CN108220219B

CN108220219B - Lactobacillus plantarum food-grade expression system and application thereof in heterologous protein expression

Info

Publication number: CN108220219B
Application number: CN201810127250.XA
Authority: CN
Inventors: 杨瑶; 陈英; 祁敏; 周思思; 徐梦秋; 刘艳容
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2018-02-08
Filing date: 2018-02-08
Publication date: 2022-02-22
Anticipated expiration: 2038-02-08
Also published as: CN108220219A

Abstract

The invention discloses a lactobacillus plantarum food-grade expression system and application thereof in heterologous protein expression. The lactobacillus plantarum food-grade expression system comprises a food-grade host lactobacillus plantarum (A)Lactobacillus plantarum）NZ5333, food-grade expression vector pSIP497, culture medium MRS-Y and culture medium MRS + N; the food grade host Lactobacillus plantarum (A)Lactobacillus plantarum) NZ5333 is knock-out on the genome of Lactobacillus plantarum WCFS1 by Cre-loxP technologyglmSThe gene thus obtained, the ability to synthesize glucosamine-6-phosphate synthase is lost. The expression system is applied to the expression of heterologous proteins, and has good expression effect. The expression system is a food-grade expression system, the DNA element of which is from a microorganism which is generally recognized as safe and does not contain an antibiotic resistance selection marker.

Description

Lactobacillus plantarum food-grade expression system and application thereof in heterologous protein expression

Technical Field

The invention relates to the field of molecular biology, in particular to a lactobacillus plantarum food-grade expression system and application thereof in heterologous protein expression.

Background

With the development of science and technology, more and more researches show that lactobacillus has significant effects on the aspects of immunoregulation, maintaining the balance of flora in intestinal tracts, promoting the absorption of nutrient substances, relieving lactose intolerance, inhibiting pathogenic bacteria, reducing the content of serum cholesterol, preventing cardiovascular diseases and the like. Since lactobacillus has generally recognized biosafety (GRAS), the development and utilization of lactobacillus as a genetic engineering vector strain has considerable potential and value in the fields of food development, nutritional health and medical health. At present, researches on the production of functional proteins, the development of oral vaccines, the development of new drugs and the like by using lactic acid bacteria genetic engineering vectors have been reported. It should be noted that the research on lactobacillus in lactobacillus is especially a hot point of research, on one hand, the reports of lactobacillus with significant probiotic function are endless, such as lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus reuteri, etc., on the other hand, the lactobacillus has wide sources and good fermentation performance, and is also an important strain in industry, especially in food industry, such as lactobacillus delbrueckii, etc.

The traditional lactobacillus expression vectors all have one or more genes for encoding the resistance of specific antibiotics (such as erythromycin and chloramphenicol), and although the resistance markers ensure the effective performance of transformant screening, the transferability and potential threat of the resistance markers obviously reduce the practical value of the molecular genetic modified lactobacillus in the fields of food and medicine, so that the development of food grade screening markers and the establishment of a food grade expression system for expressing in heterologous proteins are urgently needed.

The prior art has divided food grade selection markers into two broad categories, dominant selection markers and complementary selection markers. Dominant selection markers usually achieve the purpose of screening by using the characteristics of host bacteria such as bacteriocin resistance, heat sensitivity, saccharide utilization rate and the like, for example, the mature selection marker derived from lactococcus lactis Nisin and most widely applied at present is developed; the complementary screening marker generally utilizes the bacterial growth change caused by the deletion or mutation of certain genes of bacteria such as housekeeping genes to achieve the screening purpose, such as a lactococcus lactis thymidylate synthetase gene thyA screening marker, a lactobacillus plantarum pyruvic acid racemase alr screening marker and a lactobacillus casei Q-5/pQJ5 screening system. At present, few lactobacillus food grade screening markers are developed and applied, the requirements of food grade expression of a plurality of target proteins cannot be met, and more new screening markers are yet to be developed. The invention discloses an expression system using lactobacillus plantarum glmS gene as a screening marker, which is a novel screening marker in lactobacillus heterologous expression. Glucosamine-6-phosphate synthase (GlmS), which promotes the conversion of fructose-6-phosphate and L-glutamine to Glucosamine-6-phosphate, is the rate-limiting enzyme in the hexosamine pathway and cell wall synthesis reactions of the glucose metabolic pathway. The products of glucosamine-6-phosphate synthase catalyzed by HBP are N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), both of which are important for the living organism's life activities, polysaccharides of many biological cells such as chitin and mucopolysaccharide are finally synthesized by N-acetylglucosamine (GlcNAc), which is also an important precursor for the synthesis of bifidus factors. glmS is a gene encoding glucosamine-6-phosphate synthase, and studies have shown that without the glmS encoding gene, the strain cannot grow normally in a medium without glucosamine or N-acetylglucosamine, and cells rapidly lyse and die. In addition, as the glucosamine-6-phosphate synthase is a food-grade molecule, the glmS gene is selected as a screening marker, a food-grade expression system of lactobacillus plantarum is developed, and an effective and safe means is provided for the application research of lactobacillus in the fields of food and medicine.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a set of lactobacillus plantarum food-grade expression system and application thereof in heterologous protein expression.

