inactive Publication Date: 2012-11-28
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only affects the scope of application of the lactic acid bacteria expression system, but also is unfavorable to the research and development of oral vaccines that urgently need vaccine vectors

Method used

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  • Carrier capable of showing and expressing heterologous gene on surface of lactococcus lactis, and preparation method and application of carrier
  • Carrier capable of showing and expressing heterologous gene on surface of lactococcus lactis, and preparation method and application of carrier
  • Carrier capable of showing and expressing heterologous gene on surface of lactococcus lactis, and preparation method and application of carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of L.lactis expression vector pNZ8110-lysM

[0053] Construction of L. lactis expression vector pNZ8110-lysM see figure 1 .

[0054] 1. Preparation of L. lactis lysM gene

[0055] 1.1 Extraction of Genomic DNA of L.lactis NZ3900 Strain

[0056] ⑴ Take out the L.lactis NZ3900 strain preservation tube from the -80°C refrigerator, and inoculate 5ml GM with 100μl of the bacteria solution 17 culture medium at 30°C, 5% CO 2 Place the culture in the incubator for 12h to 14h, take 1.5ml to 3ml of bacterial liquid, centrifuge at 5000r / min for 5min, and collect the bacterial cells.

[0057] (2) Wash the bacteria once with 500μl TE buffer (10mM Tris-Cl, 1mM EDTA, pH8.0).

[0058] (3) Resuspend the cells in 50 μl TE buffer, add lysozyme to a final concentration of 10 mg / ml, and bathe in water at 37°C for 60 minutes.

[0059] (4) Add proteinase K to a final concentration of 50 μg / ml, mix well, add SDS solution to a final concentration of 10 g / L, and bat...

Embodiment 2

[0113] Example 2 Construction of recombinant strain L.lactis NZ3900 / pNZ8110

[0114] The recombinant strain L.lactis NZ3900 / pNZ8110 can be prepared according to the technical method provided by the seller of the strain L.lactis NZ3900, NIZO Food Research in the Netherlands or Mobitec in Germany, or by the following method:

[0115] 1. Preparation of L.lactis NZ3900 Competent Cells

[0116] 1.1 Inoculate 5ml liquid GSGM with 200μl bacterial solution from the L.lactis NZ3900 strain preservation tube stored at -80℃ 17 culture medium, put CO 2 Incubator, 30°C, 5% CO 2 Static culture overnight (in this specification, overnight refers to the duration of 12h ~ 14h).

[0117] 1.2 Take 5ml bacterial liquid and add 50ml GSGM 17 medium, 30°C, 5% CO 2 Static culture for 12h.

[0118] 1.3 Take 50ml of bacterial liquid and transfer it to 400ml GSGM 17 Medium, 30°C, 5% CO 2 static culture to OD 600 About 0.3.

[0119] 1.4 Centrifuge the bacterial liquid at 4000r / min, 4°C for 20min,...

Embodiment 3

[0143] Example 3 Construction of the L.lactis expression vector for surface display and expression of EGFP

[0144] For the construction of the L.lactis expression vector expressing EGFP on the surface, see Figure 4

[0145] 1. PCR amplification of EGFP gene

[0146] According to the EGFP gene sequence (GenBank: JN255744.1) in the Genbank nucleic acid database, PCR primers were designed using the biological software Primer5.0, and the upstream primer P3 sequence was 5-ATA GCCGGC ATG GTGAGCAAGGGCGAGG-3'(SEQ ID NO.6), a NaeI restriction enzyme site was introduced at its 5' end; the downstream primer P4 sequence is 5-ACAT GCATGC CTTGTACAGCTCGTCCATGCCG-3' (SEQ ID NO.7), a SphI restriction enzyme site was introduced at the 5' end of the downstream primer.

[0147] PCR reaction system: Using the plasmid pIRES2-EGFP (BD Biosciences Corporation, USA) as a template, prepare a reaction system: 1 μl of 20 μM upstream primer, 1 μl of 20 μM downstream primer, 25 μl of 2×Taq PCR Master...

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Abstract

The invention relates to a carrier capable of showing and expressing a heterologous gene on the surface of lactococcus lactis, and a preparation method and application of the carrier. The carrier has the nucleotide sequence which is shown as SEQ ID NO.1 in a sequence table, and contains promoter Pnis and lysM genes, a signal peptide secretion sequence ssusp45, a multiple cloning endonuclease site, and replicon repA and repC components. A method for constructing the carrier has the following steps of: amplifying a lactococcus lactis NZ3900 strain autolysin (AcmA) anchoring area gene lysM by applying a polymerase chain reaction (PCR) technology, performing enzyme digestion on the lysM and a carrier pNZ8110 and connecting, wherein a connection product is used for transforming a E.coli MC1061 strain, and extracting recombinant plasmid from positive transformed bacteria which are subjected to enzyme digestion and sequencing identification to obtain the expression carrier. The exogenous gene and the carrier are connected and then transferred into lactococcus lactis NZ3900, and the expression of the exogenous gene and the combination of expression protein and host cell walls can be realized by induction of nisin. The carrier can be used for expressing foreign proteins including vaccine antigens and has a wide application range.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an expression vector capable of expressing foreign protein in Lactococcus lactis and displaying the expressed protein on the surface of bacteria, and also relates to a preparation method and application of the vector. Background technique [0002] At present, for some gastrointestinal pathogen infections, such as Helicobacter pylori (H. pylori) infection, antibiotic treatment can increase bacterial resistance and has no preventive effect on repeated infection, so it is more effective for such diseases The prevention and treatment of the disease can only hope for the success of vaccine research. In vaccine research, oral vaccines have attracted attention due to their safety, rationality, and convenience [Levine MM. "IDEAL" vaccines for resource poor settings. Vaccine, 2011, 29 Suppl 4:D116-25.]. However, the current research and development of oral vaccines is restricted due to the lac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/66C12N1/21C12R1/19
Inventor 段广才张荣光程文滨范清堂张卫东郗园林陈帅印
Owner ZHENGZHOU UNIV
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