A set of Lactobacillus plantarum food-grade expression system comprises a food-grade host Lactobacillus plantarum NZ5333, a food-grade expression vector pSIP497, a culture medium MRS-Y and a culture medium MRS + N; the food-grade host Lactobacillus plantarum (Lactobacillus plantarum) NZ5333 is obtained by knocking out glmS gene on Lactobacillus plantarum WCFS1genome by Cre-loxP technology, and loses the capability of synthesizing glucosamine-6-phosphate synthase.

The food grade host Lactobacillus plantarum (Lactobacillus plantarum) NZ5333 is preserved in China general microbiological culture collection center (CGMCC) in 2017 at 12 months 08, and the preservation address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 15039.

The construction method of the food-grade expression vector pSIP497 comprises the steps of knocking out a base sequence of a coding erythromycin resistance gene on the basis of a lactobacillus expression vector pSIP409, inserting a glmS gene derived from lactobacillus plantarum WCFS1, and inserting P derived from a lactobacillus plantarum WCFS1 strain_ldhLA promoter.

The glmS gene of the Lactobacillus plantarum WCFS1 is a food-grade marker for complementary screening of food-grade host Lactobacillus plantarum (Lactobacillus plantarum) NZ5333 and a food-grade expression vector pSIP 497.

The application of the lactobacillus plantarum food-grade expression system in heterologous protein expression.

The application of the lactobacillus plantarum food-grade expression system in heterologous protein expression comprises the following steps: the method comprises the steps of adopting a PCR amplification reporter gene red fluorescent protein gene mCherry to insert a food-grade expression vector pSIP497 multiple cloning site, adopting an electrical transformation method to transfer the constructed vector into lactobacillus plantarum NZ5333, utilizing SppIP to induce peptide to induce expression, and detecting the expression of the red fluorescent protein in lactobacillus plantarum NZ5333 cells, wherein antibiotics are not added in the expression process.

Method for detecting the expression of red fluorescent protein: the fluorescence intensity value is measured by a microplate reader or the red fluorescence is observed by an inverted fluorescence microscope.

Has the advantages that:

the lactobacillus plantarum food-grade expression system provided by the invention is provided with a food-grade host lactobacillus plantarum NZ5333 and a food-grade expression vector pSIP497, the host and the vector have food-grade markers capable of completing complementary screening, antibiotics are not required to be added in the heterologous expression process of the reporter gene, and potential hazards of biological safety caused by transfer of resistance factors are avoided.

Drawings

FIG. 1 shows the validation result of knock-out plasmid pNZ5326, in which the M-DNA molecular weight is standard, the length of 1-5 'homologous recombination arm is 1064bp, the length of 2-3' homologous recombination arm is 1001 bp;

FIG. 2 shows the results of glmS knock-out PCR, where M-DNA molecular weight standard, 3-glmS gene, length 1818bp, 4-upstream lateral amplification product, length 1194 bp; 5-downstream outer amplification product with length 1283 bp;

FIG. 3 shows the PCR-verified result of the genome WCFS1 after the removal of chloramphenicol, wherein the M-DNA molecular weight standard, 6-lateral amplification product, length is 2183 bp;

FIG. 4 shows the results of amplification of the ldhL promoter and glmS gene, where M-DNA molecular weight standard, 7-P_ldhLAmplification product, length 500bp, 8-glmS gene amplification product, length 1818 bp;

FIG. 5 shows the removal verification result of food grade vector pSIP497 erythromycin, wherein M-DNA molecular weight standard, 9-P_ldhL-glmS gene, length 2318 bp; 10-erythromycin gene with a length of 602 bp;

FIG. 6 is a flow chart of the construction of the food grade expression vector pSIP497 of the present invention;

FIG. 7 shows the PCR verification result of the host bacterium into which the mCherry gene was introduced, wherein the M-DNA molecular weight is standard, and the length of the 11-mCherry gene is 711 bp;

FIG. 8 is a graph of mCherry expression strain fluorescence intensity vs. growth;

FIG. 9 is a graph showing the effect of expressing red fluorescent protein using the food-grade expression system of the present invention.

Detailed Description

The present invention will be described in further detail below with reference to specific examples.

Example 1

Bacterial strains, plasmids, main materials and reagents used and involved in the experiments of the invention:

lactobacillus plantarum WCFS1 was purchased from cikay microbiology ltd, guangdong;

a food grade host lactobacillus plantarum NZ 5333;

coli DH5 α competent cells were purchased from Beijing Quanjin Biotechnology Ltd;

plasmids pNZ5319, pNZ5348 are given by Jolanda m.lambert, dairy institute, the netherlands;

plasmid pSIP409 was purchased from Biovector NTCC plasmid vector bacterial cell protein antibody gene collection;

e.coli K12 delta glmS competent cells knocked out by the glmS gene are constructed and stored in the early stage of a laboratory;

a bacterial genome extraction kit;

a small extraction kit of SanPrep column type plasmid DNA;

an ion exchange type plasmid extraction kit;

N-acetyl-D-glucosamine (GlcNAc);

erythromycin, chloramphenicol, ampicillin, agarose, 4S nucleic acid dye, etc. were purchased from Shanghai Biotechnology engineering, Inc.; polymerases such as Ex taq and rtaq, Ex taq, PrimeSTAR Max Premix (2 ×), restriction enzymes, ligases, pMD19-T vector, DNA Marker, and DNA gel recovery kit provided by Beijing Baoji technology Limited; other main reagents are domestic analytical pure products;

MRS culture medium special for lactobacillus; mutant strains were cultured in MRS + N medium: 10g of peptone, 8g of beef powder, 4g of yeast powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diamine hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801 g of tween and 22.121g of N-acetyl-D-glucosamine (GlcNAc); LB medium for E.coli.

The recombinant lactobacillus screening culture medium MRS-Y culture medium of the invention: 10g of peptone, 8g of beef powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate and 801 g of tween.

Recombinant E.coli selection Medium MT (M9 plus 1% peptone) medium was used in the present invention.

Example 2

1. Construction of food-grade host Lactobacillus plantarum NZ 5333.

(1) Extraction of whole genome DNA of lactobacillus plantarum WCFS1

Extracting by using a bacterial genome extraction kit according to the instruction manual.

(2) PCR amplification of the 5 'and 3' homology arms of the glmS Gene and construction of the suicide plasmid pNZ5326 primer design and Synthesis

The 5 'end homologous arm and the 3' end homologous arm of the glmS gene are respectively designed by taking NCBI Lactobacillus plantarum WCFS1 gene (GenBank: AL935263.2) as a template,

primer of homologous arm at 5' end of glmS gene:

A:5’-GGGCTCGAGCAGGATACAGTCGTCACAACG-3’

B:5’-GGGATTTAAATCCACACATAAATTAATCTTCC-3’

3' end homology arm primer of glmS gene:

C:5’-CCCGAGCTCGTAAGTGTGTGGGCACCAAG-3’

D:5’-GGGAGATCTGATTGCCACAAGTAGCCAC-3’

ii.amplification, cloning and sequencing of the 5 'and 3' homology arms of the glmS Gene

The PCR reaction system is as follows:

(ii) PCR reaction conditions were as follows:

pre-denaturation at 95 ℃ for 5 min; (melt 95 ℃, 45 s; anneal 60 ℃, 45 s; extend 72 ℃, 1 min). times.30 cycles; 72 ℃ for 10 min; storing at 4 deg.C

(iii) TA cloning sequencing

The PCR product of the 5 'homology arm and the PCR product of the 3' homology arm are respectively connected with a pMD19-T simple vector for 1h at 16 ℃, then E.coli DH5 alpha chemical competent cells are transformed, then the E.coli DH5 alpha chemical competent cells are coated on an LB/Amp (100 mu g/mL) plate, and the E.coli DH5 alpha chemical competent cells are cultured at 37 ℃ until single clone formation. Single clones were picked up in 3mL of the corresponding medium and cultured with shaking at 37 ℃ and 220 rpm. The obtained clones are subjected to plasmid extraction, are cut and verified by XhoI-SwaI and Ecl136II-BglII respectively, and the correctly verified plasmids are sent to Shanghai Biometrics company Limited by Biotechnology with M13Forward Primer and M13Reverse Primer primers for sequencing.

(3) Construction of homologous recombination knockout plasmid pNZ5326

The plasmid with the upstream and downstream homologous arms of the T clone which is correctly sequenced by means of XhoI-SwaI and Ecl136II-BglII are respectively cut in a large quantity of enzyme, separated by 1% agarose gel, cut off DNA fragments under an ultraviolet lamp, and separated, purified and eluted by using a gel recovery kit. pNZ5319 is recovered in large amount by an ion exchange column kit, the plasmid concentration is about 5 mug/muL, the Ecl136II and BglII double enzyme digestion are firstly carried out to recover a large fragment vector, and the large fragment vector and a recovery product of a 3' end homology arm are connected overnight at 16 ℃ by T4 ligase. Coli DH 5. alpha. chemically competent cells were transformed, plated on LB plates (containing 10. mu.g/mL chloramphenicol and 250. mu.g/mL erythromycin), and cultured at 37 ℃. And extracting plasmids from the liquid-cultured clone, and carrying out enzyme digestion verification. Verifying that a large amount of correct clones extract plasmids, performing XhoI-SwaI double enzyme digestion treatment, connecting with a recovery product of a homologous arm at the 5' end overnight (16 ℃), transforming Escherichia coli chemically competent cells, coating the cells on an LB solid culture medium containing chloramphenicol and erythromycin, and extracting the plasmids after culture. The 5 'and 3' homology arms were verified by double digestion with XhoI-SwaI and Ecl136II-BglII, respectively, and the results are shown in FIG. 1, to obtain a knock-out plasmid pNZ5326 for homologous recombination. The molecular weight standards of the DNA used are 2000, 1000, 750, 500, 250 and 100bp in sequence from top to bottom.

(4) The glmS gene of WCFS1 was traceless knocked out using suicide plasmids pNZ5326 and pNZ5348

Preparation of competent cells of Lactobacillus plantarum WCFS1

Picking WCFS1 from an MRS plate, carrying out monoclonal inoculation on the MRS plate in 50mL of MRS culture medium, carrying out anaerobic culture at the temperature of 30 ℃ overnight, and centrifuging the culture solution at the temperature of 4 ℃ for 10 minutes to precipitate cells; cells were gently suspended with an equal volume of precooled 0.5M sucrose (containing 10% glycerol), pelleted under the same conditions, decanted, and washed once more. The cells were suspended in 100. mu.L of a 0.5M sucrose solution containing 10% glycerol and dispensed into two tubes at a volume of 40. mu.L.

Ii.electrotransfer of the knockout plasmid to competent cells

A large amount of pNZ5326 was extracted using an ion exchange column type plasmid extraction kit, and 5. mu.L of pNZ5326 was added to 40. mu.L of Lactobacillus plantarum competent cells, mixed well, transferred to a pre-cooled 2mm electric rotor (eppendorf), and electrically shocked at 1800V (eppendorf AG electrotransfer). After the electric shock was completed, the bacteria were immediately suspended in 1mL of MRS medium (containing 10mMGlcNAc), transferred to 2mL of eppendorf tubes, incubated at 30 ℃ for 3 hours, spread on MRS + N plates (containing 10. mu.g/mL of chloramphenicol), and cultured at 30 ℃. The grown monoclonals are cultured in the corresponding liquid culture medium, the whole genome is extracted, and the glmS gene is identified by using a glmS outer primer, as shown in FIG. 2.

(5) Removal of chloramphenicol resistance marker

In the first step, the glmS gene is knocked out and removed, and chloramphenicol is used as a screening marker to obtain a chloramphenicol resistant strain. The strain is prepared into competent cells (the preparation method is consistent with WCFS1 competent preparation), and 1800V electric shock is transformed into pNZ5348 extracted by a 3-L ion exchange column. Immediately, the cells were suspended in 1mL of MRS medium (containing 10mM GlcNAc), incubated at 30 ℃ for 3 hours, then spread with MRS + N solid medium, and cultured at 37 ℃ for 2 days. The target clone was verified by PCR (see FIG. 3), and the chloramphenicol gene was successfully removed, and the obtained mutant strain was named NZ 5333.

Example 3 construction of the glmS tagged food grade expression vector pSIP497

1.P_ldhLLigation cloning of glmS Gene fragments

Amplification of the ldhL promoter Gene and the glucosamine-6-phosphate synthase-encoding Gene glmS

Extracting a whole genome of Lactobacillus plantarum WCFS1 by using a bacterial genome extraction kit, searching to obtain a glmS gene sequence by referring to a whole genome sequence (GenBank: AL935263.2) published by Lactobacillus plantarum WCFS1, designing primers, and amplifying the glmS gene (1818bp) and P by PCR (polymerase chain reaction) respectively_ldhLThe fragment (500bp), shown in FIG. 4, was prepared by the following steps:

primer sequences:

Pldhl-F:5’-AGATCTAATCTTCTCACCGTCTTG-3’

Pldhl-R:5’-CACCAACAATTCCACACATTCCTACGAGAGGATGACTTATT-3’

glmSF:5’-AATAAGTCATCCTCTCGTAGGAATGTGTGGAATTGTTGGTG-3’

glmSR:5’-GGGCTCGAGTCAGTGGTGGTGGTGGTGGTGTTCAACGGTCACACTCTTG-3’

(ii) reaction system:

(iii) reaction conditions: pre-denaturation at 95 ℃ for 5 min; (melting 95 ℃, 60 s; annealing 60 ℃, 90 s; extension 72 ℃, 3 min). times.30 cycles; 72 ℃ for 10 min; storing at 4 ℃.

Ii, overlap-PCR amplification after glmS ligation of ldhL promoter Gene and glucosamine-6-phosphate synthase encoding Gene

Amplified P_ldhLAnd the glmS gene fragment is purified by a gel recovery kit respectively, and the P is amplified by connecting Pldhl-F and glmSR by using a primer pair by using the two purified fragments as templates_ldhLThe glmS fragment, PCR system and reaction conditions were as follows.

Reaction system:

(ii) reaction conditions: pre-denaturation at 95 ℃ for 5 min; (melting 95 ℃, 60 s; annealing 60 ℃, 90 s; extension 72 ℃, 3 min). times.30 cycles; 72 ℃ for 10 min; storing at 4 deg.C

After the amplification product was purified, it was ligated with T vector (16 ℃ C.), DH 5. alpha. competent cells were transformed, plated on LB solid medium containing 100. mu.g/mL Amp, and cultured at 37 ℃. Selecting single clone, culturing, extracting plasmid DNA, performing double enzyme digestion verification, and screening positive clone by sequencing comparison to verify correct plasmid.

Ii, using overlap P_ldhLRemoval of the erythromycin tag from the pSIP409 vector by the glmS fragment

BglII-XhoI enzyme digestion treatment of T clone vector to recover P_ldhLThe glmS fragment, followed by double digestion of the pSIP409 vector with BamHI and SalI, and purification to recover the linear vector fragment. P_ldhLThe ligation of the glmS fragment to the vector was performed overnight at 16 ℃. The next day is rotatedColi K12 Δ glmS chemically competent cells were lysed and incubated with 1mL MT medium for 1h after heat shock in a mixed ice bath, cultured on non-resistant MT-screening plates, overnight at 37 ℃. Selecting a single clone for culture, extracting plasmids, and carrying out PCR verification on Pldhl-F-glmSR and EryintF-EryintR by using primer pairs, wherein the sequence of the primer of the EryintF-EryintR, the PCR reaction system and the reaction conditions are as follows, the result is shown in figure 5, and the food-grade expression vector pSIP497 is successfully constructed.

2. The application of the lactobacillus plantarum food-grade expression system for heterogeneously expressing the mCherry fluorescent protein in the expression of a heterogenous protein by utilizing the lactobacillus plantarum food-grade expression system constructed by the invention comprises the following steps: the method comprises the steps of adopting a PCR amplification reporter gene red fluorescent protein gene mCherry to insert a food-grade expression vector pSIP497 multiple cloning site, adopting an electrical transformation method to transfer the constructed vector into lactobacillus plantarum NZ5333, utilizing SppIP to induce peptide to induce expression, and detecting the expression of the red fluorescent protein in lactobacillus plantarum NZ5333 cells, wherein antibiotics are not added in the expression process. As shown in FIG. 7, the red fluorescent protein was well expressed in Lactobacillus plantarum NZ5333 cells.

In addition, the present invention is not limited to the above embodiments, and may be implemented in various ways without departing from the scope of the invention.

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Claims

1. A set of lactobacillus plantarum food-grade expression system is characterized by comprising a food-grade host lactobacillus plantarum (A)Lactobacillus plantarum）NZ5333, food-grade expression vector pSIP497, culture medium MRS-Y and culture medium MRS + N; the food grade host Lactobacillus plantarum (A)Lactobacillus plantarum) NZ5333 is knock-out on the genome of Lactobacillus plantarum WCFS1 by Cre-loxP technologyglmSThe ability to synthesize glucosamine-6-phosphate synthase is lost upon acquisition of the gene, and the food-grade host Lactobacillus plantarum (A)Lactobacillus plantarum）NZ5333, which is preserved in China general microbiological culture Collection center (CGMCC) at 2017, 12 months and 08 days, and the preservation address is as follows: no. 3 Xilu No.1 Beijing, Chaoyang, with the collection number of CGMCC No. 15039; the construction method of the food-grade expression vector pSIP497 comprises the following steps: knocking out base sequence of erythromycin resistance gene based on lactobacillus expression vector pSIP409, and inserting into lactobacillus plantarum WCFS1glmSGene, P inserted into strain derived from Lactobacillus plantarum WCFS1_ldhLA promoter, theglmSThe gene is obtained by amplifying a full gene sequence GenBank: AL935263.2 of reference lactobacillus plantarum WCFS1 by using a primer glmSF and a primer glmSR,

the sequence of the primer glmSF is as follows:

5’-AATAAGTCATCCTCTCGTAGGAATGTGTGGAATTGTTGGTG-3’，

the sequence of the primer glmSR is as follows:

5’-GGGCTCGAGTCAGTGGTGGTGGTGGTGGTGTTCAACGGTCACACTCTTG-3’，

the MRS + N culture medium: 10g of peptone, 8g of beef powder, 4g of yeast powder, 20g of glucose, 2g of dipotassium phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801 g of tween and 22.121g of N-acetyl-D-glucosamine; the MRS-Y culture medium: 10g of peptone, 8g of beef powder, 20g of glucose, 2g of dipotassium phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate and 801 g of tween.

2. Use of a set of lactobacillus plantarum food-grade expression systems according to claim 1 for heterologous protein expression, comprising the steps of: PCR amplification of reporter gene red fluorescent protein genemCherryInserting a multiple cloning site of a food-grade expression vector pSIP497, transferring the constructed vector into lactobacillus plantarum NZ5333 by adopting an electrical transformation method, inducing expression by utilizing SppIP induction peptide, detecting the expression of the red fluorescent protein in lactobacillus plantarum NZ5333 cells, and adding no antibiotic in the expression process, wherein the method for detecting the expression of the red fluorescent protein comprises the following steps: the fluorescence intensity value is measured by a microplate reader or the red fluorescence is observed by an inverted fluorescence microscope.